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ONE STEP MULTIPLEX SYSTEM FOR THE DETECTION OF SALMONELLA SPP., S. TYPHI AND S. PARATYPHI A WITH AN INTERNAL CONTROL FIELD OF THE INVENTION

The present invention relates to a development of a more sensitive, specific, time- effective, as well as single step multiplex polymerase chain reaction (PCR) method for detection of Salmonella spp., S. Typhi and S. Paratyphi A. BACKGROUND OF THE INVENTION

Salmonella infections and enteric fever remain important public health problem in many parts of the world, especially in developing countries. Enteric fever is caused by either S. Typhi, S. Paratyphi A, B and C. A major increase in the incidence of Paratyphi A infection has been noted recently, particularly in Pakistan, India, Vietnam, Nepal, and China (Threlfall, E. J., Fisher, I. S. T., Berghold, C, Gerner-Smidt, P., Tschape, H., Cormican, M., Luzzi, I., Schnieder, F., Wannet, W., Machado, J. and Edwards, G. (2003). Trends in antimicrobial drug resistance in Salmonella enterica serotypes Typhi and Paratyphi A isolated in Europe, 1999-2001. International Journal of Antimicrobial Agents 22: 487-491). S. Paratyphi A is becoming predominant in some provinces in China and increasing numbers of cases have been reported from Pakistan (Girard, M. P., Steele, D., Chaignat, C. L. and Kieny, M. P. (2006). A review of vaccine research and development: human enteric infections. Vaccine 24: 2732-2750). WHO estimates of 17 million cases of TF annually. These pathogens are acquired following ingestion of faecally-contaminated food or water or directly from asymptomatic carriers and outbreaks are frequent (Girard, M. P., Steele, D., Chaignat, C. L. and Kieny, M. P. (2006). A review of vaccine research and development: human enteric infections. Vaccine 24: 2732-2750). Important vehicles of transmission in some countries include shellfish taken from sewage- contaminated beds, raw fruits, vegetables fertilized by night soil and eaten raw, milk and milk products or during preparation of food by hands (Riyaz-Ul-Hassan, S., Verma, V. and Ghulam Nabi Qazi. (2004). Rapid detection of Salmonella by polymerase chain reaction. Molecular and Cellular Probes 18: 333-339). Given the similar presentations of typhoid and paratyphoid fevers, these conditions cannot be reliably distinguished on clinical grounds alone (Vollaard, A. M., Ali, S., Widjaja, S., Henri, A. G. H.van Asten, Visser, L. G., Surjadi, C. and Jaap T. van Dissel. (2005). Identification of typhoid fever and paratyphoid fever cases at presentation in outpatient clinics in Jakarta, Indonesia. Transaction of the Royal Society of Tropical Medicine and Hygiene 99: 440-450). Although several assays for detecting S. typhi antigens or antibodies have been used for their rapidity and simplicity, no non-culture tests for typhoid fever have shown to be highly specific and/or sensitive. Confirmation of typhoid fever requires the identification of S. Typhi in clinical specimens.

Early diagnosis and prompt treatment with appropriate antimicrobial agents are crucial to reduce the morbidity and mortality associated with enteric fever. Clinical diagnosis of typhoid fever is difficult due to the overlapping of the signs and symptoms with other common febrile illness. Laboratory diagnosis of typhoid fever is conventionally based on culture and serological methods. However, these methods lack sensitivity and/or specificity and as a result of this, are of little diagnostic value. Blood culture can only detect about 45 % to 70 % of patients with typhoid fever, depending on the amount of blood sampled, the bacteremic level of S. Typhi, the type of culture medium used and the length of incubation period. The clinical usefulness of culture method is further restricted due to the fact that it takes at least 2 days to identify the microorganism. Furthermore, the likelihood of a positive blood culture and serology diagnosis declines significantly with delayed presentation or prior antibiotic use (Parry, C. M., Hien, T. T., Dougan, G., White, N. J. and Farrar, J. J. (2002). Medical progress: typhoid fever. NEngl JMed Ml: 1770-1782).

