Field of the Invention

The present invention relates to a method and kit for the detection of microorganisms, in particular for the detection of bacteria causing tuberculosis, Legionnaires' disease and/or atypical pneumonias.

Background to the Invention

Respiratory tract infections are believed to be the most common reason for patients to visit their doctor or pharmacist in the UK. Respiratory tract infections are classified into upper respiratory tract infections and lower respiratory tract infections, with lower respiratory tract infections generally the more serious. Lower respiratory tract infections are the leading cause of death for all infectious diseases. The most common lower respiratory tract infection is pneumonia, which is usually caused by viruses or bacteria and less commonly by fungi or parasites. Infective agents were historically divided into "typical" (usually bacterial pneumonia caused by Streptococcus pneumoniae and Haemophilus influenzae) and "atypical" (as a distinction from the typical pneumonia), although this classification is no longer so widely used.

Tuberculosis is another common respiratory tract infection and is caused by various strains of mycobacteria, in particular from a group of species known as the Mycobacterium tuberculosis complex ( tuberculosis, M. africanum, M. bovis,, M. microti, M. canettii, M. caprae and M. pinnipedii). The World Health Organization (Global tuberculosis report 2012) estimated 8.7 million new cases of TB (13% co-infected with HIV) and 1.4 million deaths, including almost one million among HIV-negative individuals and 430.000 among people who were HIV-positive. Also mentioned in this Report, was a concern about the progress of the disease in several regions, such as Africa and Eastern Mediterranean. Nevertheless, the incidence of the disease is highest in Asia and Africa, with South-East Asia and Western Pacific regions having about 60% of the cases. Thus, most deaths occur in developing countries, however, the disease is currently on the rise in the UK. Regarding resistance to antibiotics, 3.7% of new cases worldwide and 20% of previously treated cases were estimated to have multidrug-resistant tuberculosis (MDR-TB). The highest proportions of TB patients with MDR-TB are in eastern Europe and central Asia. The Family Legionellaceae is a pathogenic group of gram negative bacteria which includes more than 50 species of Legionella, that may cause Legionnaires' Disease. L. pneumophila, L. micdadei, L. dumoffii and L. longbeachae are the most important species from a clinical point of view.. Legionnaires' disease is a serious lung infection which was recognized as a distinct entity after 182 delegates became ill, and 28 died, at an American Legion convention in Philadelphia in 1976. Promptly treated, Legionnaires' Disease can be cured in 95 to 99% of cases occurring in otherwise healthy persons. Less than one-half of patients may respond if there is a delay in therapy, immunosuppression, or respiratory failure (1 ). Untreated disease causes death in about 15% of previously healthy patients and up to 75% of severely immunocompromised ones (2). Beta- lactam agents or aminoglycosides have no acceptable intracellular activity against Legionella, particularly for L. pneumophila.

Mycoplasma pneumoniae was first identified and described in the early 1960s. This bacteria is one of the smallest free-living organisms, having no cell wall, which makes it insensitive to beta-lactam antimicrobials, and having no Gram staining. It causes approximately 20% of all community- acquired pneumonias in the general population and up to 50% of pneumonias in certain confined groups (3). Clinical manifestations are not sufficiently unique to allow differentiation from infections caused by other common bacteria. ELISA methods have been introduced in the market, but usually have low sensitivity and present cross-reaction with other commensal Mycoplasma species, and are therefore not recommended for diagnostic purposes.

These pathogenic microorganisms cause clinical symptoms that may be similar, frequently leading to misdiagnosis and ineffective treatments, particularly since the treatments for each pathogenic groups of microorganism are distinct. Furthermore, these microorganisms are usually difficult to detect in laboratory using conventional techniques, and all them present slow growing or obligately intracellular properties. For example, the detection of Mycobacterium tuberculosis by culture usually takes 2-8 weeks and the detection of Mycoplasma pneumoniae and Legionella pneumophila by ELISA usually takes 2-3 weeks. In addition, the currently used diagnostic methods may be very specific for certain species or even serotypes of bacteria. For example, the Legionella urinary antigen test only enables the detection of the serogroup 1 of the species Legionella pneumophila.

There is therefore a need in the art for a quick and accurate method of specifically detecting bacteria that cause respiratory tract infections. Summary of the Invention

The present inventors have devised a kit and method for the detection of several microorganisms causing tuberculosis, Legionnaires' disease and/or atypical pneumonias in a biological sample. The method of the present invention is a polymerase chain reaction (PCR) method, typically a real-time PCR method, and makes use of certain primers and probes to specifically detect and differentiate the following bacteria: i) Mycobacterium tuberculosis complex, ii) members of the family Legionellaceae and iii) Mycoplasma pneumoniae. One aspect of the invention relates to a kit comprising at least some of these primers and probes. The present invention enables the rapid, accurate and simultaneous detection of certain microorganisms that cause similar clinical symptoms and are usually difficult to detect in clinical laboratories. Accordingly, in a first aspect, the present invention provides a kit comprising:

a first primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a M. tuberculosis complex nucleic acid sequence shown in SEQ ID NO: 1 , and wherein the primers are capable of amplifying the M. tuberculosis complex nucleic acid; and/or a second primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a Legionellaceae nucleic acid sequence shown in SEQ ID NO: 9, and wherein the primers are capable of amplifying the Legionellaceae nucleic acid; and/or a third primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a M. pneumoniae nucleic acid sequence shown in SEQ ID NO: 20, and wherein the primers are capable of amplifying the M. pneumoniae nucleic acid. Definitions

As used herein, a "nucleic acid" molecule or sequence is a deoxyribonucleotide or ribonucleotide polymer. The term "nucleic acid" is used interchangeably herein with the term "polynucleotide". Nucleic acids are typically the naturally occurring nucleic acids DNA and RNA, including cDNA and mRNA, and can be synthetic or natural. Nucleic acids can also be artificial nucleic acids including PNA (peptide nucleic acid), LNA (locked nucleic acid), UNA (unlocked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid). Nucleic acids can be single stranded (sense or antisense strand) or double stranded. In most cases, naturally occurring DNA sequences are double stranded and naturally occurring RNA sequences are single stranded. An "oligonucleotide" is a short single stranded nucleic acid sequence.

Nucleic acids typically consist of the naturally occurring nucleotides adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U). Nucleotides can also be modified, for example by methylation, substitution of one or more naturally-occurring nucleotides with a non-naturally occurring nucleotides. Modified nucleotides include 4-acetylcytidine, 5- (carboxyhydroxylmethyl) uridine, 2-O-methylcytidine, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino- methyluridine, dihydrouridine, 2-O-methylpseudouridine, 2-O-methylguanosine, inosine, N6- isopentyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1- methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5- methylaminomethyluridine, 5- methoxyaminomethyl-2-thiouridine, 5-methoxyuridine, 5-rnethoxycarbonylmethyl-2-thiouridine, 5- methoxycarbonylmethyluridine, 2-methylthio-N6-isopentenyladenosine, uridine-5-oxyacetic acid- methylester, uridine-5-oxyacetic acid, wybutoxosine, wybutosine, pseudouridine, queuosine, 2- thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, 2-0-methyl-5- methyluridine and 2-O-methyluridine. As used herein, the term "primer" means a short nucleic acid molecule that serves as a starting point for DNA synthesis, for example using PCR. A primer for use in the present invention is typically a DNA oligonucleotide of at least 10 nucleotides, typically around 10 to 60 nucleotides in length. In some embodiments, a primer is from 10 to 50, 1 1 to 45, 12 to 40, 15 to 30, 16 to 25, 17 to 24 or from 18 to 23 nucleotides in length, for example 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length. The skilled person will appreciate that in certain cases the specificity of a primer increases with its length. Primers for use in the present invention have a complementary sequence to that of the DNA target sequence. Primers can be annealed to such a target DNA sequence having a complementary sequence to the primer by DNA hybridization and then extended along the target DNA sequence by a polymerase enzyme to amplify the target DNA sequence.

One or more pairs of primers are typically used in PCR. A "forward" primer is a primer that is complementary to the sense strand of a double stranded nucleic acid sequence. A "reverse" primer is a primer that is complementary to the antisense strand of a double stranded nucleic acid sequence.

Methods for preparing and using nucleic acid primers for PCR are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990) and Ausubel et al. (ed.) (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). PCR primers can also be designed by using computer programs such as OLIGO (Molecular Biology Insights, Inc., Cascade, CO) and Primer (Version 0.5, Whitehead Institute for Biomedical Research, Cambridge, MA), or using programs developed in-house.

As used herein, the term "hybridization" is the process of combining two complementary single- stranded DNA or RNA molecules and allowing them to form a double-stranded molecule by base pairing. Hybridization is used in the present invention for binding of primers and/or probes to a target nucleic acid sequence from a microorganism such as M. tuberculosis complex, Legionellaceae species and M. pneumoniae. Two single-stranded sequences will hybridize to each other even if there is not 100% sequence identity between the two sequences, depending on the conditions under which the hybridization reaction occurs and the composition and length of the hybridizing nucleic acid sequences.

Generally, the temperature of hybridization and the ionic strength (such as the Mg ²⁺ concentration) of the hybridization buffer will determine the stringency of hybridization. High stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences are less similar. Calculations regarding hybridization conditions for attaining certain degrees of stringency can be readily carried out by the skilled person and are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 1 1 ). The skilled person will be able to optimise hybridization conditions according to the results from sensitivity and specificity tests.

The following is an exemplary set of hybridization conditions for use in the present invention:

Very High Stringency (detects sequences that share at least 90% identity)

Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (detects sequences that share at least 80% identity)

Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1x SSC at 55°C-70°C for 30 minutes each

Low Stringency (detects sequences that share at least 50% identity)

Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

The probes and primers disclosed herein can hybridize to nucleic acid molecules under low stringency, high stringency, and very high stringency conditions.

As used herein, the term "probe" means a short nucleic acid molecule (nucleotide sequence) that is used to detect the presence of a complementary sequence by hybridization. A probe for use in the present invention is typically a DNA sequence of at least 10 nucleotides, typically around 10 to 60 nucleotides in length. In some embodiments, a probe is from 10 to 50, 1 1 to 40, 12 to 30, 15 to 25, 16 to 20 or from 17 to 21 nucleotides in length, for example 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length. The probe is complementary to the sequence that is to be detected. Probes can be labelled for use in the methods of the present invention, for example for use in a real-time PCR method. This enables the probe to be used to detect binding of the probe to the target sequence by hybridization. The label is therefore a detectable label. Typical labels include fluorescent agents, chemiluminescent agents, radioactive isotopes, enzyme substrates, co-factors, ligands, haptens and enzymes. Methods for labeling are discussed, for example, in Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-lntersciences (1987) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).

A fluorophore is a fluorescent chemical compound that emits light (fluoresces) upon light excitation. Typically, a fluorophore is excited by a particular wavelength of light and emits light at a different wavelength on excitation.

Suitable fluorophores for use in the present invention include acridine and derivatives such as 4- amino- N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); coumarin and derivatives such as 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumaran 151 ); cyanine; eosin and derivatives such as eosin isothiocyanate; erythrosin and derivatives such as erythrosin isothiocyanate and erythrosin B; ethidium; fluorescein and derivatives such as fluorescein isothiocyanate (FITC), QFITC (XRITC), 5- carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'- dichloro-6-carboxyfluorescein (JOE), 6-carboxy- fluorescein (HEX), and TET (tetramethyl fluorescein); fluorescamine; Malachite Green isothiocyanate; nitrotyrosine; pararosaniline; Phenol Red; and rhodamine and derivatives such as 6-carboxyrhodamine (R6G), 6-carboxy-X-rhodamine (ROX), lissamine rhodamine B sulfonyl chloride, rhodamine 123, rhodamine X isothiocyanate, rhodamine B, tetramethyl rhodamine, N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), and tetramethyl rhodamine isothiocyanate (TRITC).

Additional examples of fluorophores include CAL Fluor ® dyes such as CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610 and CAL Fluor Red 635 (see for example US Patent No. 7,344.701 ), Quasar ® dyes such as Quasar 570, Quasar 670, and Quasar 705 (see for example US 2005/0214833 A1 ) and Pulsar® 650 Dyes (see for example US 2004/0146895 A1 ) (all Biosearch Technologies, Novato, CA). Other fluorophores include those commercially available from Life Technologies (Carlsbad, CA).

