QUANTITATIVE DETECTION AND DISCRIMINATION OF TARGET GENETIC MATERIAL BY HIGH-THROUGHPUT DNA ANALYSIS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Serial No. 62/715,016, filed August 6, 2018, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled“Seq-List.txt” which was created on August 1, 2019 and is 19 KB. The entire content of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Different microbial species inhabit a given subject at any time-point. Monitoring the presence of pathogens in a subject is an important process to devise strategies to reduce the negative impacts the pathogens might cause.

Also, efficacy of nucleic acid (such as DNA or RNA) based vaccines, such as viral vaccines, depends on the replication of the nucleic acids from the vaccines and their translation into the corresponding proteins. For example, recombinant viral vaccines are created by incorporating in a vector nucleic acid sequences that express antigens against which immunity is induced in a subject. The proteins produced from the nucleic acid vaccines then induce immune responses in the subject thereby protecting the subject from the infections caused by the corresponding pathogens. Thus, efficacy of nucleic acid based vaccines typically directly corresponds to the number of copies of the nucleic acids in a vaccinated subject.

Further, the sequence data about a gene of an organism can be used to identify genetic polymorphisms (e.g., SNP and indel) within different strains of the organism that may be present in a sample from a subject.

Therefore, methods are desirable of detecting, quantifying, and sequencing target nucleic acids, for example, from organisms that inhabit a subject or from nucleic acid based vaccines. BRIEF SUMMARY OF THE INVENTION

Certain embodiments of the invention provide polymerase chain reaction (PCR) based detection and quantification of a target genetic locus present in a sample obtained from a subject. In preferred embodiments, the PCR based detection and quantification is a multiplex PCR detection and quantification, thus facilitating detection and quantification of a plurality of target genetic loci present in a sample obtained from a subject. In certain embodiments, the plurality of target genetic loci is from one or more organisms that inhabit a subject or from one or more nucleic acid based vaccines administered to a subject.

In certain embodiments, the methods of the invention comprise amplifying via PCR one or more target genetic loci and one or more control genetic loci in a genetic material obtained from a sample. The one or more target genetic loci and one or more control genetic loci are amplified using one or more primer pairs. Typically, one or more control genetic loci are present in a genetic material of a subject and that provide a frame of reference for the quantification of the target genetic loci and thereby, target genetic material.

Accordingly, certain embodiments of the invention provide a method of PCR amplifying from a genetic material obtained from a subject one or more control genetic loci and one or more target genetic loci, quantifying the PCR amplicons produced from the one or more control genetic loci and the one or more target genetic loci to determine the amount of the one or more target genetic loci relative to the one or more control genetic loci in the genetic material obtained from the subject. Typically, a sample obtained from a subject contains the subject’s genetic material, for example, subject’s cells, and also the target genetic material from one or more organisms if those organisms are present in the sample obtained from the subject.

In further embodiments, different amplification products are sequenced to determine the presence of polymorphisms within the target genetic material. The sequenced genetic loci can be used to generate phylogenetic trees to illustrate evolutionary relationships within different target genetic materials, population structure and principal component analysis to group samples based on their population structure and relatedness.

Kits containing primer pairs and reagents for carrying out the methods disclosed herein are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the construct of a vaccine against Infectious Laryngotracheitis virus (ILTV), comprising incorporation in the HVT genome of the genes encoding proteins gD and gl. Primers (represented by arrows) amplify the insertion regions of the genes encoding these proteins in the vaccine against Infectious ILTV.

Figure 2 shows an example of quantitative result obtained for detection of a vaccine against ILTV. Increasing copies of the vaccine was mixed with vaccine-free chicken DNA. The normalized ratio between reads allocated to vaccine loci and reads allocated to DAGLA (chicken) are plotted. As more copies of vaccine are provided, the ratio shifts to the amplification of its loci. Test samples are observed distributed throughout this dynamic range. The values are normalized from 0 to pi, where 0 represents no detectable vaccine in the subject and pi represents saturation of vaccine relative to the subject.

Figure 3 shows schematic representation of ILTV amplification and gel showing the amplified loci. Eight loci plus DAGLA (chicken control) are amplified in several genes. The resulting data is utilized to quantify the relative abundance of ILTV as shown in Figure 2, but also to detect genetic markers.

Figure 4 shows an example of a phylogenetic tree build from ILTV samples. Strain differentiation is achieved using the markers obtained in the amplified regions.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 : A forward primer for amplification of chicken DAGLA locus.

SEQ ID NO: 2 : A reverse primer for amplification of chicken DAGLA locus.

SEQ ID NO: 3 : A forward primer for amplification of HVT-gD locus in the vaccine against ILTV.

SEQ ID NO: 4 : A reverse primer for amplification of HVT-gD locus in the vaccine against ILTV.

SEQ ID NO: 5 : A forward primer for amplification of HVT-gl locus in the vaccine against ILTV.

SEQ ID NO: 6 : A reverse primer for amplification of HVT-gl locus in the vaccine against ILTV.

SEQ ID NO: 7: A forward primer for amplification of ILTV ORFA locus.

SEQ ID NO: 8 : A reverse primer for amplification of ILTV ORFA locus.

SEQ ID NO: 9: A forward primer for amplification of ILTV UL23 locus. SEQ ID NO: 10: A reverse primer for amplification of ILTV UL23 locus.

SEQ ID NO: 11: A forward primer for amplification of ILTV UL28 locus.

SEQ ID NO: 12: A reverse primer for amplification of ILTV UL28 locus.

SEQ ID NO: 13: A forward primer for amplification of ILTV UL36 locus.

SEQ ID NO: 14: A reverse primer for amplification of ILTV UL36 locus.

SEQ ID NO: 15: A forward primer for amplification of ILTV ICP4 A locus.

SEQ ID NO: 16: A reverse primer for amplification of ILTV ICP4 A locus.

SEQ ID NO: 17: A forward primer for amplification of ILTV ICP4 B locus.

SEQ ID NO: 18: A reverse primer for amplification of ILTV ICP4 B locus.

SEQ ID NO: 19: A forward primer for amplification of ILTV US3 locus.

SEQ ID NO: 20: A reverse primer for amplification of ILTV US3 locus.

SEQ ID NO: 21: A forward primer for amplification of ILTV US5 locus.

SEQ ID NO: 22: A reverse primer for amplification of ILTV US5 locus.

SEQ ID NO: 23: A forward primer for amplification of HVT-F locus in the vaccine against ND-IBD.

SEQ ID NO: 24: A reverse primer for amplification of HVT-F locus in the vaccine against ND-IBD.

SEQ ID NO: 25: A forward primer for amplification of HVT-VP2 locus in the vaccine against ND-IBD.

SEQ ID NO: 26: A reverse primer for amplification of HVT-VP2 locus in the vaccine against ND-IBD.

SEQ ID NO: 27: A forward primer for amplification of IBDV VP2 locus.