Equally, the increasing incidence of multi-antibiotic resistant and/or fluoroquinolone- intermediate Typhi and Paratyphi A strains threatens to undermine the efficacy of empirical therapy regimens in many parts of the world (Walker, R. A., Skinner, J. A., Ward, L. R. and Threlfall, E. J. (2003). LightCycler gyrh mutation assay (GAMA) identifies heterogeneity in GyrA in Salmonella enterica serotypes Typhi and Paratyphi A with decreased susceptibility to ciprofloxacin. International Journal of Antimicrobial Agents 22: 622-625; Butt, T., Ahmad, R. N., Salman, M. and Kazmi, S. Y. (2005). Changing trends in drug resistance among typhoid salmonellae in Rawalpindi, Pakistan. La Revue de Sante de la Mediterranee orientale 11: 1038- 1044). Kawano et al (Razel L. Kawano,* Susan A. Leano, and Dorothy May A. Agdamag. (2007). Comparison of Serological Test Kits for Diagnosis of Typhoid Fever in the Philippines. Journal of Clinical Microbiology 45(1): 246-247) recently evaluated 4 serological test kits for diagnosis of typhoid fever with variable sensitivity and specificity.

The discovery of DNA in the 1950s and advances in the field of molecular biology and immunology have provided new powerful tools to enhance clinical laboratory diagnosis. The basic principle underlining DNA analysis is that all living organisms contain DNA or RNA whose nucleotide sequences differ among species and it is this species-specific sequence that can be detected.

It is known that a DNA probe specific to the Vi antigen of Salmonella Typhi has been used to detect the microorganism in blood of patients with typhoid fever. This hybridization method, however, requires concentration of bacteria from the blood samples and amplification of total bacterial DNA by overnight incubation of the bacteria on nylon filters to increase the sensitivity of the probe. This process of concentration is difficult to achieve because patients with typhoid fever usually have less than 15 S. Typhi cells per ml of blood and the probe is not able to detect when the specimen is less than 500 bacterial cells. The problem of sensitivity of DNA probe or specificity of the immuno-detection method could be circumvented by polymerase chain reaction (PCR), which can detect very little amount of DNA by way of enzymatic amplification, particularly in culture-negative cases.

Several PCR assays using different sets of primers targeting different sites of the Salmonella Typhi genome have been developed to detect the microorganism. However, these methods are not fully specific for S. Typhi or involve multiple primers that increase the complexity of PCR systems. Song et al (J H Song, H Cho, M Y Park, D S Na, H B Moon and C H Pai. (1993). Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction. J Clin Microbiol 31(6): 1439-1443) reported the development of a PCR-based assay that can detect S. Typhi DNA by amplification of flagelin gene of S. Typhi in the blood of typhoid patients. In this process, two pairs of oligonucleotide primers were designed to amplify a 343 bp fragment of the flagelin gene. This flagelin gene targeting primers were subsequently used by many other researchers (R Chaudhry, B V Laxmi, N Nisar, Ray, and D Kumar. (1997). Standardisation of polymerase chain reaction for the detection of Salmonella typhi in typhoid fever. J Clin Pathol. 50(5): 437^139; Abdul Haque; Janbaz Ahmed; Javed A. Qureshi. (1999). Early Detection Of Typhoid By Polymerase Chain Reaction. Annals of Saudi Medicine, Vol 19, No 4). However, this PCR assay was not completely specific for S. Typhi as it also detects other Salmonella strains containing the dH flagelin such as S. Stanley, S. Livingston and S. Schwarzengrund. Hashimoto et al (Y Hashimoto, Y Itho, Y Fujinaga, A Q Khan, F Sultana, M Miyake, K Hirose, H Yamamoto, and T Ezaki. (1995). Development of nested PCR based on the ViaB sequence to detect Salmonella typhi. J Clin Microbiol. 33(3): 775-777) reported the development of nested PCR based on the Via B sequence to detect S. Typhi. All the S. Typhi strains isolated from blood specimens possess the Vi antigens and the DNA sequence encoding the Vi antigen that is called the Via B regions. However, this method was not 100 % specific as it also detected S. Paratyphi C. A major drawback of nested PCR is the carryover contamination which often results in false positivity. More recently, a multiplex PCR targeting the tyv, prt, viaB and fliC genes was developed for the simultaneous detection of S. Typhi and S. Paratyphi A (Hirose, K., Itoh, K. I., Nakajima, H., Kurazono, T., Yamaguchi, M., Moriya, K., Ezaki, T., Kawamura, Y., Tamura, K. and Watanabe, H. (2002). Selective amplification of tyv (rfbE), prt (r/bS), viaB, and fliC genes by multiplex PCR for identification of Salmonella enterica serovars Typhi and Paratyphi A. Journal of Clinical Microbiology 40: 633-636) The fliC-a gene was probably selected because of its presumed restricted distribution to Paratyphi A strains. However, the prt gene had been reported and was found to be more widely distributed (Hirose, K., Itoh, K. I., Nakajima, H., Kurazono, T., Yamaguchi, M., Moriya, K., Ezaki, T., Kawamura, Y., Tamura, K. and Watanabe, H. (2002). Selective amplification of tyv (r ¾E), prt (rfbS), viaB, and fliC genes by multiplex PCR for identification of Salmonella enterica serovars Typhi and Paratyphi A. Journal of Clinical Microbiology 40: 633-636). However, this method utilizes 5 pairs of primers in 1 assay and therefore is complex and requires optimization of each amplified product.