As used herein, a "donor fluorophore" is a fluorophore capable of transferring energy to a quencher, thus preventing the emission of a signal detectable by the equipment. Donor fluorophores are also referred to herein as "reporters".

As used herein, a quencher is a molecule that absorbs energy from a donor fluorophore. A quencher reduces or quenches the emission of a donor fluorophore, typically when the quencher is in close proximity to the donor fluorophore.

Quenchers for use in the invention include dark quenchers. Dark quenchers absorb excitation energy from a fluorophore and dissipate the energy as heat; in contrast to typical (fluorescent) quenchers which re-emit this energy as light. In a particular embodiment, an acceptor fluorophore is a dark quencher, for example Black Hole Quenchers™ such as BHQ-0, BHQ-1 , BHQ-2, and BHQ-3 (Biosearch Technologies; see for example US Patent No. 7,019,129), Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), ECLIPSE™ Dark Quencher (Epoch Biosciences), or IOWA BLACK™ (Integrated DNA Technologies).

In one embodiment, a probe for use in the invention includes at least one fluorophore, such as a donor fluorophore . For example, a fluorophore can be attached at the 5'- or 3'-end of the probe, for example by attachment to the base at the 5'- or 3'-end of the probe or to the phosphate group at the 5'-end or to a modified base. Typically, a probe for use in the invention is dual labelled with both a reporter and a quencher. For example, a probe for use in the invention can be labelled with a reporter such as a Quasar ® dye (for example Quasar 570, Quasar 670, and Quasar 705) or a Pulsar® dye (for example Pulsar® 650) and a quencher such as a Black Hole Quencher (for example BHQ-0, BHQ-1 , BHQ-2, and BHQ-3).

As used herein, the term "identity" is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences, typically along their whole length. In the art, identity also means the degree of sequence relatedness between polynucleotide or polypeptide sequences, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polynucleotide or two polypeptide sequences, methods commonly employed to determine identity are codified in computer programs.

Computational approaches to sequence alignment generally fall into two categories: global alignments and local alignments. A global alignment attempts to align every nucleotide or residue in every sequence and thus forces the alignment to span the entire length of all query sequences. Global alignments are most useful when the sequences in the query set are similar and of approximately equal size. A general global alignment technique is the Needleman-Wunsch algorithm, which is based on dynamic programming. In contrast, local alignments identify regions of similarity within long sequences that can be widely divergent overall. Local alignments are often preferable, but can be more difficult to calculate because of the additional challenge of identifying the regions of similarity. Local alignments are more useful for dissimilar sequences that are suspected to contain regions of similarity or similar sequence motifs within a larger sequence. The Smith-Waterman algorithm is a general local alignment method and is also based on dynamic programming. With sufficiently similar sequences, there is no difference between local and global alignments. Hybrid methods, known as semiglobal or "glocal" (short for global-local) methods, attempt to find the best possible alignment that includes the start and end of one or the other sequence. This can be especially useful when the downstream part of one sequence overlaps with the upstream part of the other sequence. Suitable computer programs to determine identity between two sequences include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215, 403 (1990), available at http://blast.ncbi.nlm.nih.gov/Blast.cgi), including BLASTp (for proteins), BLASTn and BLASTx (for nucleotides), gapped BLAST and PSI-BLAST (for proteins, Altschul et al., Nucleic Acids Research 25 (17): 3389-402, 1997), FASTA (available at http://www.ebi.ac.uk/Tools/sss/). ClustalW/ClustalX (Thompson et al., Nucleic Acids Research 22 (22): 4673-4680 (1994), latest version is 2.1 ) and the GCG program package (Devereux et al., Nucleic Acids Research, 12, 387 (1984)).

The Clustal program can be used to compare both nucleotide and amino acid sequences. This program compares sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.

The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the one sequence for best alignment with the other sequence) and comparing the nucleotides or amino acid residues at corresponding positions. The "best alignment" is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical nucleotides or amino acid residues in the sequences being compared (i.e., % identity = number of identical positions/total number of positions x 100). As described above, determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). The BLASTn and BLASTx programs of Altschul et al., J. Mol. Biol. 215, 403 (1990) have incorporated such an algorithm. BLAST nucleotide searches can be performed with the BLASTn program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See http://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. Detailed Description of the Invention

The present invention provides a kit and a method for detecting the presence of one or more of a Mycobacterium tuberculosis complex nucleic acid, a Legionellaceae nucleic acid and a Mycoplasma pneumoniae nucleic acid in a sample. The method is a PCR method which makes use of various primers and probes which are present in the kit of the invention. The present invention uses the RNA polymerase beta subunit (rpoB) gene to detect Mycobacterium tuberculosis complex, the 16S ribosomal RNA (16S) gene to detect Legionellaceae species and the 16S ribosomal RNA (16S) gene to detect Mycoplasma pneumoniae.

In a first aspect, the present invention provides a kit comprising:

a first primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a M. tuberculosis complex nucleic acid sequence shown in SEQ ID NO: 1 , and wherein the primers are capable of amplifying the M. tuberculosis complex nucleic acid; and/or a second primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a Legionellaceae nucleic acid sequence shown in SEQ ID NO: 9, and wherein the primers are capable of amplifying the Legionellaceae nucleic acid; and/or a third primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a M. pneumoniae nucleic acid sequence shown in SEQ ID NO: 20, and wherein the primers are capable of amplifying the M. pneumoniae nucleic acid.

The kit of the first aspect of the invention therefore comprises one or more of the first, second and third primer set, in any combination. For example, a kit of the first aspect of the invention can comprise only the first primer set, second primer set or third primer set. In other embodiments, the kit of the first aspect of the invention comprises the first and second primer set, the second and third primer set or the first and third primer set. In another embodiment, the kit of the first aspect of the invention comprises the first, second and third primer sets. Accordingly, the primer sets of the invention may be sold separately or in combination.

The present invention enables the detection of a group of species known as the Mycobacterium tuberculosis complex (M. tuberculosis, M, africanum, M, bovis, M, microti, M, canettii, M, caprae and M. pinnipedii). Nucleic acid and protein sequences for M. tuberculosis species are publically available. For example, GenBank Accession Nos. NC_020559.1 , AP012340.1 , NZ_DF126614.1 and DF126614.1 provide exem lary genome sequences for Mycobacterium tuberculosis. The present invention uses the RNA polymerase beta subunit (rpoB) gene to detect Mycobacterium tuberculosis complex. A representative nucleotide sequence of the M. tuberculosis rpoB gene is as follows:

[ttggcagattcccgccagagcaaaacagccgctagtcctagtccgagtcgcccgca aagttcctcgaataactccgtacccggagcgcc aaaccgggtctccttcgctaagctgcgcgaaccacttgaggttccgggactccttgacgt ccagaccgattcgttcgagtggctgatcggttc gccgcgctggcgcgaatccgccgccgagcggggtgatgtcaacccagtgggtggcctgga agaggtgctctacgagctgtctccgatcg aggacttctccgggtcgatgtcgttgtcgttctctgaccctcgtttcgacgatgtcaagg cacccgtcgacgagtgcaaagacaaggacatg acgtacgcggctccactgttcgtcaccgccgagttcatcaacaacaacaccggtgagatc aagagtcagacggtgttcatgggtgacttcc cgatgatgaccgagaagggcacgttcatcatcaacgggaccgagcgtgtggtggtcagcc agctggtgcggtcgcccggggtgtacttc gacgagaccattgacaagtccaccgacaagacgctgcacagcgtcaaggtgatcccgagc cgcggcgcgtggctcgagtttgacgtcg acaagcgcgacaccgtcggcgtgcgcatcgaccgcaaacgccggcaaccggtcaccgtgc tgctcaaggcgctgggctggaccagc gagcagattgtcgagcggttcgggttctccgagatcatgcgatcgacgctggagaaggac aacaccgtcggcaccgacgaggcgctgtt ggacatctaccgcaagctgcgtccgggcgagcccccgaccaaagagtcagcgcagacgct gttggaaaacttgttcttcaaggagaag cgctacgacctggcccgcgtcggtcgctataaggtcaacaagaagctcgggctgcatgtc ggcgagcccatcacgtcgtcgacgctgac cgaagaagacgtcgtggccaccatcgaatatctggtccgcttgcacgagggtcagaccac gatgaccgttccgggcggcgtcgaggtgc cggtggaaaccgacgacatcgaccacttcggcaaccgccgcctgcgtacggtcggcgagc tgatccaaaaccagatccgggtcggcat gtcgcggatggagcgggtggtccgggagcggatgaccacccaggacgtggaggcgatcac accgcagacgttgatcaacatccggcc ggtggtcgccgcgatcaaggagttcttcggcaccagccagctgagccaattcatggacca gaacaacccgctgtcggggttgacccaca agcgccgactgtcggcgctggggcccggcggtctgtcacgtgagcgtgccgggctggagg tccgcgacgtgcacccgtcgcactacgg ccggatgtgcccgatcgaaacccctgaggggcccaacatcggtctgatcggctcgctgtc ggtgtacgcgcgggtcaacccgttcgggttc atcgaaacgccgtaccgcaaggtggtcgacggcgtggttagcgacgagatcgtgtacctg accgccgacgaggaggaccgccacgtg gtggcacaggccaattcgccgatcgatgcggacggtcgcttcgtcgagccgcgcgtgctg gtccgccgcaaggcgggc gaggtggagtacgtgccctcgtctgaggtggactacatggacgtctcgccccgccagatg gtgtcggtggccaccgcgatgattcccttcct ggagcacgacgacgccaaccgtgccctcatgggggcaaacatgcagcgccaggcggtgcc gctggtccgtagcgaggccccgctggt gggcaccgggatggagctgcgcgcggcgatcgacgccggcgacgtcgtcgtcgccgaaga aagcggcgtcatcgaggaggtgtcgg ccgactacatcactgtgatgcacgacaacggcacccggcgtacctaccggatgcgcaagt ttgcccggtccaaccacggcacttgcgcc aaccagtgccccatcgtggacgcgggcgaccgagtcgaggccggtcaggtgatcgccgac ggtccctgtactgacgacggcgagatg gcgctgggcaagaacctgctggtggccatcatgccgtgggagggccacaactacgaggac gcgatcatcctgtccaaccgcctggtcg aagaggacgtgctcacctcgatccacatcgaggagcatgagatcgatgctcgcgacacca agctgggtgcggaggagatcacccgcg acatcccgaacatctccgacgaggtgctcgccgacctggatgagcggggcatcgtgcgca tcggtgccgaggttcgcgacggggacat cctggtcggcaaggtcaccccgaagggtgagaccgagctgacgccggaggagcggctgct gcgtgccatcttcggtgagaaggcccg cgaggtgcgcgacacttcgctgaaggtgccgcacggcgaatccggcaaggtgatcggcat tcgggtgttttcccgcgaggacgaggac gagttgccggccggtgtcaacgagctggtgcgtgtgtatgtggctcagaaacgcaagatc tccgacggtgacaagctggccggccggca cggcaacaagggcgtgatcggcaagatcctgccggttgaggacatgccgttccttgccga cggcaccccggtggacattattttgaacac ccacggcgtgccgcgacggatgaacatcggccagattttggagacccacctgggttggtg tgcccacagcggctggaaggtcgacgcc gccaagggggttccggactgggccgccaggctgcccgacgaactgctcgaggcgcagccg aacgccattgtgtcgacgccggtgttcg acggcgcccaggaggccgagctgcagggcctgttgtcgtgcacgctgcccaaccgcgacg gtgacgtgctggtcgacgccgacggca aggccatgctcttcgacgggcgcagcggcgagccgttcccgtacccggtcacggttggct acatgtacatcatgaagctgcaccacctggt ggacgacaagatccacgcccgctccaccgggccgtactcgatgatcacccagcagccgct gggcggtaaggcgcagttcggtggcca gcggttcggggagatggagtgctgggccatgcaggcctacggtgctgcctacaccctgca ggagctgttgaccatcaagtccgatgacac cgtcggccgcgtcaaggtgtacgaggcgatcgtcaagggtgagaacatcccggagccggg catccccgagtcgttcaaggtgctgctca aagaactgcagtcgctgtgcctcaacgtcgaggtgctatcgagtgacggtgcggcgatcg aactgcgcgaaggtgaggacgaggacct ggagcgggccgcggccaacctgggaatcaatctgtcccgcaacgaatccgcaagtgtcga ggatcttgcgtaa]

(SEQ ID N0: 1 ). Genbank Gl number 444893469,

http://www.ncbi. nlm.nih.aov/nuccore/444893469?from=759807&to=763325& sat=2&sat kev=33681 433

The target region is a sequence of around 380 nucleotides of the rpoB gene, corresponding to nucleotides 1 192 to 1574 of SEQ ID NO: 1. This is the region coding for amino acids where mutations are associated with resistance to rifamycins, which is one of the main groups of antibiotics used for the treatment of tuberculosis. The target region includes the three most important mutations, which according to the literature are associated with about 85% of the total cases. The present invention therefore enables the detection of mutations associated with resistance of M. tuberculosis to antibiotics.