SEQ ID NO: 28: A reverse primer for amplification of IBDV VP2 locus.

SEQ ID NO: 29: A forward primer for amplification of IBDV VP4-3(l) locus. SEQ ID NO: 30: A reverse primer for amplification of IBDV VP4-3(l) locus.

SEQ ID NO: 31: A forward primer for amplification of IBDV VP4-3(2) locus. SEQ ID NO: 32: A reverse primer for amplification of IBDV VP4-3(2) locus.

SEQ ID NO: 33: A forward primer for amplification of IBDV VP5 locus.

SEQ ID NO: 34: A reverse primer for amplification of IBDV VP5 locus.

SEQ ID NO: 35: A forward primer for amplification of IBDV VP4-3(3) locus. SEQ ID NO: 36: A reverse primer for amplification of IBDV VP4-3(3) locus.

SEQ ID NO: 37: A forward primer for amplification of IBDV VP1 locus.

SEQ ID NO: 38: A reverse primer for amplification of IBDV VP1 locus. SEQ ID NO: 39: Sequence of the chicken DAGLA locus.

SEQ ID NO: 40: Sequence of the HVT-gD locus in the vaccine against ILTV.

SEQ ID NO: 41: Sequence of the HVT-gl locus in the vaccine against ILTV.

SEQ ID NO: 42: Sequence of the ILTV ORFA locus.

SEQ ID NO: 43: Sequence of the ILTV UL23 locus.

SEQ ID NO: 44: Sequence of the ILTV UL28 locus.

SEQ ID NO: 45: Sequence of the ILTV UL36 locus.

SEQ ID NO: 46: Sequence of the ILTV ICP4 A locus.

SEQ ID NO: 47: Sequence of the ILTV ICP4 B locus.

SEQ ID NO: 48: Sequence of the ILTV US3 locus.

SEQ ID NO: 49: Sequence of the ILTV US5 locus.

SEQ ID NO: 50: Sequence of the HVT-F locus in the vaccine against ND-IBD. SEQ ID NO: 51: Sequence of the HVT-VP2 locus in the vaccine against ND-IBD. SEQ ID NO: 52: Sequence of the IBDV VP2 locus.

SEQ ID NO: 53: Sequence of the IBDV VP4-3(l) locus.

SEQ ID NO: 54: Sequence of the IBDV VP4-3(2) locus.

SEQ ID NO: 55: Sequence of the IBDV VP5 locus.

SEQ ID NO: 56: Sequence of the IBDV VP4-3(3) locus.

SEQ ID NO: 57: Sequence of the IBDV VP1 locus.

DETAILED DISCLOSURE OF THE INVENTION

As used herein, the singular forms“a”,“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that the terms“including”,“includes”,“having”,“has”,� ��with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”. The transitional terms/phrases (and any grammatical variations thereof)“comprising”,“comprises”,“comprise”, include the phrases “consisting essentially of’,“consists essentially of’,“consisting”, and“consists”.

The phrases“consisting essentially of’ or“consists essentially of’ indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim.

The term“about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Where particular values are described in the application and claims, unless otherwise stated the term“about” meaning within an acceptable error range for the particular value should be assumed.

In the present disclosure, ranges are stated in shorthand, to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 1-10 represents the terminal values of 1 and 10, as well as the intermediate values of 2, 3, 4, 5, 6, 7, 8, 9, and all intermediate ranges encompassed within 1-10, such as 2-5, 2-8, and 7-10. Also, when ranges are used herein, combinations and sub-combinations of ranges ( e.g ., subranges within the disclosed range) and specific embodiments therein are intended to be explicitly included.

The term“nucleic acid” refers to polynucleotide such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term also includes analogs of RNA or DNA made from nucleotide analogs. Nucleic acids can be single or double-stranded. Non-limiting examples of nucleic acids include chromosomes, cDNAs, mRNAs, and rRNAs.

An exogenous genetic material is any genetic material that originates outside a subject. For example, genetic material from a pathogen that has infected a subject constitutes exogenous genetic material.

An endogenous genetic material is any genetic material that originates from the subject. An endogenous genetic material of a subject includes the subject’s genomic DNA, such as chromosomal DNA, and mitochondrial DNA.

The term“probe” refers to an oligonucleotide that hybridizes to a target sequence contained within a target oligonucleotide. A probe hybridizes to a target sequence due to complementarity of a sequence in the probe with the target sequence. Generally, the probe and target sequence complementarity will be in sense-anti-sense configuration. The nucleotides of any particular probe may be deoxyribonucleotides, ribonucleotides, and/or synthetic nucleotide analogs. A probe is complementary or substantially complementary to the target sequence.

“Complementary” and“substantially complementary” nucleic acids refer to base pairing between the two strands of a double-stranded nucleic acid molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid. Complementary nucleotides are, generally, A and T (or A and U), and G and C. The specific sequence lengths of complementary sequences are illustrative and not limiting and that sequences covering the same positions, but having slightly fewer or greater numbers of bases are deemed to be equivalents of the sequences and fall within the scope of the invention. Because it is understood that nucleic acids do not require complete complementarity to hybridize, the probe and primer sequences disclosed herein may be modified without loss of utility as specific primers and probes. Typically, two sequences having more than 80% sequence homology hybridize with each other. Hybridization of complementary and partially complementary nucleic acid sequences may be obtained by adjustment of the hybridization conditions to increase or decrease stringency. Under high stringency conditions, more sequence complementarity is necessary for two sequences to hybridize with each other and under low stringency conditions even the sequences that have low sequence complementarity may hybridize with each other. Hybridization stringency can be adjusted by modifying, among other conditions, temperature or salt content of the buffer. Such minor modifications of the disclosed sequences and any necessary adjustments of hybridization conditions to maintain specificity are within the skill of a person of ordinary skill in the art.

“Hybridizing conditions” refer to conditions of temperature, pH, and concentrations of reactants that allow at least a portion of complementary sequences to anneal with each other. Conditions required to accomplish hybridization depend on the size of the oligonucleotides to be hybridized, the degree of complementarity between the oligonucleotides and the presence of other materials in the hybridization reaction admixture. The actual conditions necessary for each hybridization step are well known in the art or can be readily determined by a person of ordinary skill in the art. Typical hybridizing conditions include the use of solutions buffered to a pH from about 7 to about 8.5 and temperatures of from about 30°C to about 60°C. Hybridization conditions may also include a buffer that is compatible, i.e., chemically inert, with respect to the oligonucleotides and other components, yet still allows for hybridization between complementary base pairs.

A“primer” is an oligonucleotide capable of initiating synthesis of nucleic acid sequence in a PCR reaction. A primer initiates PCR when placed under conditions in which synthesis is induced of a primer extension product that is complementary to a template nucleic acid strand. Such conditions include provision of appropriate nucleotides, an enzyme for polymerization such as a DNA polymerase, an appropriate buffer and a suitable temperature. Primers are synthesized based on the sequence of a target locus. For example, based on the sequence of a target locus and the sequences flanking the target locus, a skilled artisan can determine the sequence of a primer or a primer pair for amplification of the target locus.