In WIPO patent (WO201 1/001449A2), two pairs of primers have been developed for amplification of genes of Salmonella species. However, after amplification, both S. Typhi and & Paratyphi A are having a same PCR product at 384 bp band. This may cause confusion to the analyst.

In general, the infections of S. Typhi and S. Paratyphi A can be distinguished effectively through the genomic approach. The application of this approach directly to clinical specimen dramatically reduces the time to final identification. The specificity of the PCR developed will become a better control and surveillance of important pathogens, such as S. Typhi and Paratyphi A in the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a single step multiplex PCR system for the diagnosis of Salmonella spp., S. Typhi and S. Paratyphi A.

Another object of the present invention is to provide a nucleotide fragment pair comprising base sequence (SEQ ID NO:l) and (SEQ ID NO:2) represented by the following sequence (SEQ ID NO:l) and (SEQ ID NO:2), a base sequence pair complementary thereto or a mutation sequence thereof and a primer pair for detecting the Salmonella spp. comprising the following nucleotide fragments:

5 ' - ATTACCGGA ACGTTATTTGCGCC ATGCTGA-3 ' (SEQ ID NO:l)

5'-TTATAGCATGGATCCCCGCCGGCGAGA-3' (SEQ ID NO:2)

Further object of the present invention is to provide a nucleotide fragment pair comprising a base sequence (SEQ ID NO:3) and (SEQ ID NO:4) represented by the following sequence (SEQ ID NO:3) and (SEQ ID NO:4), a base sequence pair complementary thereto or a mutation sequence thereof and a primer pair for detecting the S. Paratyphi A comprising the following nucleotide fragments:

5 ' - AATTCCCC AAT ATCGTCTCCCGCC-3 ' (SEQ ID NO:3)

5'-GTACACTTGTACTTCGAGCCATGGAG-3' (SEQ ID NO:4)

Still further object of the present invention is to provide a nucleotide fragment pair comprising a base sequence (SEQ ID NO:5) and (SEQ ID NO:6) represented by the following sequence (SEQ ID NO:5) and (SEQ ID NO:6), a base sequence pair complementary thereto or a mutation sequence thereof and a primer pair for detecting the S. Typhi comprising the following nucleotide fragments:

5 '-CATC AC ATCCTGCCGGAAAG-3 ' (SEQ ID NO:5)

5'-GTACTGACGAATTTGCTCCA-3' (SEQ ID NO:6)

Yet still further object of the present invention is to provide a nucleotide fragment pair comprising a base sequence (SEQ ID NO:7) and (SEQ ID NO:8) represented by the following sequence (SEQ ID NO: 7) and (SEQ ID NO: 8), a base sequence pair complementary thereto or a mutation sequence thereof and a primer pair for internal amplification control comprising the following nucleotide fragments:

5 ' -TGCATGTGAGCGG ATAAC AATTC A-3 ' (SEQ ID NO:7)

5 ' - ATCGTGTTCTGAGGTC ATTACT-3 ' (SEQ ID NO:8)

Yet still further object of the present invention is to provide a specific PCR premix ingredients with specific PCR cycling conditions have been optimized and obtained to carry out the detection. The uniqueness of the present invention is the premix assay only requires one step PCR to be carried out for rapid and simultaneous detection and differentiation of S. Typhi and S. Paratyphi A. It takes only 4 hours for the entire detection, in which 3 hours is for the preparation and PCR amplification and 1 hour is for gel detection.