The nucleotide sequences of exemplary primers and probes for the detection of M. tuberculosis complex are as follows: Primer forward (MtubF3) - 5'- ATGACCACCCAGGACGTGG-3' (SEQ ID NO: 2)

Primer reverse (MtubRev2) - 5'-AGGTACACGATCTCGTCGCTA-3' (SEQ ID NO: 3)

Probe (PforMtubl )- 5'-ATCAACATCCGGCCGGTGGTCG-3' (SEQ ID NO: 4)

Additional primers

Primer forward (MtubF2) - 5'-ATGACCACCCAGGACGTGGA-3' (SEQ ID NO: 5)

Primer forward (MtubFI ) - 5'-GGCGATCACACCGCAGACGT-3' (SEQ ID NO: 6)

Primer reverse (MtubRevl ) - 5'-CCACCTTGCGGTACGGCGTT-3' (SEQ ID NO: 7)

Additional probe

PforMtub2 - 5'-CAACATCCGGCCGGTGGTCG-3' (SEQ ID NO: 8)

The first primer set for use in the kit of the present invention comprises at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a M. tuberculosis complex nucleic acid sequence shown in SEQ ID NO: 1 , and wherein the primers are capable of amplifying the M. tuberculosis nucleic acid. Typically, the primers are capable of hybridizing under very high stringency conditions to a sequence between 1 192 and 1574 of SEQ ID NO: 1.

Any of the above forward primers for the detection of M. tuberculosis complex can be used in combination with any of the reverse primers for the detection of M. tuberculosis complex. Accordingly, in one embodiment, the first primer set comprises primers comprising nucleic acid sequences at least 75% identical to: a. SEQ ID NO: 2 or SEQ ID NO: 5 or SEQ ID NO: 6; and

b. SEQ ID NO: 3 or SEQ ID NO: 7;

or a sequence complementary to any thereof.

Bacteria of the family Legionellaceae are responsible for various infections including Legionnaires' disease. Of the family, the species L. pneumophila is the most extensively studied and responsible for the majority of the infections. The present invention allows the detection of any bacteria of the family Legionellaceae, which includes about 50 different species, about 20 of them previously isolated from human patients. These species include Legionella adelaidensis, Legionella anisa, Legionella beliardensis, Legionella birminghamensis, Legionella bozemanae, Legionella brunensis, Legionella busanensis, Legionella cardiac, Legionella cherrii, Legionella cincinnatiensis, Legionella drancourtii, Legionella dresdenensis, Legionella drozanskii, Legionella dumoffii, Legionella erythra, Legionella fairfieldensis, Legionella fallonii, Legionella feeleii, Legionella geestiana, Legionella gormanii, Legionella gratiana, Legionella gresilensis, Legionella hackeliae, Legionella impletisoli, Legionella israelensis, Legionella jamestowniensis, Legionella jordanis, Legionella lansingensis, Legionella londiniensis, Legionella longbeachae, Legionella lytica, Legionella maceachernii, Legionella massiliensis, Legionella micdadei, Legionella moravica, Legionella nagasakiensis, Legionella nautarum, Legionella oakridgensis, Legionella parisiensis, Legionella pittsburghensis, Legionella pneumophila, Legionella quateirensis, Legionella quinlivanii, Legionella rowbothamii, Legionella rubrilucens, Legionella sainthelensi, Legionella santicrucis, Legionella shakespearei, Legionella spiritensis, Legionella steelei, Legionella steigerwaltii, Legionella taurinensis, Legionella tucsonensis, Legionella tunisiensis, Legionella wadsworthii, Legionella waltersii

Legionella worsleiensis and Legionella yabuuchiae.

Very few in vitro diagnostic products allow the detection of other species of Legionella besides L pneumophila. Nucleic acid and protein sequences for species of the family Legionellaceae are publicly available. For example, GenBank Accession Nos. NC_006368.1 , CR628336.1 and NC_006369.1 provide exemplary L. pneumophila genome sequences.

The present invention uses the 16S ribosomal RNA (16S) gene to detect Legionella species. A representative nucleotide sequence of the Legionellaceae 16S gene is as follows:

[aactnaagagtnnnatcctggctcagatnnaacgctngcggcatgctnaacacatg caagtcgaacggcagcatngtctagctngctn gacagatgcgagtggcgaacgggtgagtaacgcgtaggaatatgcctngaagagggggac aacttggggaaactnaagctnatacc gcataatgtctgaggacgaaagctggggaccttcgggcctngcgctnnaagatnagcctg cgtccgatnagctngttggtggggtaagg gcctaccaaggcgacgatcggtagctggtctgagaggatgaccagccacactggaactga gacacggtccagactcctacgggaggc agcagtggggaatattggacaatgggggcaaccctratccagcaatgccgcgtgtgtgaa gaaggcctnagggttgtaaagcactttcag tggggaggagggttnataggtnaagagctgattaactngacgttacccacagaagaagca ccggctnactccgtgccagcagccgcgg taatacggagggtgcgagcgttaatcggaattactgggcgtcaagggtgcgtaggtggtn nattaagtnatctgtgaaattcctrggctnaa cctgggacggtcagatnatactngttgactcgagtatgggagagggtagtggaatttccg gtgtagcggtnaaatgcgtagagatcggaa ggaacaccagtggcgaaggcggctacctngcctaatactgacactgaggcacgaaagcgt ggggagcaaacaggattagataccctg gtagtccacgctgtaaacgatgtcaactagctrttggttatatgaaaataattagtggcg cagcaaacgcgataagttgaccgcctggggag tacggtcgcaagatyaaaactcaaaggaattgacgggggcccgcacaagcggtggagcat gtngtnnnattcgatgcaacgcgaaga acctnacctaccctngacatacagtgaattrtgcagagatgcataagtgccttcgggaac actnatacaggtgctgcatggctgtcgtcagc tcgtgtcgtgagatgttgggtnaagtcccgtaacgagcgcaaccctngtccttagttgcc agcatgtgatggtggggactctaaggagactg ccggtgacaaaccggaggaaggcggggatgacgtcaagtcatcatngccntnacgggtag ggctacacacgtgctacaatggccgat acagagggcggcgaaggggcgacctngagcaaatcctnaaaagtcggtcgtngtccggat tggagtctgcaactcgactccatgaagt cggaatcgctagtaatcgcgaatcagcatgtcgcggtgaatacgttcccgggccttgtac acaccgcccgtcacaccatgggagtgggttg caccagaagtagatagtctaaccttcggggggacgtntaccacggtgt]

(SEQ ID N0:9).

Genbank Gl number 175189, http://www.ncbi.nlm.nih.gOv/nuccore/M36023.1

The target region is a sequence of around 320 nucleotides of the 16S gene, corresponding to nucleotides 401 to 722 of SEQ ID NO: 9. The nucleotide sequences of exemplary primers and probes for the detection of Legionellaceae are as follows:

Primer forward (LegF3) - 5'- CGTGTGTGAAGAAGGCCTGA-3' (SEQ ID NO: 10)

Primer reverse (LegRevl ) - 5'- CACTGGTGTTCCTTCCGATC-3' (SEQ ID NO: 1 1 )

Probe (PrevLeg3)- 5'-CGGAAATTCCACTACCCTCTCC-3' (SEQ ID NO: 12)

Additional primers

Primer forward (LegF1 ) - 5'-CACTTTCAGTGGGGAGGAG-3' (SEQ ID NO: 13)

Primer forward (LegF2) - 5'-CTTTAAGATTAGCCTGCGTCC-3' (SEQ ID NO: 14)

Primer forward (LegF4) - 5'-GTGTGTGAAGAAGGCCTGAG-3' (SEQ ID NO: 15)

Primer reverse (LegRev2) - 5'-CACTGGTGTTCCTTCCGATCT-3' (SEQ ID NO: 16)

Primer reverse (LegRev3) - 5'-ACTGGTGTTCCTTCCGATCT-3' (SEQ ID NO: 17)

Additional probes

PforLegl - 5'-CCCACAGAAGAAGCACCGGCT-3' (SEQ ID NO: 18)

Pforl_eg2 - 5'-CCACAGAAGAAGCACCGGCT-3' (SEQ ID NO: 19)

The second primer set for use in the kit of the present invention comprises at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a Legionellaceae nucleic acid sequence shown in SEQ ID NO: 9, and wherein the primers are capable of amplifying the Legionellaceae nucleic acid. Typically, the primers are capable of hybridizing under very high stringency conditions to a sequence between nucleotides 401 and 722 of SEQ ID NO: 9.

Any of the above forward primers for the detection of a Legionellaceae nucleic acid can be used in combination with any of the reverse primers for the detection of a Legionellaceae nucleic acid. Accordingly, in one embodiment, the second primer set comprises primers comprising nucleic acid sequences at least 75% identical to:

a. SEQ ID NO: 10 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15; and b. SEQ ID NO: 1 1 or SEQ ID NO: 16 or SEQ ID NO: 17;

or a sequence complementary to any thereof.

Mycoplasma pneumoniae is a bacterium that causes atypical pneumonia. Multiple strains of M. pneumoniae have been identified such as Ml 29 and FH. Nucleic acid and protein sequences for M. pneumoniae are publicly available. For example, GenBank Accession Nos. NC_020076.1 , CP003913.1 , NC_016807.1 and NC_000912.1 provide exemplary M. pneumoniae genome sequences.

The present invention uses the 16S ribosomal RNA (16S) gene to detect Mycoplasma pneumoniae. A representative nucleotide sequence of the M. pneumoniae 16S gene is as follows:

[nnttttctgagagtttgatcctggctcaggattaacgctggcggcatgcctaatac atgcaagtcgatcgaaagtagtaatactttagaggcg aacgggtgagtaacacgtatccaatctaccttataatgggggataactagttgaaagact agctaataccgcataagaactttggttcgcat gaatcaaagttgaaaggacctgcaagggttcgttatttgatgagggtgcgccatatcagc tagttggtggggtaacggcctaccaaggcaa tgacgtgtagctatgctgagaagtagaatagccacaatgggactgagacacggcccatac tcctacgggagncagcagtagggaattttt cacaatgagcgaaagcttgatggagcaatgccgcgtgaacgatgaaggtctttaagattg taaagttcatttatttgggaagaatgactttag caggtaatggctagagtttgactgtaccattttgaataagtgacgactaactatgtgcca gcagtcgcggtaatacataggtcgcaagcgtta tccggatttattgggcgtaaagcaagcgcaggcggattgaaaagtctggtgttaaaggca gctgcttaacagttgtatgcattggaaactatt aatctagagtgtggtagggagttttggaatttcatgtggagcggtgaaatgcgtagatat atgaaggaacaccagtggcgaaggcgaaaa cttaggccattactgacgcttaggcttgaaagtgtgggnagcaaataggattagataccc tagtagtccacaccgtaaacgatagatacta gctgtcggggcgatcccctcggtagtgaagttaacacattaagtatctcgcctgggtagt acattcgcaagaatgaaactcaaacggaattg acggggacccgcacaagtggtggagcatgttgcttaattcgacggtacacgaaaaacctt acctagacttgacatccttggcaaagttatg gaaacataatggaggttaaccgagtgacaggtggtgcatggttgtcgtcagctcgtgtcg tgagatgttgggttaagtcccgcaacgagcg caacccttatcgttagttac

attgtctagcgagactgctaatgcaaattggaggaaggaagggatgacgtcaaatca tcatgccccttatgtctagggctgcaaacgtgct acaatggccaatacaaacagtcgccagcttgtaaaagtgagcaaatctgtaaagttggtc tcagttcggattgagggctgcaattcgtcctc atgaagtcggaatcactagtaatcgcgaatcagctatgtcgcggtgaatacgttctcggg tcttgtacacaccgcccgtcaaactatgaaag ctggtaatatttaaaaacgtgttgctaaccattaggaagcgcatgtcaaggatagcaccg gtgattggag]

(SEQ ID NO:20).