A primer pair is a pair of oligonucleotides, each typically having about 10 to 30 nucleotides, and designed to amplify a specific locus from a template genetic material. Guidelines for designing a primer pair to amplify a specific locus to in a template genetic material are well known in the art.

A singleplex PCR is a reaction where only one primer pair is used per reaction; whereas, a multiplex reaction is one that uses multiple primer pairs per PCR reaction.

The term“amplification of a genetic locus” refers to enzyme-mediated synthesis of many copies of a genetic locus. Examples of enzyme-mediated target amplification procedures known in the art include PCR, nucleic acid-sequence-based amplification (“NASBA”), strand displacement amplification (“SDA”), ligase chain reaction (“LCR”), and rolling circle amplification (“RCA”). PCR is preferred in the methods disclosed herein.

A typical PCR designed to amplify a double stranded genetic locus involves contacting a template genetic material containing the double stranded genetic locus with a primer pair containing about 10-30 base pairs each and that are complementary to the 3’ end of each strand of the double stranded genetic locus; a molar excess of unattached nucleotide bases (i.e., dNTPs); and DNA polymerase, preferably Taq polymerase, which is heat stable and which catalyzes the formation of DNA from the oligonucleotide primers and dNTPs. One of the two primers in a primer pair is called a forward primer and the other is a reverse primer. Each primer binds in the 5’ -3’ direction to the 3’ end of one strand of the denatured double stranded genetic locus. The solution is heated to 94-96°C to denature the double- stranded nucleotides into single-stranded nucleotide. When the solution cools, the primers bind to the separated strands and the polymerase catalyzes a new nucleotide strand by joining the dNTPs to the primers. When the process is repeated an extension product synthesized from a forward primer, upon separation, would serve as a template for a complementary extension product synthesized from the reverse primer. Similarly, the extension product synthesized from a reverse primer, upon separation, would serve as a template for a complementary extension product synthesized from the forward primer. In this way, a genetic locus located between the binding sites for the forward and the reverse primers is selectively replicated with each repetition of the process. Since the sequence being amplified doubles after each cycle, an exponential amplification copies is produced within a short amount of time. For example, after PCR amplification having 30 cycles each copy of the template DNA would produce about a billion copies of the amplified product.

Certain embodiments of the invention provide a method for detecting and/or quantifying a target genetic material in a sample obtained from a subject. The sample can be treated to isolate genetic material by removing non-genetic material, such as proteins, carbohydrates, lipids, etc. to produce a sample comprising the genetic material from the subject and if present in the subject, the target genetic material to be detected and quantified. The target genetic material is exogenous to the genetic material of the subject and the quantification of the target genetic material is performed based on the amount of the target genetic material relative to the genetic material of the subject.

The amount of a target genetic material present in a sample obtained from a subject is quantified based on PCR amplification of one or more target genetic loci present in the target genetic material. PCR amplification of two or more target genetic loci can be performed in a multiplex reaction. The amount of a subject’s genetic material present in a sample is quantified based on PCR amplifying one or more control genetic loci present in the genetic material of the subject. PCR amplification of two or more control genetic loci can also be performed in a multiplex reaction. In certain embodiments, PCR amplification of the one or more target genetic loci and the one or more control genetic loci is performed together in a multiplex reaction.

Accordingly, an embodiment of the invention provides a method for quantifying a target genetic material in a sample obtained from a subject, wherein the target genetic material is exogenous to the genetic material of the subject, the method comprising the steps of:

a) subjecting the sample to a PCR amplification using one or more target primer pairs that amplify one or more target genetic loci present in the target genetic material and one or more control primer pairs that amplify one or more control genetic loci present in the genetic material of the subject, and

b) quantifying the PCR amplicons produced in step a) from the one or more target genetic loci and the one or more control genetic loci.

In certain embodiments, before the step of subjecting the sample to a PCR amplification, the sample can be processed to isolate the genetic material from the sample by removing non-genetic material, such as proteins, carbohydrates, lipids, etc. The term“target genetic locus” refers to a nucleic acid sequence present in a target genetic material to be characterized, for example, by way of amplification, quantification or identification. For example, a target genetic locus can be present in the genome of an organism whose presence and/or amount in a sample is to be detected and/or quantified. A target genetic locus can also be present in a nucleic acid vaccine whose presence and/or amount in a subject is to be detected and/or quantified. A target genetic locus is absent from the genetic material of a subject.

The term“control genetic locus” refers to a nucleic acid sequence present in the genetic material of a subject. For example, in a human tissue sample, a control genetic locus can be a sequence present in the human genetic material, including human nuclear genome or mitochondrial genome. In a chicken tissue sample, a control genetic locus can be a sequence present in the chicken genetic material, including chicken nuclear or mitochondrial genome. A control genetic locus can be any sequence known to be present in a known number of copies per genome of a subject. For example, a control genetic locus can be a gene or a specific portion of a gene known to be present in one copy per haploid genome of a human. Thus, two copies of the gene or the specific portion of the gene are present in a diploid human cell. Alternatively, a control genetic locus can be a gene or a specific portion of a gene known to be present in two copies per haploid genome of a human. Thus, four copies of the gene or the specific portion of the gene are present in a diploid human cell. As a skilled artisan can envision, a control genetic locus can be any sequence located anywhere in the genetic material of a subject as long as the number of copies of the control genetic locus within the entire genetic material of a diploid cell of a subject is known. A control genetic locus is absent from the target genetic material.

As noted above, a control genetic locus can be present in mitochondrial genetic material of a subject. However, because the amount and number of mitochondrial genomes within different cells may vary, it is preferable to use as genetic locus within the chromosomal DNA of a cell as a control genetic locus. However, a control genetic locus can be a sequence located in the mitochondrial genetic material of a subject as long as the number of copies of the control genetic locus within the entire genetic material of a diploid cell of a subject is known.

A skilled artisan can identify a control genetic locus to be used for a subject based on the genome sequence information of the subject. Certain non-limiting examples are provided below. A control genetic locus provides, among other benefits, two purposes in the methods of the instant invention. First, it acts as a positive control and allows for distinguishing between a true negative result and a false negative result, i.e., between a sample lacking the target genetic material (true negative) from a failed reaction (false negative). A failed reaction can be caused by improper collection of sample, DNA degradation, lab failures, etc. In other words, when a target genetic material is absent in a sample, a target genetic locus should not be amplified, but the control genetic locus is amplified. In a failed reaction, no amplification occurs. Second, the control genetic locus serves as internal normalization to identify the relative abundance of the target genetic material. Thus, amount of the target genetic material in a sample can be determined based on the relative amplification of the one or more target genetic loci and the one or more control genetic loci (Figure 2). For a given amount of nucleic acids used as an input nucleic acid, a part corresponds to the subject’s genetic material (typically, the vast majority) and a part corresponds to the target genetic material (typically, a small portion). A sample with large amounts of the target genetic material will have greater amplification of the one or more target genetic loci compared to a sample with low amounts of the target genetic material. The ratio of the amplified amounts of the one or more target genetic loci to the one or more control genetic loci can be used to quantify the amount of target genetic material in a sample.