Overall, this multiplex PCR system could be applied directly to bacterial DNA obtained from blood and faecal specimens, dramatically reducing time to final identification. Furthermore, based on parallels with S. Typhi infections, such assay could significantly increase the number of laboratory-confirmed Paratyphi A infections. BRIEF DESCRIPTION OF THE DRAWING/FIGURES

Figure 1 : Representative gel showing the presence of the amplified bands for strains tested. M: lOObp marker, Lanes 1 : Positive Control, Lane 2-6: S. Paratyphi A, Lane 7- 10: S. Typhi, Lane 1 1-17: Non-Typhoidal Salmonella strains, Lane 18-20: Non- Salmonella strains.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

A single step multiplex PCR system for the diagnosis of Salmonella spp., S. Typhi and S. Paratyphi A in accordance with the present invention will herein below be described in further detail.

In the present invention, 4 pairs of specific primers represented by the following sequences (SEQ ID NO.l to 8) have been developed:

5 ' -ATT ACCGG AACGTTATTTGCGCC ATGCTGA-3 ' (SEQ ID NO: l)

5'-TTATAGCATGGATCCCCGCCGGCGAGA-3' (SEQ ID NO:2)

5 ' -AATTCCCC AAT ATCGTCTCCCGCC-3 ' (SEQ ID NO:3)

5'-GTACACTTGTACTTCGAGCCATGGAG-3' (SEQ ID NO:4)

5 ' -C ATC AC ATCCTGCCGG A AAG-3 ' (SEQ ID NO:5)

5'-GTACTGACGAATTTGCTCCA-3 ' (SEQ ID NO:6)

5 ' -TGC ATGTG AGCGGAT AAC AATTC A-3 ' (SEQ ID NO:7)

5 ' - ATCGTGTTCTGAGGTC ATTACT-3 ' (SEQ ID NO:8) The said primers (SEQ ID N0:1 to 8) are especially adapted to be used in single step multiplex PCR system for the diagnosis of Salmonella spp., S. Typhi and S. Paratyphi A. Indeed, the primers having SEQ ID NO:l and 2 is used for detecting the Salmonella spp. with the predicted PCR product of 784 bp; wherein the primers having SEQ ID NO:3 and 4 is used for detecting the S. Paratyphi A with the predicted PCR product of 496 bp; wherein the primers having SEQ ID NO: 5 and 6 is used for detecting the 5. Typhi with the predicted PCR product of 332 bp; wherein the primers having SEQ ID NO: 7 and 8 is used for internal amplification control to show the PCR is working with the predicted PCR product of 187 bp respectively.

The function and predicted PCR product of each primer are summarized in table 1.

Table 1

The present invention also developed a PCR reaction mixture with a preferably total reaction volume of 25 μΐ which comprise of IX Promega buffer, 1.8 mM MgCl ₂ , 120μΜ of each dNTP, 1.5 U Taq DNA polymerase (Promega), 0.4 μΜ of each primer (SEQ ID NO:l to 8), 11.8 μΐ sterile distilled water and 5 μΐ of DNA template. To further illustrate the present invention in greater detail and not by the way of limitation, the following examples will be given. Example

Specimen Preparation

Bacterial culture: A single bacterial colony was picked with a sterile tooth pick directly into a microfuge tube containing 50 μΐ sterile distilled water and boiled at 99 °C for 10 min in a thermocycler and immediately cooled on ice. After a brief centrifugation, 5 μΐ of the supernatant was used in the PCR assay.

Reaction Mixture PCR PCR amplification was carried out in a total reaction volume of 25 μΐ containing IX Promega buffer, 1.8 mM MgCl ₂ , 120 μΜ of each dNTP, 1.5 U Taq DNA polymerase (Promega), 0.4 μΜ of each primer, 11.8 μΐ sterile distilled water and 5 μΐ of DNA template.

5X PCR buffer: Proprietary formulation supplied at pH 8.5 containing blue and yellow dyes. In a 1 % agarose gel, the blue dye migrates at the same rate as 3-5 kb DNA fragments and the yellow dye migrates at a rate faster than primers (<50 bp). Green Go Taq® Flexi Buffer also increases the density of the sample, so, it will sink into the well of the agarose gel, allowing reactions to be loaded directly onto gels without loading dye.