Genbank Gl number 175479, http://www.ncbi.nlm.nih.gOv/nuccore/M29061.1

The target region is a sequence of around 620 nucleotides of the 16S gene, corresponding to nucleotides 828 to 1448 of SEQ ID NO: 20.

The nucleotide sequences of exemplary primers and probes for the detection of M. pneumoniae are as follows: Primer forward (MpneuF5) - 5'- CTGTCGGGGCGATCCCC-3' (SEQ ID NO: 21 ) Primer reverse (MpneuRev2) - 5'- TGACATGCGCTTCCTAATGG-3' (SEQ ID NO: 22)

Probe (PforMpneu2) - 5'-CCAATACAAACAGTCGCCAGCTT-3' (SEQ ID NO: 23) Additional primers

Primer forward (MpneuFI ) - 5'-CTAGCGAGACTGCTAATGC-3' (SEQ ID NO: 24)

Primer forward (MpneuF2) - 5'-CTAGCGAGACTGCTAATGCA-3' (SEQ ID NO: 25)

Primer forward (MpneuF3) - 5'-CATGCAAGTCGATCGAAAGTAGT-3' (SEQ ID NO: 26)

Primer reverse (MpneuRevl ) - 5'-CTTGACATGCGCTTCCTAAT-3' (SEQ ID NO: 27)

Primer reverse (MpneuRev3) - 5'-CTTTGATTCATGCGAACCAAAGTT-3' (SEQ ID NO: 28) Additional probes

PforMpneu3 - 5'-ACTAGTTGAAAGACTAGCTAATACCG-3' (SEQ ID NO: 29)

PrevMpneu4 - 5'-CCCCGACAGCTAGTATCTATCGTT-3' (SEQ ID NO: 30) The third primer set for use in the present invention comprises at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a M. pneumoniae nucleic acid sequence shown in SEQ ID NO: 20, and wherein the primers are capable of amplifying the M. pneumoniae nucleic acid. Typically, the primers are capable of hybridizing under very high stringency conditions to a sequence between nucleotides 828 and 1448 of SEQ ID NO: 20.

In general, any of the above forward primers for the detection of a M. pneumoniae nucleic acid can be used in combination with any of the reverse primers for the detection of a M. pneumoniae nucleic acid. Accordingly, in one embodiment, the third primer set comprises primers comprising nucleic acid sequences at least 75% identical to:

a. SEQ ID NO: 21 or SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NO: 26; and b. SEQ ID NO: 22 or SEQ ID NO: 27 or SEQ ID NO: 28;

or a sequence complementary to any thereof. In one embodiment, the forward primer comprising a nucleic acid sequence at least 75% identical to SEQ ID NO: 26 is used in combination with the reverse primer comprising a nucleic acid sequence at least 75% identical to SEQ ID NO: 28. In one embodiment, these primers are used in combination with a probe comprising a nucleic acid sequence at least 75% identical to SEQ ID NO: 29.

In one embodiment, the forward primer comprising a nucleic acid sequence at least 75% identical to SEQ ID NO: 21 is used in combination with a probe comprising a nucleic acid sequence at least 75% identical to SEQ ID NO: 30. In some embodiments, the methods and/or kits of the invention can be used for the detection of Chlamydia pneumonia, also known as Chlamydophila pneumonia and Taiwan acute respiratory agent (TWAR). C. pneumoniae is a major cause of pneumonia in humans. Nucleic acid and protein sequences for C. pneumoniae are publicly available. For example, GenBank Accession No. HV214386.1 provides an exemplary C. pneumoniae genome sequence.

The present invention uses the 16S gene to detect C. pneumoniae.

A representative nucleotide sequence of the C. pneumoniae 16S gene is as follows:

[ggaataatgacttcggttgttatttagtggcggaagggttagtagtacatagataa tctgccctcaacttggggataacggttggaaacgatc gctaataccgaatgtagtgtaattaggcatctaatatatattaaagaaggggatcttcgg acctttcggttgaggaagagtttatgcgatatca gcttgttggtggggtaaaagcccaccaaggcgatgacgtctaggcggattgagagattga ccgccaacactgggactgagacactgccc agactcctacgggaggctgcagtcgagaatctttcgcaatggacgaaagtctgacgaagc gacgccgcgtgtgtgatgaaggccttagg gttgtaaagcactttcgcctgggaataagagagattggctaatatccaatcgatttgagc gtaccaggtaaagaagcaccggctaactccg tgccagcagctgcggtaatacggagggtgctagcgttaatcggatttattgggcgtaaag ggcgtgtaggcggaaaggaaagttagatgtt aaattttggggctcaaccccaagtcagcatttaaaactatctttctagaggatagatggg gaaaagggaattccacgtgtagcggtgaaatg cgtagatatgtggaagaacaccagtggcgaaggcgcttttctaatttatacctgacgcta aggcgcgaaagcaaggggagcaaacagg attagataccctggtagtccttgccgtaaacgatgcatacttgatgtggatggtctcaac cccatccgtgtcggagctaacgtgttaagtatgc cgcctgaggagtacactcgcaagggtgaaactcaaaagaattgacgggggcccgcacaag cagtggagcatgtggtttaattcgatgc aacgcgaaggaccttacctggacttgacatgtatttgacaactgtagaaatacagctttc cgcaaggacagatacacaggtgctgcatggc tgtcgtcagctcgtgccgtgaggtgttgggttaagtcccgcaacgagcgcaacccttatc gttagttgccagcacttagggtgggaactctaa cgagactgcctgggttaaccaggaggaaggcgaggatgacgtcaagtcagcatggccctt atgtccagggcgacacacgtgctacaat ggttagtacagaaggtagcaagatcgtgagatggagcaaatcctaaaagctagccccagt tcggattgtagtctgcaactcgactacatg aagtcggaattgctagtaatggcgtgtcagccataacgccgtgaatacgttctcgggcct tgtacacaccgcccgtcacatcatgggagttg gttttaccttaagtcgttgactcaacctatttataggagagaggcgcccaaggtgaggct gatgactgggatgaagtcgtaacaaggtagcc ctaccggaaggtggggctggatca]

(SEQ ID NO:31 ).

Genbank Gl number 219846935, http://www.ncbi.nlm.nih.gov/nuccore/219846935

The target region is a sequence of around 500 nucleotides of the 16S gene, corresponding to nucleotides 93 to 596 of SEQ ID NO: 31 .

The nucleotide sequences of exemplary primers and probes for the detection of C. pneumoniae are as follows:

Primers

Primer forward (CpneuF2) - 5'-CGCTAATACCGAATGTAGTGTAA -3' (SEQ ID NO: 32)

Primer reverse (CpneuRev4) - 5'-TCTAGAAAGATAGTTTTAAATGCTGA-3' (SEQ ID NO: 33) Probe PrevCpneul - 5'-TCGCATAAACTCTTCCTCAACCG-3' (SEQ ID NO: 34) Additional primers

Primer forward (CpneuFI ) - 5'-CTAATACCGAATGTAGTGTAA-3' (SEQ ID NO: 35)

Primer reverse (CpneuRev4-5) - 5'-CGAAAGTGCTTTACAACCCTAAG-3' (SEQ ID NO: 36) Primer reverse (CpneuRev3) - 5'-GTCATCGCCTTGGTGGGCTT-3' (SEQ ID NO: 37)

In these embodiments, the kit of the invention comprises a fourth primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a C. pneumoniae nucleic acid sequence shown in SEQ ID NO: 31 , and wherein the primers are capable of amplifying the C. pneumoniae nucleic acid. Typically, the primers are capable of hybridizing under very high stringency conditions to a sequence between nucleotides 93 and 596 of SEQ ID NO: 31.

Any of the above forward primers for the detection of a C. pneumoniae nucleic acid can be used in combination with any of the reverse primers for the detection of a C. pneumoniae nucleic acid. Accordingly, in one embodiment, the fourth primer set comprises primers comprising nucleic acid sequences at least 75% identical to:

a. SEQ ID NO: 32 or SEQ ID NO: 35; and

b. SEQ ID NO: 33 or SEQ ID NO: 36 or SEQ ID NO: 37;

or a sequence complementary to any thereof.

In some embodiments, the methods and/or kits of the invention can be used for the detection of Coxiella burnetii, a pathogenic bacteria known to cause disease in humans, such as Q-fever. Nucleic acid and protein sequences for C. burnetii are publicly available. For example, GenBank Accession No. AE016828.2 provides an exemplary C. burnetii genome sequence.

The present invention uses the 16S gene to detect C. burnetii.

A representative nucleotide sequence of the C. burnetii 16S gene is as follows:

[gattgaacgctagcggcatgcttaacacatgcaagtcgaacggcagcgcggggagc ttgctccctggcggcgagtggcggacgggtg agtaatgcgtaggaatctaccttgtagtgggggataacctggggaaactcgggctaatac cgcataatctctttggagcaaagcgggggat cttcggacctcgtgctataagatgagcctacgtcggattagcttgttggtggggtaatgg cctaccaaggcgacgatccgtagctggtctgag aggacgatcagccacactgggactgagacacggcccagactcctacgggaggcagcagtg gggaatattggacaatgggggaaacc ctgatccagcaatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcggtgg ggaagaaattctcaagggtaatatccttgggc gttgacgttacccacagaagaagcactggctaactctgtgccagcagccgcggtaataca gagagtgcaagcgttaatcggaatcactg ggcgtaaagcgcgcgtaggtggatatttaagtcggatgtgaaagccctgggcttaacctg ggaattgcacccgatactgggtatcttgagta tggtagagggaagtggaatttccggtgtagcggtgaaatgcgtagatatcggaaagaaca ccagtggcgaaggcgacttcctggaccaa tactgacactgaggcgcgaaagcgtggggagcaaacaggattagagaccctggtagtcca cgccgtcaacgatgagaactagctgttg ggaagttcacttcttagtagcgaagctaacgcgttaagttctccgcctggggagtacggc cgcaaggttaaaactcaaagaaattgacgg gggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaaaaaccttacct acccttgacatcctcggaacttgtcagagat gatttggtgccttcgggaaccgagtgacaggtgctgcatggctgtcgtcagctcgtgtcg tgagatgttgggttaagtcccgtaacgagcgca accctcgtccttagttgccagcgagtcaagtcgggaactctaaggagactgccggtgata aaccggaggaaggtggggatgatgtcaag tcatcatggcccttacgggtagggctacacacgtgctacaatgggcagtacaaagggttg ccaagccgcgaggtggagctaatcccaga aaactgctcgtagtccggattggagtctgcaactcgactccatgaagttggaatcgctag taatcgcgaatcagcatgtcgcggtgaatacg ttctcgggccttgtacacaccgcccgtcacaccatgggagtgaattgtaccagaagcggg taggctaaccttcgggaggccgctcaccac ggtatgatccatgactggggtgaa]

(SEQ ID NO:38).

Genbank Gl number 559795323, http://www.ncbi.nlm.nih.gOv/nuccore/NR_104916.1

The target region is a sequence of around 360 nucleotides of the 16S gene, corresponding to nucleotides 90 to 454 of SEQ ID NO: 38.