The amounts of the target genetic material relative to the amount of the genetic material of the subject is calculated based on the relative amounts of the amplicons obtained from one or more target genetic loci and the amplicons obtained from the one or more control genetic loci.

Many techniques are available in the art for quantifying the amount of an amplicon produced in a PCR amplification reaction and any such method can be used to quantify the amplicons obtained from the target genetic loci and the amplicons obtained from the control genetic loci. Such quantification of amplicons can be performed at the end of the PCR amplification or during the PCR amplification, i.e., real-time quantification.

In one embodiment, the amplicons obtained from the target genetic loci and the amplicons obtained from the control genetic loci can be separated on a gel, for example, an agarose gel. The amplicons can then be quantified based on the intensity of the bands corresponding to the amplicons. If this method is used for quantification, the target genetic loci and the control genetic loci are designed so that each genetic locus has a different size and thus, can be separated on a gel from the other loci. In another embodiment, specific probes can be used to identify and quantify amplicons produced from different genetic loci. Each of the different probes can be conjugated to a different label and the amplicons can be quantified based on the amount of each label conjugated to the amplicons. Such labels include fluorescent and chemiluminescent dyes. Examples of fluorescent and chemiluminescent dyes include xanthene, anthracene, cyanine, porphyrin and coumarin dyes. Examples of xanthene dyes include fluorescein, 6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein (5-Fam), 5- or 6- carb oxy-4, 7, 2’ , 7’ -tetrach 1 orofl uore scei n (TET), 5- or 6-carboxy-4’5’2’4’5’7’ hexachlorofluorescein (HEX), 5’ or 6 ^, -carboxy-4 ^, ,5 ^, -dichloro-2 ^, ,7 ^, -dimethoxyfluorescein (JOE), 5-carboxy-2 ^, ,4 ^, ,5 ^, ,7’-tetrachlorofluorescein (ZOE) rhodol, rhodamine, tetramethylrhodamine (TAMRA), 4,7-dichlorotetramethyl rhodamine (DTAMRA), rhodamine X (ROX) and Texas Red. Examples of cyanine dyes include Cy 3, Cy 3.5, Cy 5, Cy 5.5, Cy 7 and Cy 7.5. Other dyes that can be used in the methods described herein include energy transfer dyes, composite dyes and other aromatic compounds that give fluorescent signals. Chemiluminescent compounds that may be used in the instant methods include dioxetane and acridinium esters. Additional dyes suitable for use in the methods disclosed herein are known in the art and such embodiments are within the purview of the invention.

In a further embodiment, specific labels can be conjugated to one or both primers designed to amplify each of the plurality of amplified genetic loci. Amplicons produced from the specifically labeled primers can be quantified based on the presence of fluorescence incorporated into the amplicons. For such quantification, the free primers are removed from the reaction mixture to ensure that only the primers incorporated into the amplicons are quantified. Alternatively, one or both primers of different primer pairs can be labeled with the same dye and at the end of the PCR amplification, different amplicons are separated from each other, for example, based on their sizes. The dyes described above in connection with the probes envisioned in this disclosure can also be used in the primer pairs described herein.

In an even further embodiment, a real-time quantification of amplicons is performed. Several techniques of real-time quantification of PCR amplicons are known in the art and such methods can be implemented in the methods disclosed herein. One such method comprises using amplicon specific probes labeled with a specific fluorescent label and quencher. The fluorescent label emits the fluorescence only upon hybridization to the target amplicon. Thus, the amount of fluorescence emitted from a probe indicates the amount of the corresponding amplicon.

Direct sequencing of the amplicons can also be used to quantify their abundance. The resulting sequencing reads are aligned to the amplicons and the count of sequencing reads in each amplicon region is used as a signal for quantification. By sequencing a pool of amplicons, the more abundant amplicons will result in more sequencing reads, whereas low abundant ones will receive proportionally fewer sequencing reads.

Another such method comprises using amplicon specific probes labeled with a specific fluorescent and quencher, which hybridizes to the template strand and emits the fluorescence only upon degradation of the probe caused by polymerization of the newly formed strand. Thus, the amount of fluorescence emitted from a probe indicates the amount of the corresponding amplicon.

Additional methods of quantifying different amplicons, either at the end of the PCR amplification or in real-time, are known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

In one embodiment, different amplification products can be sequenced to determine the presence of polymorphisms within the target genetic material. The sequenced genetic loci can be used to generate phylogenetic trees to illustrate evolutionary relationships within different target genetic materials, population structure and principal component analysis to group samples based on their population structure and relatedness.

As used herein“polymorphism” refers to a difference in the nucleotide sequence between different individuals or strains of a same species. Polymorphism can be a single base pair change or insertion, deletion, indel (insertion and deletion), or a change in the number of copies of a given sequence. Single nucleotide polymorphism (SNP) is the most common DNA polymorphism in humans.

Many techniques are available in the art for sequencing an amplicon, particularly, a plurality of amplicons, produced in a PCR amplification reaction and any such method can be used to sequence the amplicons obtained from the target genetic loci. Such sequencing of amplicons can be performed at the end of the PCR amplification or during the PCR amplification. In preferred embodiments, next generation sequencing is conducted to sequence a plurality of amplicons obtained from the target genetic loci.

Non-limiting examples of sequencing technologies that can be used in the methods of the invention are described by Lin et al. (2008), Recent Patents and Advances in the Next- Generation Sequencing Technologies, Recent Patents on Biomedical Engineering 2008, 7, 60-67. The Lin et al. reference is incorporated herein in its entirety. Some of the technology discussed by Lin et al. is described in the United States patent numbers 7211390, 7244559, 7264929, 7232656, 7282370, 7279563, 7105300, 7264934, 7169560, 7244567, 7270951, 7057026, 7282337, 6013445, 7220549 and 7276338 and the United States patent application publication numbers US20070172869, US20070172860, US20070172819, US20070105131, US20060252923, US20060240439, US20060281109, US20070207482, US20060024681,

US20070172839, US20060292611, US20070087362, US20070202521, US20070184475,

US20070190542, US20070042366, US20070048745, US20060231419, US20070178507,

US20070178516, US20060287833, US20060246497, US20070194225, and

US20060275779. Each of these patents and publications is incorporated by reference in its entirety.

Different combinations of a subject and a target genetic material are envisioned. Typically, the methods of the invention can be carried out to quantify in a sample from a subject the amount of a target genetic material that is exogenous to the genetic material of the subject.