MgCte: having final concentration of 25 mM.

dNTP solution: A mixture of dATP, dCTP, dGTP, and dTTP each having final concentration of 10 mM.

Taq DNA Polymerase: 5 U Taq DNA polymerase / μΐ.

Primers: Aqueous solution of the above described synthesized and purified oligonucleotides, each having a final concentration of 100 μΜ.

PCR Cycling Conditions

The PCR cycling conditions are as follow: - 1. Initial denaturation 95 °C for 5 minutes; 2. Denaturation at 95 °C for 30 seconds;

3. Annealing at 60.0 °C for 30 seconds;

4. Extension at 72 °C for 1 minutes; and

5. Final extension at 72 °C for 7 minutes.

The cycle (2), (3), and (4) are repeated for 30 times. Any thermocycler can be used to carry out the amplification process.

Detection and Confirmation of PCR Product

The products of PCR, in the amount of 5 μΐ are subjected to agarose gel electrophoresis at a voltage of 90 V on a 1.5 % agarose gel, stained with ethidium bromide and examined under ultra violet (UV) illumination. The 784 bp was present in all Salmonella spp., while the 496 bp and 332 bp was only present in S. Paratyphi A and S. Typhi respectively. The 187 bp band for IAC was present in all reactions included non-Salmonella spp. This band was to ensure that the PCR is working. In Figure 1 , a representative gel showing the presence of the amplified bands for strains tested. M: lOObp marker, Lanes 1 : Positive Control, Lane 2-6: S. Paratyphi A, Lane 7- 10: S. Typhi, Lane 11-17: Non-Typhoidal Salmonella strains, Lane 18-20: Non- Salmonella strains.

Result

41 species of bacterial has been tested with the above method and the results are shown in Table 2. Table 2 shows the list of strains used to test the specificity of the multiplex PCR and the results obtained.

No. Species Total of Strains Multiplex Result

Tested

784bp 496bp 332bp 187bp

1. Salmonella Paratyphi A 56 + + - +

2. Salmonella Agona 1 + - - +

3. Salmonella Albany 5 + - - +

4. Salmonella Bereilly 3 + - - +

5. Salmonella Blockley 1 + - - +

6. Salmonella Bovismorficans 1 + - - +

7. Salmonella Braenderup 1 + - - + 8. Salmonella Chingola 1 + - - +

9. Salmonella Corvalis 4 + - - +

10. Salmonella Dublin 4 + - - +

11. Salmonella Enteritidis 20 + - - +

12. Salmonella Hadar 1 + - - +

13. Salmonella Haifa 1 + - - +

14. Salmonella Houten 1 + - - +

15. Salmonella Hvittingfoss 1 + - - +

16. Salmonella Infantis 1 + - - +

17. Salmonella Kentucky 8 + - - +

18. Salmonella Lomita 1 + - - +

19. Salmonella Matopeni 3 + - - +

20. Salmonella Muenchen 2 + - - +

21. Salmonella Newport 1 + - - +

22. Salmonella Okerara 1 + - +

23. Salmonella Paratyphi B 15 + - - +

24. Salmonella Paratyphi C 4 + - - +

25. Salmonella Raus 1 + - - +

26. Salmonella Tshiongwe 1 + - - +

27. Salmonella Thompson 1 + - - +

28. Salmonella Typhi 120 + - + +

29. Salmonella Typhimurium 25 + - - +

30. Salmonella Virchow 5 + - - +

31. Salmonella Weltevreden 6 + - - +

32. Burkholderia pseudomallei 2 - - - +

33. Escherichia coli 10 - - - +

34. Klebsiella pneumoniae 10 - - - +

35. Listeria monocytogenes 2 - - - +

36. Pseudomonas aeruginosa 8 - - ^■ - +

37. Shigella bodyii 5 - - - +

38. Shigella dysentery 2 5 - - - +

39. Shigella flexneri 5 - - - +

40. Shigella sonnei 5 - - - +

41. Staphylococcus aureus 3 - - - +

The result in Table 2 shows that the present invention is capable to detect Salmonella spp., S. Typhi and S. Paratyphi A in a single step multiplex PCR system.