The nucleotide sequences of exemplary primers and probes for the detection of C. burnetii are as follows:

Primers

Primer forward (CburFI ) - 5'- AATGCGTAGGAATCTACCTTGT-3' (SEQ ID NO: 39)

Primer reverse (CburRevl ) - 5'- TCAACGCCCAAGGATATTACC-3' (SEQ ID NO: 40)

Probe PforCburl - 5'- CTATAAGATGAGCCTACGTCGGA-3' (SEQ ID NO: 41 )

Additional primers and probe

Primer forward (CburF2) - 5'- CACTTTCGGTGGGGAAGAAATT-3' (SEQ ID NO: 42)

Primer reverse (CburRev2) - 5'- GATACCCAGTATCGGGTGCAA-3' (SEQ ID NO: 43)

Probe (PforCbur2) - 5'- TCCTTGGGCGTTGACGTTACCC-3' (SEQ ID NO: 44)

In these embodiments, the kit of the invention comprises a fifth primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a C. burnetii nucleic acid sequence shown in SEQ ID NO: 38, and wherein the primers are capable of amplifying the C. burnetii nucleic acid. Typically, the primers are capable of hybridizing under very high stringency conditions to a sequence between nucleotides 90 and 454 of SEQ ID NO: 38.

Accordingly, in one embodiment, the fifth primer set comprises primers comprising nucleic acid sequences at least 75% identical to:

c. SEQ ID NO: 39 or SEQ ID NO: 42; and

d. SEQ ID NO: 40 or SEQ ID NO: 43;

or a sequence complementary to any thereof. In some embodiments, the methods and/or kits of the invention use a human gene as an internal positive control (IPC). In particular, the human thyroglobulin (TG) gene is used as an internal positive control for amplification. The internal positive control amplification relies on the detection of human DNA naturally present in biological samples, thus validating the nucleic acid extraction procedure as well as the presence or absence of PCR inhibitors.

A representative nucleotide sequence of the human thyroglobulin gene is shown in

SEQ ID NO:45.

Genbank Gl number 263191878, http://www.ncbi.nlm.nih.gov/nuccore/NG 015832.1

The target region is a sequence of around 200 nucleotides of the TG gene, corresponding to nucleotides 79393 to 79603 of SEQ ID NO: 45.

The nucleotide sequences of exemplary primers and probes for the detection of the human TG gene used as an internal positive control are as follows:

Primer forward Exon26-T F1 - 5'-CCAAGGATCCACCACAACACT-3' (SEQ ID NO: 46)

Primer reverse lntron26-27Rev - 5'-GCCCTCACTGCCAACATTAC-3' (SEQ ID NO: 47)

Probe PforExon26 2 - 5'-ATGGCTTCGTCCTCACACAGGTT-3' (SEQ ID NO: 48)

In these embodiments, the kit of the invention comprises a sixth primer set comprising at least two primers between 10 and 60 nucleotides in length, wherein the primers are capable of hybridizing under very high stringency conditions to a human nucleic acid sequence shown in SEQ ID NO: 45, and wherein the primers are capable of amplifying the human nucleic acid. Typically, the primers are capable of hybridizing under very high stringency conditions to a sequence between nucleotides 79393 and 79603 of SEQ ID NO: 45.

In one embodiment, the sixth primer set comprises primers comprising nucleic acid sequences at least 75% identical to SEQ ID NO: 46 and SEQ ID NO: 47 or a sequence complementary to either thereof.

In some embodiments, the methods and/or kits of the invention also use an external positive control (EPC). For the external positive control amplification, a long oligonucleotide, of about 1 10 bases, with recognition sites for the respective primers and probe allows the PCR reaction. The nucleotide sequences of exemplary EPCs are as follows:

CPE MtubF3U - for M. tuberculosis complex AGGTACACGATCTCGTCGCTAACCACGCCGTCGATCCTTGATCGCGGCGACCACCGGCCG G ATGTTGATTTACGTCTGCGGTGGTGTGATCGCCTCCACGTCCTGGGTGGTCAT (SEQ ID NO: 49) CPE LegF3U - for Legionellaceae

CACTGGTGTTCCTTCCGATCTCTACGCATTTCATTCACCGCTACACCGGAAATTCCACTA CCCT CTCCCATACTCGAGTCATGCTTTACAACCCTCAGGCCTTCTTCACACACG (SEQ ID NO: 50)

CPE MpneuF5U - for Mycoplasma pneumoniae

TGACATGCGCTTCCTAATGGTTAGCAACACGTTTGCTCACTTTTACAAGCTGGCGAC TGTTTGT ATTGGTTATTGTAGCACGACTTCACTACCGAGGGGATCGCCCCGACAG (SEQ ID NO: 51 )

CPE CpneuF2U - for C. pneumoniae

TCTAGAAAGATAGTTTTAAATGCTGACTTGGGATTGAGCCAACAAGCTGATATCGCATAA ACTC TTCCTCAACCGAAAGGTCCGAAGAATTAGATGCCTAATTACACTACATTCGGTATTAGCG (SEQ ID NO: 52)

CPE CburFI U - for C. burnetii

TCAACGCCCAAGGATATTACCCTTGAGAATTTCTACCAACAAGCTAATCCGACGTAGGCT CAT CTTATAGCACGAGGTCCGAAGTTATCCCCCACTACAAGGTAGATTCCTACGCATT (SEQ ID NO: 53)

The Ts may be replaced by Us, partially or totally, in any of these sequences.

The kit of the invention may also comprise one or more probes for the detection of target nucleic acid molecules. This embodiment of the invention is particularly of use when the kit is to be used for the conduction of real-time PCR experiments. The probes are useful to detect the production of target DNA molecules using PCR and are thus targeted to (i.e. complementary to and hybridize to) the M. tuberculosis complex nucleic acid sequence shown in SEQ ID NO: 1 ; the Legionellaceae nucleic acid sequence shown in SEQ ID NO: 9; and the M. pneumoniae nucleic acid sequence shown in SEQ ID NO: 20.

Accordingly, in one embodiment, the kit of the first aspect of the invention further comprises:

a first probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the first probe is capable of hybridising under very high stringency conditions to a M. tuberculosis complex nucleic acid sequence shown in SEQ ID NO: 1 ; and/or

a second probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the second probe is capable of hybridising under very high stringency conditions to a Legionellaceae nucleic acid sequence shown in SEQ ID NO: 9; and/or a third probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the third probe is capable of hybridising under very high stringency conditions to a M. pneumoniae nucleic acid sequence shown in SEQ ID NO: 20.

The kit of the first aspect of the invention may therefore comprises one or more of the first, second and third probe, in any combination. This will typically be in line with the primers that form part of the kit of the first aspect of the invention, in other words, the kit of the first aspect of the invention will typically further comprise the probe that corresponds to the primer sets contained within the kit. For example, if a kit of the first aspect of the invention comprises only the first primer set, it will typically only contain the first probe. In other embodiments, if the kit of the first aspect of the invention comprises the second and third primer set. It will typically further comprise the second and third probe and so on. In another embodiment, the kit of the first aspect of the invention comprises the first, second and third probes.

The probes are typically targeted to sequences between nucleotides 1 192 and 1574 of SEQ ID NO: 1 , nucleotides 401 and 722 of SEQ ID NO: 9 and nucleotides 828 and 1448 of SEQ ID NO: 20 respectively. In some embodiments, the first probe has a sequence at least 75% identical to SEQ ID NO: 4 or SEQ ID NO: 8 or a sequence complementary to either thereof. In some embodiments, the second probe has a sequence at least 75% identical to SEQ ID NO: 12, SEQ ID NO: 18 or SEQ ID NO: 19 or a sequence complementary to any thereof. In some embodiments, the third probe has a sequence at least 75% identical to SEQ ID NO: 23, SEQ ID NO: 29 or SEQ ID NO: 30 or a sequence complementary to any thereof.

In some embodiments, the kit of the first aspect of the invention can also be used for detection of C. pneumoniae and therefore further comprises a fourth probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the fourth probe is capable of hybridising under very high stringency conditions to a Chlamydia pneumoniae nucleic acid sequence shown in SEQ ID NO: 31. The fourth probe is typically targeted to a sequence between nucleotides 93 and 596 of SEQ ID NO: 31. In some embodiments, the fourth probe has a sequence at least 75% identical to SEQ ID NO: 34 or a sequence complementary thereto. In some embodiments, the kit of the first aspect of the invention can also be used for detection of C. burnetii and therefore further comprises a fifth probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the fifth probe is capable of hybridising under very high stringency conditions to a Coxiella burnetii nucleic acid sequence shown in SEQ ID NO: 38. The fifth probe is typically targeted to a sequence between nucleotides 90 and 454 of SEQ ID NO: 38. In some embodiments, the fifth probe has a sequence at least 75% identical to SEQ ID NO: 41 or SEQ ID NO: 44 or a sequence complementary thereto.

In some embodiments, the kit of the first aspect of the invention can also be used for detection of an internal positive control as described herein and therefore further comprises a sixth probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the sixth probe is capable of hybridising under very high stringency conditions to a human nucleic acid sequence shown in SEQ ID NO: 45. The sixth probe is typically targeted to a sequence between nucleotides 79393 and 79603 of SEQ ID NO: 45. In some embodiments, the sixth probe has a sequence at least 75% identical to SEQ ID NO: 48 or a sequence complementary thereto.

In some embodiments, typically when the kit of the first aspect of the invention is intended for use in carrying out real-time PCR, one or more of the probes present in the kit is detectably labelled. Typically, each of the probes present in the kit is detectably labelled.

Suitable labels are described in detail herein. Typically, the label is a fluorophore. In one embodiment, each probe is dual-labelled with a reporter and a quencher. Typically, each probe is labelled with a different set of labels. This enables the detection of a plurality of amplified DNA sequences at the same time in the same PCR reaction.

The kit of the first aspect of the invention optionally comprises further reagents required to carry out PCR. For example, the kit can further comprise one or more further reagents for the PCR reaction such as enzymes (including a DNA polymerase and optionally a reverse transcriptase), buffers, dNTPs, RNase inhibitors and external positive controls (EPCs).

In one embodiment, the kit of the invention further comprises one or more external positive controls comprising nucleic acid sequences at least 75% identical to one or more of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52 and SEQ ID NO: 53. The kit of the first aspect of the invention is a kit for detecting the presence of one or more of a Mycobacterium tuberculosis complex nucleic acid, a Legionellaceae nucleic acid and a Mycoplasma pneumoniae nucleic acid in a sample. In some aspects, the kit is further useful for detecting the presence of a C. pneumoniae or C. burnetii nucleic acid sequence in a sample. In some aspects, the kit of the first aspect of the invention also comprises instructions for use, for example in a PCR reaction, typically a real-time PCR reaction.

Typically, the nucleotide sequences of the primers, probes and other nucleic acids used in the invention (such as internal and external positive controls) have at least 75% identity, using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403-410 (1990)) provided by HGMP (Human Genome Mapping Project), at the nucleotide level, to the nucleotide sequences described herein. More typically, the nucleotide sequence has at least 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, at the nucleotide level, to the nucleotide sequences described herein. This means that where the nucleotide sequence of any nucleic acid used in the invention is referred to herein as having at least 75% identity to a particular nucleotide sequence, it may have at least 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to that nucleotide sequence. The nucleic acids referred to herein comprise the specified nucleotide sequence and in some embodiments the nucleic acids referred to herein consist of the specified nucleotide sequence.

The present invention also relates to a method which in some embodiments is a PCR method. The polymerase chain reaction (PCR) is a well-known tool used in molecular biology to amplify specific DNA sequences and was first described for the amplification of DNA by Mullis ef al in US Patent No. 4,683, 195 and Mullis in US Patent No. 4,683,202. PCR is carried out in a thermal cycler, and consists of cycles or repeated heating (for DNA denaturation) and cooling (for enzymatic replication of the DNA using a DNA polymerase enzyme such as Taq polymerase). PCR requires the use of at least one primer, typically at least one pair of primers, which are short DNA fragments containing sequences complementary to a target region of a DNA sequence. The reaction mixture also includes deoxynucleotide triphosphates (dNTPs), which are the building blocks for synthesis of a new DNA strand by the DNA polymerase. At the reaction progresses, the DNA that has been amplified in the reaction is itself used as a template for replication, which results in a chain reaction in which the DNA template is exponentially amplified.