In some embodiments, two or more target genetic materials can be quantified. For example, a first target genetic material can be from a first organism that is suspected to inhabit a subject and a second target genetic material can be from a second organism that is suspected to inhabit the subject. In such embodiments, a first group of one or more target primer pairs are designed to amplify a first group of one or more target genetic loci and a second group of one or more target primer pairs are designed to amplify a second group of one or more target genetic loci. A multiplex PCR can then be performed on a sample obtained from a subject using one or more control primer pairs and the first group of one or more target primer pairs and the second group of one or more target primer pairs. The first target genetic material can be quantified by quantifying the amplicons produced from the first group of one or more target genetic loci relative to amplicons produced from the control genetic loci. The second target genetic material can be quantified by quantifying the amplicons produced from the second group of one or more target genetic loci relative to amplicons produced from the control genetic loci. As a skilled artisan can envision, more than two, for example, three to ten or more, different target genetic materials can be detected and quantified by using multiple primer pairs. Thus, two or more target genetic materials can be detected and/or quantified in a multiplex reaction. For example, a subject can be suspected of being inhabited by an organism and the target genetic material comprises the genetic material from the organism. The organism inhabiting the subject can belong to the normal microflora of the subject, for example, normal gut microflora, or a pathogen infecting and causing a disease in the subject. Quantifying an organism that belongs to the normal microflora of a subject can be used to determine whether the subject’s normal microflora is disturbed and needs to be manipulated. Quantifying a pathogenic organism that has infected the subject can be used to determine the status of the infection and/or the load of the pathogen, which can then be used to administer appropriate treatments to treat the infection in the subject.

An organism, either belonging to a subject’s normal flora or a pathogen, can be a microorganism. For the purposes of the invention, a microorganism can be a virus, a prokaryote, namely a bacterium or an archaea; a eukaryote, for example, a protozoan; a fungus, an alga, or a microscopic plant (green algae). The microorganisms of the current invention are typically microscopic, i.e. not visible with the naked eye; however, certain microorganisms, for example, certain filamentous fungi, can be macroscopic and visible to the naked eye.

A virus can be a DNA virus or an RNA virus. Virus can also contain a protein capsid. In preferred embodiments, a virus is pathogenic virus or an attenuated virus, for example, used for immunization.

A bacterium is a microscopic, single-celled organism belonging to Kingdom Monera that possess a prokaryotic type of cell structure, which means their cells are not compartmentalized, and their DNA (usually circular) can be found throughout the cytoplasm rather than within a membrane-bound nucleus. A bacterium typically reproduces by fission or by forming spores.

Yeast is typically a microscopic single-celled fungus that reproduces asexually by budding or binary fission, produces ascospores, and is capable of fermenting carbohydrates. For the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. 10 M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The fungus can be a filamentous fungus.“Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth el al ., Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). Typically, a filamentous fungus is multinucleated, filamentous microorganism composed of hyphae. A hypha is a branching tubular structure approximately 2-10 microns in diameter which is usually divided into cell-like units by cross- walls called septa. Filamentous fungus has a cell wall usually composed of chitin, sometimes cellulose, occasionally both chitin and cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Filamentous fungus is obligate aerobe and grows by elongation at apical tips of their hyphae and thus is able to penetrate the surfaces on which they begin growing.

A protozoan is a heterotrophic single-celled eukaryote. A protozoan can be parasitic, such as amoeba.

An organism can also be an attenuated organism or a genetically modified organism administered to immunize a subject against the pathogenic version of the organism.

When the methods of the invention are performed to quantify the presence of an organism in a sample obtained from a subject, the target genetic loci are present in the organism’s genetic material.

The subject can be immunized with a nucleic acid vaccine. A nucleic acid vaccine is designed to express in a subject an antigen against which the subject develops immunity. The antigen is typically an antigen from a pathogenic organism and developing immunity against the antigen immunizes the subject against the disease caused by the pathogen. Detecting a nucleic acid vaccine as the target genetic material in a subject immunized with the nucleic acid vaccine can be used to determine the efficacy of immunization. Typically, higher amount of a nucleic acid vaccine in an immunized subject would indicate better expression of the antigen encoded by the nucleic acid vaccine and consequently, better immunization of the subject against the pathogen. On the other hand, lower amount of a nucleic acid vaccine in an immunized subject would indicate lower expression of the antigen encoded by the nucleic acid vaccine and consequently, ineffective immunization of the subject against the pathogen. Therefore, quantifying a nucleic acid vaccine in a subject can be used to modulate the immunization, for example, by administering additional doses of the vaccine or using alternative methods of immunization in subjects that are not sufficiently immunized. In preferred embodiments, the nucleic acid vaccine is a viral vaccine, which typically comprises genetically modified viral nucleic acid which encodes one or more antigens and is not capable of causing a disease in the subject.

The subject can also be a subject to whom a nucleic acid is administered, for example, as a therapeutic. In such embodiments, the target genetic material is present in the administered nucleic acid. Detecting a nucleic acid as the target genetic material in a subject administered with the nucleic acid can be used to determine the efficacy of the delivery of the nucleic acid to the subject. Particularly, higher amount of a nucleic acid in a subject would indicate better delivery of the nucleic acid and lower amount of a nucleic acid in a subject would indicate lower delivery of the nucleic acid. Therefore, quantifying a nucleic acid in a subject can be used to modulate the administration, for example, by administering additional doses of the nucleic acid or using alternative methods of therapy in subjects in which nucleic acid is not efficiently delivered.

Additionally, the subject can be part of a study to define the minimal protective dose of a vaccine. In such a case, different doses of the vaccine are administered to subjects that are then exposed to the pathogen to which the vaccine is expected to protect against. One or more strains of the pathogen can be used as well as different quantities of pathogen challenge agent, such as a vaccine. Quantifying the nucleic acids of the vaccine in the subjects provide a measure of how well different doses of the vaccine are able to replicate in the subjects, referred to as vaccine-take levels. These vaccine-take levels are then correlated with the subjects’ response to the pathogen exposure, thus allowing the definition at the molecular level of a minimum protective dose, that needs to be administered to each subject to protect it against the pathogen.

The methods disclosed herein can be performed in any subject, for example, any animal or a plant. An animal can be a bird, primate, rodent, feline, canine, porcine, bovine or equine. Preferred subjects include chicken, non-human primate, human, mouse, rat, cat, dog, pig, cattle or horse.

In certain embodiments, the subject is a human and the target genetic material is a pathogen suspected of infecting the human. Such a pathogen could be the human immunodeficiency virus (HIV); infectious laryngotracheitis virus (ILTV) in chicken; Campylobacter jejuni in chicken, dairy milk or other infected matters; porcine epidemic diarrhea virus in pig; Salmonella spp. in meat, processed food, surfaces for food processing or other infected matters.

The sample from a subject containing the subject’s genetic material can be obtained from any organ or tissue. Non-limiting examples of the organ or tissue which can be used as samples are placenta, brain, eyes, pineal gland, pituitary gland, thyroid gland, parathyroid glands, thorax, heart, lung, esophagus, thymus gland, pleura, adrenal glands, appendix, gall bladder, urinary bladder, large intestine, small intestine, kidneys, liver, pancreas, spleen, stoma, ovaries, uterus, testis, trachea, skin, blood or buffy coat sample of blood. For plants, non-limiting examples of the tissues which can be used as samples are roots, stem, leaves, flowers or seeds. Additional examples of organs and tissues that contain a subject’s genetic material are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

In certain other embodiments, the sample containing a subject’s genetic material is a body fluid. The source of subject’s genetic material in a body fluid sample can be cells present in the fluid. Non-limiting examples of the body fluids which can be used as samples include amniotic fluid, aqueous humor, vitreous humor, bile, blood, cerebrospinal fluid, chyle, endolymph, perilymph, female ejaculate, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sputum, synovial fluid, vaginal secretion, semen, or blood. Additional examples of body fluids that can contain a subject’s genetic material are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

In one embodiment of the invention, the subject is chicken and the target genetic material is a viral genetic material or a viral vaccine. The viral genetic material can be from a pathogenic virus which has inhabited or infected the chicken. The viral vaccine can be a nucleic acid viral vaccine designed to immunize the chicken against one or more diseases. In preferred embodiments, the viral vaccine is against infectious bursal disease (IBD), Newcastle disease (ND), Infectious Laryngotrachetitis (ILT), Marek’s disease (MD), or infectious bronchitis (IB).

A viral vaccine can be Vectormune™ AI, which is a viral vaccine against Avian Influenza virus (AIV). Vectormune™ AI is a live vector frozen vaccine, for the active immunization of chickens against AIV H5 subtype and MD. Vectormune™ AI vaccine is based on live serotype 3 Marek virus vaccine, HVT and comprises a genetically engineered live HVT vaccine expressing the HA gene of an H5N 1 AIV. Therefore, in the methods of the invention for quantification and detection of Vectormune™ AI vaccine, one or more target primer pairs amplify one or more target genetic loci within the HA gene of an H5N1 AIV.

A viral vaccine can be Vectormune™ ND, which is a viral vaccine against Newcastle disease virus (ND). Vectormune™ ND is a live vectored frozen vaccine for in-ovo and subcutaneous administration for the active immunization of chickens against ND and MD. Vectormune™ ND contains a live frozen serotype 3 Marek (HVT) vector vaccine virus indicated for use in chickens. The HVT in Vectormune™ ND expresses key protective NDV antigen. Therefore, in the methods of the invention for quantification and detection of Vectormune™ ND vaccine, one or more target primer pairs amplify one or more target genetic loci within the NDV gene encoding the protective NDV antigen.

A viral vaccine can also be Vectormune™ ND-IBD, which is a viral vaccine against NDV as well as IBDV. Vectormune™ ND-IBD comprises the use of two HVT vector at the same time for the same bird. One HVT vector can contain a gene encoding an NDV antigen and the other HVT vector can contain a gene encoding an IBDV antigen. Therefore, in the methods of the invention for quantification and detection of Vectormune™ ND-IBD vaccine, one or more target primer pairs can amplify one or more target genetic loci within the NDV gene encoding the protective NDV antigen and/or the IBDV gene encoding the protective IBDV antigen.

Further, a viral vaccine can be Vectormune™ IBD, which is a viral vaccine against IBDV. Vectormune™ IBD is a live vectored frozen vaccine for in-ovo and subcutaneous administration for the active immunization of chickens against ND and MD. Vectormune™ IBD contains a live frozen serotype 3 Marek (HVT) vector vaccine virus indicated for use in chickens. The HVT in Vectormune™ IBD expresses key protective IBDV antigens. This vaccine can be presented in a frozen cell-associated form. Therefore, in the methods of the invention for quantification and detection of Vectormune™ IBD vaccine, one or more target primer pairs amplify one or more target genetic loci within the IBDV gene encoding the protective IBDV antigen.

Furthermore, a viral vaccine can be Vectormune™ ILT, which is a viral vaccine against ILTV. Vectormune™ ILT is a live vectored frozen vaccine for in-ovo and subcutaneous administration for the active immunization of chickens against ILTV and MD. Vectormune™ ILT contains a live frozen serotype 3 Marek (HVT) vector vaccine virus indicated for use in chickens. The HVT in Vectormune™ ILT expresses key protective ILTV antigens. This vaccine can be presented in a frozen cell-associated form. Therefore, in the methods of the invention for quantification and detection of Vectormune™ ILT vaccine, one or more target primer pairs amplify one or more target genetic loci within the ILTV gene encoding the protective ILTV antigen.

Even further, a viral vaccine can be VAXXITEK™ HVT-IBD. VAXXITEK™ HVT- IBD is a viral vaccine providing protection against IBD and MD. To stimulate IBD immunity, VAXXITEK™ HVT-IBD uses only the viral protein 2 (VP2), a major protective antigen of IBDV. VAXXITEK™ HVT-IBD provided as a live, whole HVT used as the vaccine vector. HVT is non-pathogenic to chickens, but closely related to the Marek's virus, thus stimulating immunity against MD. The HVT vector replicates systemically in the subject, such as chicken, and induces protection against two important diseases in a single vaccine. Both IBD VP2 and HVT experience minimal interference from inherited maternal antibodies in chickens. Therefore, in the methods of the invention for quantification and detection of VAXXITEK™ HVT -IBD vaccine, one or more target primer pairs amplify one or more target genetic loci within the VP2 gene encoding the protective IBDV antigen.

Additionally, a viral vaccine can be INNOVAX™-ND-IBD, which is a multivalent live vaccine comprising a live HVT containing one NDV gene and one IBDV gene inserted into the genome. INNOVAX™-ND-IBD provides life-long immunity against ND and IBD. INNOVAX-ND-IBD does not contain live NDV or IBDV and cannot induce virus spread, revert to virulence or interfere with other live respiratory vaccines. Therefore, in the methods of the invention for quantification and detection of INNOVAX™ ND-IBD vaccine, one or more target primer pairs can amplify one or more target genetic loci within the NDV gene encoding the protective NDV antigen and/or the IBDV gene encoding the protective IBDV antigen.

In a specific embodiment, a viral vaccine can be INNOVAX™-ILT, which is a frozen, cell associated, live virus vaccine that contains the recombinant serotype 3 HVT with genes from ILTV. INNOVAX™-ILT is indicated for vaccination by the in-ovo route or by subcutaneous route as an aid in the prevention of MD and ILT. Therefore, in the methods of the invention for quantification and detection of INNOVAX™-ILT vaccine, one or more target primer pairs can amplify one or more target genetic loci within the ILTV genes encoding the protective ILTV antigen.