Real-time PCR (also referred to as quantative PCR, qPCR) is a variant of PCR which is used to amplify and simultaneously detect or quantify a targeted DNA molecule. Real-time PCR follows the same general principle as PCR but involves detection of the amplification products whilst the reaction is ongoing. This can be done, for example, by adding to the reaction mixture sequence- specific DNA probes (as described herein) that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence. In this embodiment, different DNA probes that are labelled with different fluorescent reporters can be used to enable the detection of multiple different amplification products simultaneously. Suitable fluorescent labels for use in this embodiment are described above. Detection can alternatively be carried out using non-specific fluorescent dyes that intercalate with any double-stranded DNA. Suitable double-stranded DNA binding dyes include SYBR® Green 1 or EvaGreen®. In a second aspect, the present invention provides a method for detecting the presence of one or more of Mycobacterium tuberculosis complex, Legionellaceae and Mycoplasma pneumoniae in a sample, comprising:

a. amplifying one or more of a M. tuberculosis complex nucleic acid, a Legionellaceae nucleic acid and a M. pneumoniae nucleic acid in a sample,

wherein amplifying the M. tuberculosis complex nucleic acid comprises contacting the sample with at least one first primer between 10 and 60 nucleotides in length, wherein the primer is capable of hybridizing under very high stringency conditions to a M. tuberculosis complex nucleic acid sequence shown in SEQ ID NO: 1 , and wherein the primer is capable of amplifying the M. tuberculosis complex nucleic acid;

wherein amplifying the Legionellaceae nucleic acid comprises contacting the

sample with at least one second primer between 10 and 60 nucleotides in length, wherein the primer is capable of hybridizing under very high stringency conditions to a Legionellaceae nucleic acid sequence shown in SEQ ID NO: 9, and wherein the primer is capable of amplifying the Legionellaceae nucleic acid; and

wherein amplifying the M. pneumoniae nucleic acid comprises contacting the

sample with at least one third primer between 10 and 60 nucleotides in length, wherein the primer is capable of hybridizing under very high stringency conditions to a M. pneumoniae nucleic acid sequence shown in SEQ ID NO: 20, and wherein the primer is capable of amplifying the M.

pneumoniae nucleic acid; and

b. detecting amplification of one or more of a Mycobacterium tuberculosis complex nucleic acid, a Legionellaceae nucleic acid and a Mycoplasma pneumoniae nucleic acid if present in the sample. In the method of the present invention, detection of amplification of a M. tuberculosis complex nucleic acid is indicative of the presence of M. tuberculosis complex in the sample, detection of amplification of a Legionellaceae nucleic acid is indicative of the presence of Legionellaceae in the sample and detection of amplification of a M. pneumoniae nucleic acid is indicative of the presence of M. pneumoniae in the sample.

In some embodiments, the method of the invention is used to detect the presence of C. pneumoniae in the sample. In these embodiments, the method further comprises amplifying a C. pneumoniae nucleic acid sequence shown in SEQ ID NO: 31 , comprising contacting the sample with at least one fourth primer between 10 and 60 nucleotides in length, wherein the primer is capable of hybridizing under very high stringency conditions to a C. pneumoniae nucleic acid sequence shown in SEQ ID NO: 31 , and wherein the primer is capable of amplifying the C. pneumoniae nucleic acid. In these embodiments, detection of amplification of a C. pneumoniae nucleic acid is indicative of the presence of C. pneumoniae in the sample. In some embodiments, the method of the invention is used to detect the presence of C. burnetii in the sample. In these embodiments, the method further comprises amplifying a C. burnetii nucleic acid sequence shown in SEQ ID NO: 38, comprising contacting the sample with at least one fifth primer between 10 and 60 nucleotides in length, wherein the primer is capable of hybridizing under very high stringency conditions to a C. burnetii nucleic acid sequence shown in SEQ ID NO: 38, and wherein the primer is capable of amplifying the C. burnetii nucleic acid. In these embodiments, detection of amplification of a C. burnetii nucleic acid is indicative of the presence of C. burnetii in the sample. In some embodiments, an internal positive control is used. In these embodiments, the method further comprises amplifying a human nucleic acid sequence shown in SEQ ID NO: 45, comprising contacting the sample with at least one sixth primer between 10 and 60 nucleotides in length, wherein the primer is capable of hybridizing under very high stringency conditions to a human nucleic acid sequence shown in SEQ ID NO: 45, and wherein the primer is capable of amplifying the human nucleic acid. In these embodiments, detection of amplification of a human nucleic acid is indicative of the presence of human nucleic acid in the sample.

The primers used in the method of the second aspect of the invention are as described in relation to the first aspect of the invention. Single primers as described in relation to the first aspect of the invention can be used in combination with other suitable primers, or in pairs as described herein in relation to the first aspect of the invention.

Step a of the method of the second aspect of the invention (the amplification step) is typically carried out using polymerase chain reaction (PCR) but can also be carried out using nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA) or ligase chain reaction (LCR). The method of the second aspect of the invention is typically a real-time PCR method. However, other types of DNA-based techniques, can also be used.

The results of the amplification step (e.g. the PCR reaction) can be determined using any suitable method known to the person skilled in the art, for example using agarose gel electrophoresis.

Typically, however, probes are used to hybridize to the amplification products (e.g. PCR products). The probes are typically labelled for this purpose and suitable labels are described herein.

In one embodiment, the method of the second aspect of the invention further comprises:

contacting the sample with a first probe comprising a nucleic acid molecule between

10 and 40 nucleotides in length, wherein the first probe is capable of hybridising under very high stringency conditions to a M. tuberculosis complex nucleic acid sequence shown in SEQ ID NO: 1 ; and/or

contacting the sample with a second probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the second probe is capable of hybridising under very high stringency conditions to a Legionellaceae nucleic acid sequence shown in SEQ ID NO: 9; and/or

contacting the sample with a third probe comprising a nucleic acid molecule between

10 and 40 nucleotides in length, wherein the third probe is capable of hybridising under very high stringency conditions to a M. pneumoniae nucleic acid sequence shown in SEQ ID NO: 20; and detecting hybridization between one or more probe and a nucleic acid in the sample.

In the method of the invention, detection of hybridization between the first probe and a nucleic acid in the sample indicates the presence of M. tuberculosis complex in the sample, detection of hybridization between the second probe and a nucleic acid in the sample indicates the presence of Legionellaceae in the sample and detection of hybridization between the third probe and a nucleic acid in the sample indicates the presence of M. pneumoniae in the sample. In some embodiments, the method of the invention is used to detect the presence of C. pneumoniae in the sample. In these embodiments, the method further comprises contacting the sample with a fourth probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the fourth probe is capable of hybridising under very high stringency conditions to a Chlamydia pneumoniae nucleic acid sequence shown in SEQ ID NO: 31 ; and detecting hybridization between the fourth probe and a nucleic acid in the sample. In these embodiments, detection of hybridization between the fourth probe and a nucleic acid in the sample indicates the presence of C. pneumoniae in the sample.

In some embodiments, the method of the invention is used to detect the presence of C. burnetii in the sample. In these embodiments, the method further comprises contacting the sample with a fifth probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the fifth probe is capable of hybridising under very high stringency conditions to a Coxiella burnetii nucleic acid sequence shown in SEQ ID NO: 38; and detecting hybridization between the fifth probe and a nucleic acid in the sample. In these embodiments, detection of hybridization between the fourth probe and a nucleic acid in the sample indicates the presence of C. burnetii in the sample.

In some embodiments, an internal positive control is used. In these embodiments, the method further comprises contacting the sample with a sixth probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length, wherein the sixth probe is capable of hybridising under very high stringency conditions to a human nucleic acid sequence shown in SEQ ID NO: 45; and detecting hybridization between the sixth probe and a nucleic acid in the sample. In these embodiments, detection of hybridization between the sixth probe and a nucleic acid in the sample indicates the presence of human nucleic acid in the sample. In some embodiments, the probes used in the method of the second aspect of the invention are as described in relation to the first aspect of the invention. The probes are typically labelled, as described herein. In one embodiment, when the method of the invention is a real-time PCR method, each probe used in the reaction is labelled with a reporter and a quencher, typically a different reporter/quencher pair for each probe in the same reaction. In another embodiment, one probe used in the reaction is labelled with a reporter and an adjacent probe used in the reaction is labelled with a quencher. Such energy transfer between adjacent molecules is known as fluorescence resonance energy transfer (FRET) and occurs when the reporter and the quencher are located very close together, typically at a distance of from 10 to 100 Angstroms.

When the method of the invention is a PCR method it can be carried out using any appropriate PCR programme. One specific PCR programme which corresponds to that used in the Examples is as follows: 37°C - 15min

95°C - 5min

94°C - 15s, denaturation step

66°C - 10s, annealing step 45 cycles

59°C - 30s, annealing step

72°C - 15s, extension step

40°C - 30s, cooling step

However, alternative PCR programmes can also be used, with variations in the above-mentioned programme. In one embodiment, any of the annealing or extension steps can be increased by up to 4°C compared to the programme set out above, for example by +1°C, +1.5°C, +2°C, +2.5°C, +3°C or +3.5°C, or decreased by up to 9°C. compared to the programme set out above, for example by - 1°C, -1 ,5°C, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C or -8°C. In another embodiment, a different number of cycles can be used. For example, from 25 to 55 cycles can be used, such as from 30 to 50 cycles, from 32 to 48, cycles, from 35 to 47 cycles, from 36 to 46 cycles, or from 38 to 45 cycles. For example, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54 or 55 cycles can be used.

In one embodiment, a hot start DNA polymerase is used in the PCR reaction. In these embodiments, the reaction components are heated e.g. to a temperature of 95°C prior to commencing the cycles of denaturation, annealing and extension. This step can be omitted if a different DNA polymerase is used.

In some embodiments, the method of the invention is a multiplex PCR method, i.e. one in which a single PCR reaction is carried out to amplify a number of different DNA sequences. In one embodiment, the method is a "one pot" method in which all 3 of a Mycobacterium tuberculosis complex nucleic acid, a Legionellaceae nucleic acid and a Mycoplasma pneumoniae nucleic acid are detected in a single reaction, optionally together with one or more internal or external positive controls. For example, the method can be a real-time PCR method that uses probes labelled with a different reporter and quencher for each nucleic acid, enabling the detection of 3 or more target nucleic acids from different organisms in a single reaction. In other embodiments, the method is split into a number of reactions. For example, the method can comprise or consist of two reactions, for example a duplex reaction and a triplex reaction. In one embodiment, the method comprises a duplex for the detection of Mycobacterium tuberculosis complex and an internal positive control (IPC) and a triplex for the detection of Legionellaceae and Mycoplasma pneumoniae and an IPC. In yet another embodiment, the method further comprises an external positive control (EPC) for Mycobacterium tuberculosis complex, Legionellaceae and Mycoplasma pneumoniae.

The method of the second aspect of the invention is carried out on a sample. Typically, the sample used in the method of the invention is a respiratory sample such as sputum, bronchial secretion, respiratory fluid, tracheal secretion. Bronchoalveolar lavage (BAL) fluid, bronchial washing, bronchoalveolar aspirates (such as lung fine-needle aspirates - LFNA), cough swabs/plates or a tissue biopsy such as a lung biopsy. Other types of samples suitable for use in the present invention include clinical samples such as blood, bone marrow, gastric washing, urine, stool or from post-mortem specimens. Also suitable for use in the invention are sterile site body fluids such as cerebrospinal fluid (CSF), pleural fluids etc.

The method of the invention typically finds use in the diagnosis of disease in humans. However, the method equally finds use in veterinary medicine. Samples may therefore be obtained from humans or animals, such as cows (particularly for the detection of bovine tuberculosis), sheep, goats, cats, dogs, rabbits etc.