In another embodiment, a viral vaccine can be INNOVAX™-ND, which is a frozen, cell associated, live virus vaccine that contains the recombinant serotype 3 HVT with the F gene from NDV. INNOVAX™-ND is indicated for vaccination by the in-ovo route or by subcutaneous route as an aid in the prevention of MD and ND. Therefore, in the methods of the invention for quantification and detection of INNOVAX™-ND vaccine, one or more target primer pairs can amplify one or more target genetic loci within the F gene from NDV encoding the protective NDV antigen.

In an even further embodiment, a viral vaccine can be INNOVAX™-ND-SB, which is a frozen, cell associated, live virus vaccine that contains the recombinant serotype 3 HVT with the F gene from NDV and the SB-l strain of chicken herpesvirus serotype 2. INNOVAX™-ND-SB is indicated for vaccination by the in-ovo route to aid in the prevention of MD and ND. Therefore, in the methods of the invention for quantification and detection of INNOVAX™-ND vaccine, one or more target primer pairs can amplify one or more target genetic loci within the F gene from NDV encoding the protective NDV antigen and/or one or more one or more target primer pairs can amplify one or more target genetic loci within the SB-l strain of chicken herpesvirus serotype 2.

In certain embodiments, one or more one or more target primer pairs can also amplify one or more target genetic loci within portion of the viral vaccine genetic material that does not encode the proteins that induce a protective immune response in a subject.

In certain embodiments, when a chicken is used as a subject, the gene encoding diacylglycerol lipase alpha (DAGLA) or a portion thereof can be used as a control genetic locus. As apparent to a person of ordinary skill in the art, any gene or locus with a known sequence and known number of copies within a diploid chicken cell can be used as a control genetic locus.

One embodiment of the invention provides a method of quantifying a vaccine against ILTV in a sample obtained from a chicken, the method comprising the steps of:

a) subjecting the sample to a PCR amplification using one or more control primer pairs designed based on the gene encoding DAGLA in chicken and one or more target primer pairs designed to amplify the recombinant insertion of target genetic loci that are genetically modified to contain genes gD and gl in the vaccine against ILTV, and

b) quantifying the PCR amplicons produced in step a) from the one or more target genetic loci and the one or more control genetic loci to quantify the amount of the vaccine against ILTV.

In certain such embodiments, the PCR amplification is performed using one or more target primer pairs selected from the primer pairs of SEQ ID NOs: 3 and 4; and 5 and 6; and a control primer pair comprising SEQ ID NOs: 1 and 2.

Another embodiment of the invention provides a method of quantifying ILTV in a sample obtained from a chicken, the method comprising the steps of:

a) subjecting the sample to a PCR amplification using one or more control primer pairs designed based on the gene encoding DAGLA in chicken and one or more target primer pairs designed to amplify the target genetic loci selected from ILTV genes ORFA, UL23, UL28, UL36, ICP4A, ICP4B, US3, and US5, and b) quantifying the PCR amplicons produced in step a) from the one or more target genetic loci and the one or more control genetic loci to quantify the amount of ILTV.

In certain such embodiments, the PCR amplification is performed using one or more target primer pairs selected from the primer pairs of SEQ ID NOs: 7 and 8; 9 and 10; 11 and 12; 13 and 14; 15 and 16; 17 and 18; 19 and 20; and 21 and 22; and a control primer pair comprising SEQ ID NOs: 1 and 2.

A further embodiment of the invention provides a method of quantifying a vaccine against ND-IBD virus in a sample obtained from a chicken, the method comprising the steps of:

a) subjecting the sample to a PCR amplification using one or more control primer pairs designed based on the gene encoding DAGLA in chicken and one or more target primer pairs designed to amplify the recombinant insertion of target genetic loci that are genetically modified to contain genes F and VP2 in the vaccine against NDV and IBDV, respectively, and

b) quantifying the PCR amplicons produced in step a) from the one or more target genetic loci and the one or more control genetic loci to quantify the amount of the vaccine against ND-IBD.

In certain such embodiments, the PCR amplification is performed using one or more target primer pairs selected from the primer pairs of SEQ ID NOs: 23 and 24; and 25 and 26; and a control primer pair comprising SEQ ID NOs: 1 and 2.

An even further embodiment of the invention provides a method of quantifying IBD virus (IBDV) in a sample obtained from a chicken, the method comprising the steps of:

a) subjecting the sample to a PCR amplification using one or more control primer pairs designed based on the gene encoding DAGLA in chicken and one or more target primer pairs designed to amplify the target genetic loci are selected from IBDV genes VP1 to VP5, and

b) quantifying the PCR amplicons produced in step a) from the one or more target genetic loci and the one or more control genetic loci to quantify the amount of IBDV.

In one embodiment, the target genetic loci from IBDV genes VPl to VP5 are selected from nucleotide positions in the IBDV genome as follows: VP2: nucleotides 620 to 1262 (SEQ ID NO: 52); VP4-3Q): nucleotides 1050 to 1580 (SEQ ID NO: 53); VP4-3(2): nucleotide numbers 2602 to 3053 (SEQ ID NO: 54); VP5: nucleotides from 1 to 339 (SEQ ID NO: 55), and VP4-3(3): nucleotides from 2197 to 2637 (SEQ ID NO: 56). Accordingly, in certain embodiments, the PCR amplification is performed using one or more target primer pairs selected from the primer pairs of SEQ ID NOs: 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 36; and 37 and 38; and a control primer pair comprising SEQ ID NOs: 1 and 2.

Further embodiments of the invention provide a kit for quantifying a target genetic material in a sample obtained from a subject, wherein the target genetic material is exogenous to the genetic material of the subject. Typically, the kit comprises:

a) one or more target primer pairs that amplify one or more target genetic loci present in the target genetic material and one or more control primer pairs that amplify one or more control genetic loci present in the genetic material of the subject, and optionally,

b) one or more reagents for PCR amplifying the one or more target genetic loci and the one or more control genetic loci.

The aspects of the methods of the invention discussed above are also applicable to the kits of the invention. Particularly, the target primer pairs and the control primer pairs are designed based on the sequences of the one or more control genetic loci in a subject and the sequence of the one or more target genetic loci in the target genetic material to be detected.

For example, a kit is provided comprising one or more control primer pairs designed based on the gene encoding DAGLA in chicken and one or more target primer pairs designed to amplify the target genetic loci are selected from ILTV genes ORFA, EIL23, EIL28, ETL36, ICP4A, ICP4B, ETS3, and ETS5. Thus, the one or more target primer pairs can be one or more primer pairs selected from SEQ ID NOs: 7 and 8; 9 and 10; 11 and 12; 13 and 14; 15 and 16; 17 and 18; 19 and 20; and 21 and 22; and a control primer pair can be SEQ ID NOs: 1 and 2. Such kit can be used to quantify ILTV load in an ILTV infected chicken.