In order to carry out the method of the present invention, nucleic acids such as DNA are extracted from the sample. This can be carried out by any suitable method known in the art, for example using commercially available kits. For example, DNA can be extracted using commercially available kits with spin-columns, but any method may be used including methods using phenol- chloroform or magnetic beads, based on manual or robotic systems. Such methods are within the common general knowledge of a skilled person.

In one specific embodiment, the present invention provides a method for detecting the presence of one or more of Mycobacterium tuberculosis complex, Legionellaceae and a Mycoplasma pneumoniae in a sample, comprising:

amplifying one or more of the M. tuberculosis complex rpoB gene using a forward primer comprising SEQ ID NO: 2 and a reverse primer comprising SEQ ID NO: 3; the Legionellaceae 16S gene using a forward primer comprising SEQ ID NO: 10 and a reverse primer comprising SEQ ID NO: 1 1 ; the M. pneumoniae 16S gene using a forward primer comprising SEQ ID NO: 21 and a reverse primer comprising SEQ ID NO: 22;

contacting a sample with a first probe comprising SEQ ID NO: 4, a second probe comprising SEQ ID NO: 12 and a third probe comprising SEQ ID NO: 23; and

detecting hybridization between one or more probe and a nucleic acid in the sample.

In one embodiment, the method further comprises amplifying the C. pneumoniae 16S gene using a forward primer comprising SEQ ID NO: 32 or SEQ ID NO: 35 and a reverse primer comprising SEQ ID NO: 33, SEQ ID NO: 36 or SEQ ID NO: 37 and contacting a sample with a fourth probe comprising SEQ ID NO: 34.

In one embodiment, the method further comprises amplifying the C. burnetii 16S gene using a forward primer comprising SEQ ID NO: 39 or SEQ ID NO: 42 and a reverse primer comprising SEQ ID NO: 40 or SEQ ID NO: 43, and contacting a sample with a fifth probe comprising SEQ ID NO: 41 or SEQ ID NO: 44.

In another embodiment, the method further comprises amplifying the human TG gene using a forward primer comprising SEQ ID NO: 46 and a reverse primer comprising SEQ ID NO: 47 and contacting a sample with a sixth probe comprising SEQ ID NO: 48.

In these embodiments, the method is typically a real-time PCR method. In these embodiments, each probe used in the reaction is labelled with a reporter and a quencher, typically a different reporter/quencher pair for each probe in the same reaction. In one specific embodiment, the method of the second aspect of the invention comprises the following reactions that may be run in a single experiment:

Reaction 1 : Duplex for the detection of Mycobacterium tuberculosis complex and an Internal Positive Control (IPC).

Reaction 2: Triplex for the detection of members of the Family Legionellaceae, Mycoplasma pneumoniae and an Internal Positive Control.

Reaction 3: External Positive Control (EPC) for M. tuberculosis complex, Legionellaceae and Mycoplasma pneumoniae.

The present invention allows a rapid and accurate detection of the bacteria mentioned herein, and is structured in such way that requires minimum handling, preventing the risk of contamination of the reactions. In some embodiments, the kit of the first aspect of the invention includes primers and probes for the amplification of internal and external positive controls, which can be used in the method of the second aspect of the invention. Preferred features of the second aspect of the invention are as described for the first aspect mutatis mutandis. The invention will now be further described with reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as limiting on the invention. Reference is made to a number of Figures, in which:

Figure 1 is an example of the preparation of the PCR reaction. In the Figure, the following abbreviations are used:

Mix A - Detection of Mycobacterium tuberculosis complex and IPC

Mix B - Detection of Legionellaceae, Mycoplasma pneumoniae and IPC

IPC - Internal Positive Control

UDG - Uracyl DNA Glycosylase

CPEs - External Positive Controls

Results are shown in Figure 2.

Figure 2 gives examples of the results according to different real-time PCR experiments, as follows:

A

Equipment: LightCycler 2.0

Reaction: Triplex for Legionellaceae, M. pneumoniae and Internal Positive Control Shown in chart results for Legionellaceae

Probes in the reaction:

PrevLeg3- Labelled with FAM at 5' and BHQ1 at 3'

PforMpneu2 - Labelled with ROX at 5' and BHQ2 at 3'

PforExon26 2 - Labelled with BHQ2 at 5' and Pulsar 650 at 3'

Samples: Samples from EQAS (External Quality Assessment Schemes)

B

Equipment: ABI 7500 Fast

Reaction: Duplex for M. tuberculosis complex (Gray) and Internal Positive Control (Black) Probes in the reaction:

PforMtuM- Labelled with FAM at 5' and BHQ1 at 3'

PforExon26 2 - Labelled with Quasar 670 at 5' and BHQ2 at 3'

Samples: spiked samples for Analytical sensitivity

C

Equipment: Rotor-Gene 6000

Reaction: Duplex for M. tuberculosis complex and Internal Positive Control

Shown in chart results for M. tuberculosis complex

Probes in the reaction: PforMtubl- Labelled with FAM at 5' and BHQ1 at 3'

PforExon26 2 - Labelled with Quasar 670 at 5' and BHQ2 at 3'

Samples: Respiratory samples Example 1 - Detection of Tuberculosis and Atypical Pneumonia by Real-Time PCR caused by Mycobacterium tuberculosis complex. Mycoplasma pneumoniae and bacteria belonging to Family Legionellaceae.

The test can be applied to several clinical samples used for detection of the pathologies described above, including sputum, bronchial secretions, bronchoalveolar lavage (BAL), bronchoalveolar aspirates (lung fine-needle aspirates - LFNA) and biopsies. The test consisted of two amplification reactions of bacterial agents:

i) a duplex which allows the detection of both Mycobacterium tuberculosis complex and an Internal Positive Control (IPC) that amplifies human DNA naturally present in samples mentioned above. ii) a triplex which allows the detection of Mycoplasma pneumoniae and Legionellaceae, as well as an internal positive control as described above.

IPC allows the evaluation of the extraction of the sample to be analyzed, several of the reagents, the PCR program, as well as the detection of false negatives due to inhibitors present in the sample.

There is an additional reaction triplex, which acts as External Positive Control (EPC), for evaluation of the specific oligos regarding the detection of bacteria. Equipment Used - The development phase was essentially carried out in a Roche LightCycler 2.0. During the validation phase several tests were conducted, allowing the validation of the following Real-Time PCR equipments: ABI 7500 FAST, Rotor-Gene 6000, LightCycler 480 and IQ5.

Primer design

Mycobacterium tuberculosis complex

Target region - Sequence of about 380 bases of RNA polymerase B gene (rpo B).

Legionellaceae

Target region - Sequence of about 320 bases of ribosomal 16S gene (16S).

Mycoplasma pneumoniae

Target region - Sequence of about 620 bases of ribosomal 16S gene (16S). Internal Positive Controls - Sequence of about 200 bases of thyroglobulin gene (TG). External Positive Controls - Sequence of DNA synthesized with about 1 10 bases, including the sequences of primers and probes.

Reporters and Quenchers Equipments

Mycobacterium tuberculosis complex detection

PforMtubl- FAM-5'-ATCAACATCCGGCCGGTGGTCG-3'-BHQ1

and

PforExon26 2 - BHQ2-5'-ATGGCTTCGTCCTCACACAGGTT-3'- P650 LightCycler 2.0

or

PforExon26 2 - Q670-5'-ATGGCTTCGTCCTCACACAGGTT-3'- BHQ2 other equipments

Legionellaceae / Mycoplasma pneumoniae detection

PrevLeg3- FAM-5'-CGGAAATTCCACTACCCTCTCC-3'-BHQ1

PforMpneu2- ROX-5'-CCAATACAAACAGTCGCCAGCTT-3'-BHQ2

and

PforExon26 2 - BHQ2-5'-ATGGCTTCGTCCTCACACAGGTT-3'- P650 LightCycler 2.0

or

PforExon26 2 - Q670-5'-ATGGCTTCGTCCTCACACAGGTT-3'- BHQ2 other equipments

Reactions for External Positive Controls

PforMtubl - FAM-5'-ATCAACATCCGGCCGGTGGTCG-3'-BHQ1

PforMpneu2- ROX-5'-CCAATACAAACAGTCGCCAGCTT-3'-BHQ2

and

PrevLeg3- BHQ2-5'-CGGAAATTCCACTACCCTCTCC-3'-P650 LightCycler 2.0

or

PrevLeg3- Q670-5'-CGGAAATTCCACTACCCTCTCC-3'-BHQ2 other equipments Legend

BHQ - Black Hole Quencher (Biosearch)

Q670 - Quasar 670 (Biosearch)

P650 - Pulsar 650 (Biosearch)

Methods

Samples used were bacterial and fungal isolates, clinical samples, samples from EQAS and clinical samples inoculated with E. coli transformed with the target region for detecting each bacterial target.

DNA was extracted using commercial kits with spin-col

Specificity assays were performed using 20ng of DNA whenever possible, or 4 or 5ul of sample, with the aim of increasing the difficulty of the reaction. In order to perform rigorous trials, were selected phylogenetically closely related species, and species with the highest DNA homogeneity to the primer and probe sequences, and usually described as present in respiratory flora, or causing respiratory infections. Experiments were conducted on several different types of equipment, using the standard protocol as well as a protocol with a decrease of the temperatures in the PCR cycle, thus, decreasing stringency and increasing the difficulty of the specificity. Analytical sensitivity tests were performed in order to estimate the minimum number of equivalents of each genome to be detected, assuming 3 copies of 16S gene per genome and only one copy of rpoB gene per genome. For this experiment the fragments to detect were amplified, for each of the agents, using the following strains: i) MB275 - Mycoplasma pneumoniae (DSM2291 1 ^T ), ii) MB274 - Legionella pneumophila subsp. pneumophila (DSM7513 ^T ) and MB108 - Legionella micdadei, iii) MB244 - Mycobacterium tuberculosis and MB343 - Mycobacterium bovis. Amplified fragments were cloned into E. coli and subsequent counts were made of colonies' suspensions, in order to add before extraction different quantities of equivalents of copies of genomes to negatives samples of sputum. Comparisons were also conducted in order to evaluate the sensitivity between different types of Real-Time PCR equipment.

For clinical sensitivity tests, samples from several Portuguese Hospitals were analyzed. These samples were also submitted to tests for the identification of mutations associated with rifampicin resistance in those positive for Mycobacterium tuberculosis complex. The results were compared with those obtained by diagnostic procedures performed by technicians in several Hospitals, including Ziehl-Neelsen staining technique (AFB) and culture, considered the Gold Standard.

Additionally, results from different commercial IVD kits, based in Real-Time PCR, were used to complete the comparison between different tests.

Results

Theoretical

Sensitivity - Mycobacterium tuberculosis complex DNA sequences were analyzed, concerning the RNA polymerase B gene (rpo B) deposited in the public database Genbank (length of the sequences: 700-4000 bases). For 68 DNA sequences having the region corresponding to the forward primer and 79 the reverse primer region, no mismatches were detected between the oligo sequences and respective sequences that theoretically could inhibit its amplification. 62 published Legionellaceae DNA sequences, regarding the 16S ribosomal DNA, and deposited in the public database Genbank (size of the sequences: 1250-1750 bases) were analyzed. No mismatches were detected that theoretically could inhibit its amplification.

4 DNA sequences of Mycoplasma pneumoniae were analyzed, for the 16S ribosomal deposited in the public database Genbank (size of the sequences: 1250-1750 bases), and no differences between the oligo sequences and respective sequences were detected, that theoretically could inhibit its amplification.

Specificity - 670 DNA sequences related to the rpoB gene of Mycobacterium spp. outside the M. tuberculosis complex were analyzed. No theoretical cases were detected that could indicate the possibility of amplification.

Related to Corynebacterineae except Mycobacterium spp., 70 sequences were analyzed concerning the rpoB gene, and no cases were detected that theoretically indicate the possibility of amplification. Within Actinobacteria except Corynebacterineae, 105 sequences were analyzed concerning the rpoB gene, and no cases have been detected that theoretically indicate the possibility of amplification. We analyzed 1975 published sequences of Bacteria except Actinobacteria, for the rpoB gene, with a limit of 3 sequences per species, and no cases were detected that theoretically indicate the possibility of amplification.