Another embodiment of the invention provides a kit comprising one or more control primer pairs designed based on the gene encoding DAGLA in chicken and one or more target primer pairs designed to amplify the target genetic loci are selected from genes gD and gl in the vaccine against ILTV. Thus, the one or more target primer pairs can be selected from the primer pairs of SEQ ID NOs: 3 and 4; and 5 and 6; and a control primer pair comprises SEQ ID NOs: 1 and 2. Such kit can be used to quantify the vaccine load in a chicken immunized with the vaccine against ILTV.

A further embodiment of the invention provides a kit comprising one or more control primer pairs designed based on the gene encoding DAGLA in chicken and one or more target primer pairs designed to amplify the target genetic loci are selected from genes F and VP2 from a vaccine against ND-IBD. Thus, the one or more target primer pairs can be selected from the primer pairs of SEQ ID NOs: 23 and 24; and 25 and 26; and a control primer pair comprises SEQ ID NOs: 1 and 2. Such kit can be used to quantify a vaccine against BD-IBD in a chicken immunized against such vaccine.

An even further embodiment provides a kit comprising one or more control primer pairs designed based on the gene encoding DAGLA in chicken and one or more target primer pairs designed to amplify the target genetic loci are selected from IBDV genes VP1 to VP5. Thus, the one or more target primer pairs can be one or more primer pairs selected from SEQ ID NOs: 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 36; and 37 and 38; and a control primer pair can be SEQ ID NOs: 1 and 2. Such kit can be used to quantify IBDV load in an IBDV infected chicken.

In further embodiments, the kit comprises one or more reagents, for example, reagents for treating a sample, reagents for isolating cells from the sample, reagents for isolating genetic material from the sample, reagents for conducting PCR, and reagents for quantifying PCR amplicons.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 - QUANTIFYING ILTV OR A VACCINE AGAINST ILTV IN CHICKEN

Two multiplex assays were developed for quantifying the amount of different target genetic materials in chicken. One multiplex assay was designed for quantifying the amount of a vaccine against ILTV present in a chicken sample and the other multiplex assay was designed for quantifying the amount of ILTV present in a chicken sample.

An exemplary vaccine against ILTV contains a live recombinant turkey herpesvirus (strain HVT/ILT-138) expressing the glycoproteins gD and gl of ILTV. Because the vaccine against ILTV comprises genes gD and gl recombined into HVT genome, the one or more target genetic loci are selected to amplify the junction between HVT genome and genes gD, gl or a portion thereof. For example, two target primer pairs are used, namely, SEQ ID NOs: 3 and 4 and SEQ ID NOs: 5 and 6. A control primer pair designed to amplify a control genetic locus present in the chicken and the control primer pair comprises SEQ ID NOs: 1 and 2, located in the gene DAGLA gene of chicken.

For the ILTV multiplex assay, eight regions of the ILTV genome were amplified as target genetic loci and chicken DAGLA was amplified as a control genetic locus.

Therefore, for the assay of a vaccine against ILTV three loci are amplified, two specific to the vaccine (Figure 1) and one for chicken (DAGLA). For the assay of ILTV, nine loci are amplified, eight ILTV specific (Figure 3) and one for chicken (DAGLA).

The following table provides forward and reverse primers for the amplification of the two specific target genetic loci in the vaccine against ILTV, eight ILTV specific target genetic loci, and one chicken specific control locus.

As noted above, the multiplex reaction designed to amplify and quantify a vaccine against ILTV, HVT-gD and HVT-gl target genetic loci can be used to specifically quantify the abundance of vaccine in chicken. Also, the multiplex reaction designed to amplify and quantify ILTV genes as target genetic loci can be used to quantify the abundance of ILTV in chicken infected with this virus.

Moreover, the multiplex reaction designed to amplify and quantify ILTV genes as target genetic loci can be used to identify evolutionary relationships between various ILTV strains, particularly, based on sequence information obtained from a number of different chicken samples containing ILTV. The results of the multiple reactions using these two multiplex assays are provided in Figures 2 to 4.

Exemplary PCR amplification reaction conditions for a vaccine against ILTV are provided below:

One step of 96°C for 3 minutes; 35 cycles of: 96°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1 minute; and one step of 72°C for 3 minutes.

Exemplary PCR amplification reaction conditions for ILTV are provided below:

One step of 96°C for 3 minutes; 35 cycles of: 96°C for 30 seconds, 67°C for 50 seconds, and 72°C for 1 minute; and one step of 72°C for 3 minutes. EXAMPLE 2 - QUANTIFYING IBDV OR THE VACCINE AGAINST NDV-IBDV IN CHICKEN

Two multiplex assays were developed for quantifying the amount of different target genetic materials in chicken. One multiplex assay was designed for quantifying the amount of a vaccine against NDV-IBDV present in a chicken sample and the other multiplex assay was designed for quantifying the amount of IBDV present in a chicken sample.

Because the vaccine against NDV-IBDV comprises genes F and VP2 recombined into HVT genome, the one or more target genetic loci are selected to amplify the junction between HVT genome and from F, VP2 or a portion thereof. For example, two target primer pairs are used, namely, SEQ ID NOs: 23 and 24 and SEQ ID NOs: 25 and 26. A control primer pair designed to amplify a control genetic locus present in the chicken and the control primer pair comprises SEQ ID NOs: 1 and 2, located in the gene DAGLA gene of chicken.

For the IBDV multiplex assay, six regions of the IBDV genome were amplified as target genetic loci and chicken DAGLA was amplified as a control genetic locus.

Therefore, for the assay of a vaccine against ND-IBD three loci are amplified, two specific to the vaccine and one for chicken (DAGLA). For the assay of IBDV, eleven loci are amplified, ten IBDV specific and one for chicken (DAGLA).

The following table provides forward and reverse primers for the amplification of the two specific target genetic loci in the vaccine against ND-IBD, six IBDV specific target genetic loci, and one chicken specific control locus.

As noted above, the multiplex reaction designed to amplify and quantify a vaccine against NDV-IBDV, HVT-F and HVT-VP2 target genetic loci can be used to quantify the abundance of vaccine in chicken. Also, the multiplex reaction designed to amplify and quantify IBDV genes as target genetic loci can be used to quantify the abundance of IBDV in chicken infected with this virus.

Moreover, the multiplex reaction designed to amplify and quantify IBDV genes as target genetic loci can be used to identify evolutionary relationships between various IBDV strains, particularly, based on sequence information obtained from a number of different chicken samples containing IBDV.

Exemplary PCR amplification reaction conditions for the vaccine against ND-IBD are provided below:

One step of 96°C for 3 minutes; 35 cycles of: 96°C for 30 seconds, 64°C for 30 seconds, and 72°C for 1 minute; and one step of 72°C for 3 minutes. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated within the scope of the invention without limitation thereto.