Concerning Legionellales except Legionellaceae, 55 sequences were analyzed in the 16S gene region, and no cases were detected that theoretically indicate the possibility of amplification.

In Gammaproteobacteria, except Legionellales, Alteromonadales, Enterobacteriales and Pseudomonadales, 1809 sequences related to the 16S gene region, were analyzed, and no cases were detected that indicate the possibility of amplification with the theoretical following exceptions corresponding to about 6% of the sequences mentioned above. These sequences belong to genera essentially living in extreme environmental conditions and therefore with no previous records of occurrence in the type of samples to which this test is intended. The genera are: Alcanivorax, Bathy modiolus, Caedibacter, Clonothrix, Ectothiorhodosinus, Halomonas, Methylomicrobium, Natronocella and Thiothrix.

We analyzed 5171 sequences, concerning Alteromonadales, Enterobacteriales and Pseudomonadales, in the 16S region, but did not detect any cases that theoretically indicate the possibility of amplification, with the following exceptions of two genera of marine bacteria Order Alteromonadales, corresponding to 0.4 % of the sequences analyzed: Paraferrimonas and Marinobacterium .

1616 sequences belonging to Tenericutes, except Mycoplasma pneumoniae, regarding the 16S gene region were analyzed, but no theoretical cases of amplification were detected.

Experimental

1. Mycobacterium tuberculosis complex

1.1 Specificity - 123 samples were used, comprising 39 microbial isolates samples (34 bacteria and 5 fungi), 76 clinical respiratory samples (12 were extensively analyzed to evaluate microbial diversity) and 8 samples of EQAS (Instand E.V.) (Tables 1 and 3). Analyzed samples include 70 different species of microorganisms. There were no false positive cases, even in cases in which we decreased PCR program temperature in 1 °C or 2°C. 1.2 Limit of Detection - A detection limit was determined corresponding to the equivalent of 1 copy of the genome, using for that Mycobacterium tuberculosis and Mycobacterium bovis species. Assays were performed comparing dilutions of Mycobacterium tuberculosis DNA to assess the sensitivity in different types of equipment. 1.3 Clinical Sensitivity - We compared results of respiratory specimens with suspected TB infection, coming from hospital centers. In total, 1 18 samples were analyzed, consisting of sputum, bronchial secretions, broncho-alveolar lavages (BAL), bronchoalveolar aspirate (LFNA) and purulent material (Table 3). Culture results were considered as reference test. In discrepant results when culture was negative and a positive test was obtained by Biopremier, we proceeded to DNA sequencing for confirmation; when the opposite occured, and a positive culture was obtained, the confirmation was carried out by identifying the isolate by DNA sequencing. The result of the clinical sensitivity was also better using Biopremier's kit, than in cases of Q or S1 commercial kits.

1.4 Comparison of Molecular Biology Kits - Results using Biopremier's test were compared with the test results using two other commercial kits, designated for that purpose Q and S1. The comparison allowed us to analyze results in terms of specificity (Tables 2 and 3) and clinical sensitivity (Table 3). Both Q and S1 kits were used in the LightCycler 2.0 equipment. Q and S1 Molecular Biology kits to compare Clinical Sensitivity and Specificity, applied to samples that were theoretically more complicated to distinguish, or in cases of clinical samples in which the intensity of the result signal of Biopremier's test was weaker. Therefore, it may be concluded a higher or lower clinical sensitivity and specificity in comparison between the different molecular biology tests, not quantifying this difference, because sampling for other kits was not randomly executed but rather according to the criteria mentioned above. 1.4.1 Specificity Comparison - There were no nonspecific results using the Biopremier's test, with specificity results superior to those obtained with the kits Q and S1. Experiments were conducted using bacterial isolates and samples from EQAS.

1.4.2 Sensitivity Comparison - There was one False Negative for Biopremier's test in 1 18 samples and no False Positives. For Q and S1 kits there was one and two False Positives, respectively, and no False Negatives were observed.

2. Leqionellaceae

2.1 Specificity - 75 samples were analyzed, comprising 34 samples of microbial isolates (28 bacteria, 6 fungi), 23 clinical respiratory samples (extensively analyzed to evaluate microbial diversity) and 18 samples for EQAS (Instand E.V.) (Tables 4 and 5). Samples analyzed include 66 different species of microorganisms. We excluded samples MB183 and CL8, because the first was contaminated and the second was found to have the presence of L. pneumophila. There were no false positive cases, even when decreasing the temperature of the PCR program on 1 °C or 2° C for different types of Real-Time PCR equipment.

2.2 Limit of Detection - It was estimated a detection limit corresponding to the equivalent of 1 copy of the genome, using for that determination both the species L. pneumophila (type strain) and T. micdadei.

2.3 Clinical Sensitivity - Respiratory samples from Hospitals, with suspected infection of Tuberculosis, were analyzed also for evaluation of infection with Legionellaceae or Mycoplasma pneumoniae. A total of 44 samples were analyzed, consisting of sputum, bronchial secretions and one broncho-alveolar lavages (BAL), as well as samples from EQAS (Table 5).

2.4 Comparison of Molecular Biology Kits - Biopremier's test results were compared with those of two other commercial kits designated for this purpose T and M1 , with the difference that both detect just L. pneumophila species and not the Legionellaceae Family, as the former test does. The comparison allowed comparison of results in terms of Inclusiveness, Specificity, and Clinical Sensitivity (Tables 4 and 5). In the case of Clinical Sensitivity and Specificity, we used Molecular Biology kits T and M1 in cases of samples theoretically more complicated to distinguish, or in cases of clinical samples in which the results of the test corresponded to Biopremier's weaker signals. Therefore, it may be concluded a higher or lower clinical sensitivity and specificity in comparison between the different Molecular Biology tests, not quantifying this difference, because sampling for other kits was not randomly executed but rather according to the criteria mentioned above. Kits T and M1 were used in the LightCycler 2.0 equipment.

2.4.1 Inclusiveness Comparison - To assess inclusiveness, 18 different positive samples were analyzed (17 bacterial isolates and one sample from an EQAS), representing 5 species belonging to the Legionellaceae Family. All samples were positive using Biopremier's test. With T kit, the 6 analyzed samples, corresponding to five different species, showed unexpected positive results to Fluoribacter dumoffii, Legionella anisa, and Tatlockia micdadei, and an expected negative result to Fluoribacter gormanii. 2.4.2 Specificity comparison - The comparison of specificity outside the Legionellaceae Family, yielded no nonspecific results using Biopremier's test or T kit. For this comparison, five samples were analyzed, corresponding to five different species of Gammaproteobacteria, present as pathogens or commensals of the airways. 2.4.3 Sensitivity Comparison - No False Negatives or False Positives were obtained using Biopremier's test. Some False results were obtained using kit T and M1. Kit T was used in 17 clinical samples and in 8 samples from EQAS, while kit M1 was only used in 6 clinical samples. 3. Mycoplasma pneumoniae

3.1 Specificity - 70 samples were analyzed, comprising 36 samples of microbial isolates (30 bacteria and 6 fungi), 12 clinical respiratory samples from CHLN (analyzed to evaluate microbial diversity) and 22 samples from clinical inter-laboratory (Instand E.V.) (Tables 6 and 7). Samples analyzed include 69 different species of microorganisms. There were no false positive cases, even when the PCR program temperature was decreased by 1 °C or 2°C in different types of Real-Time PCR equipment.

3.2 Limit of Detection - We estimated a detection limit corresponding to the equivalent of 5 copies of the genome, using Mycoplasma pneumoniae (type strain) for this determination.

3.3 Clinical Sensitivity - Experiments were conducted using mainly samples from EQAS (21 ) and one clinical sample (sputum). Biopremier's results were compared with those from the commercial kit S2, which enables also the detection of Chlamydia pneumoniae. The S2 kit was tested in the equipment Rotor-Gene 6000. The Clinical Sensitivity results were similar when using Biopremier's test and S2 kit in LightCycler 2.0 (Table 7).

4. Performance

The evaluation of the performance of Biopremier's Test is shown in Table 8, describing results of Specificity, Analytical and Clinical Sensitivity, Confidence Intervals when applicable, Negative and Positive Predictive Values and Accuracy.

Table 1. Specificity of the Test for all the detections

Mycoplasma lipophilum CL4 Table 1 (cont.) Specificity of the Test for all the detections

Influenza A CL4

Table 1 (cont.) Specificity of the Test for all the detections

Table 2. Comparison of Specificity between Molecular Biology (MB) Tests - M. tuberculosis complex

Table 3. Clinical Sensitivity for Mycobacterium tuberculosis complex

Clinical Sensitivity Mycobacterium tuberculosis complex

Clinical samples Type of samples Origin PT Kit S1 Kit Q ampli Seq BAAR Cult.

Table 3 (cont.). Clinical Sensitivity for Mycobacterium tuberculosis complex

Legend Table 4. Comparison of Specificity between Molecular Biology Tests - Leqionellaceae

Table 5. Comparison of Sensitivity between Molecular Biology Tests - Leqionellaceae

EIL71 D lEQAS samp Instand

Table 6. Comparison of Specificity between Molecular Biology Tests - M. pneumoniae

Specificity comparison between MB kits Mycoplasma pneumoniae

Species Code n° Ref PT Kit M2 Kit S2 Origin Confirm.

M. hominis, M. salivarium,... CL31 N N N CHL

M.faucium, M.spermatophilum,... CL4 N N N CHL

Mycoplasma orale EIL39M1 N N N Instand

Mycoplasma penetrans MB38-40 1 N N Isolate

Ureaplasma parvum MB26 N N U Isolate

Ureaplasma urealyticum MB31 1 N Isolate Table 7. Comparison of Sensitivity between Molecular Biology Tests - M. pneumoniae

Table 8. Performance characteristics of Biopremier's Test for all targets

M. tuberculosis complex Leeionellaceae Mycoplasma pneumoniae

VN 17Γ VN VN W

VP 54 VP 23 VP 12

RN E 58 RN E 58 RN E 58

R SC 64 RN SC 20 RN SC 12

RP 52 RP 22 RP 9

IN 1 FN 0 FN 1

EP 0 FP 0 FP 0

U E 1 U E 1 U E 0 use 1 u se 1 use 2

E - RN/(RN+FP) 100,0% E - RN/(RN+FP) 100,0% E - RN/(RN+FP) 100,0% SC - RP/(RP+FN) 98,1% SC - RP/(RP+FN) 100,0% SC - RP/(RP+FN) 90,0% IC95% SC 0,981+0,037 IC95% SC IC95% SC 0,900+0,186 SA . 1 copy genome SA 1 copy genome SA eq. 5 cop. genome

VPN - (RN SC/RN SC+FN) 98,5% VPN - (RN SC RN SC+FN) 100,0% VPN - (RN SC/RN SC+FN) 92,3%

VPP - (RP/RP+FP) 100,0% VPP - (RP RP+FP) 100,0% VPP - (RP/RP+FP) 100,0%

99,4% P 100,0% P 98,8%

IC95% SC - SC±l,96V[(SC*(l-SC))/n] Interpretation of the Results

Results are shown in Figure 2. For all target agents, namely: M. tuberculosis complex, Legionellaceae and Mycoplasma pneumonia should be observed a sigmoidal curve, with Ct or Cp values normally ranging from 25 to 37 cycles. When the target is detected, the IPCs may have no amplification. The Ct or Cp for the IPC usually is between cycles 18 and 33.

References

1. Edelstein P.H. and Cianciotto N.P., 2005. Legionella, p. 271 1-2724. In Mandell G.L., Bennett J.E. and Dolin R. (ed.), Principles and Practice of Infectious Diseases. Elsevier Churchill Livingstone, Philadelphia, Pa.

2. Edelstein P.H., 2007. Legionella, p. 835-849. In Murray P.R. (ed.), Manual of Clinical Microbiology, 9 ^th Edition. ASM Press, Washington, DC.

3. Waites K.B. and Talkington D.F., 2004. Mycoplasma pneumoniae and its role as a human pathogen. Clin. Microbiol. Rev. 17:697-728.