Development of multiplex RT-PCR for detection and differentiation of foot-and-mouth disease virus O and A serotypes in Turkey

In this study, a two-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and evaluatedusing designed primers for the simultaneous detection and differentiation of Turkish foot-and-mouth disease virus (FMDV) serotypesA and O from clinical samples. In the method, a mix of one universal reverse primer designed from the 2B gene and two serotype- specific forward primers designed from the hypervariable regions of the capsid VP1-1D coding gene of FMDV were used. Totally, 272FMDV-infected samples collected between 2006 and 2008 constituted the material of the study. mRT-PCR was compared to ELISA interms of positivity percentage (%) and the difference between the two tests was found to be statistically significant (P < 0.05). In thestudy, 148 (54.4%) of 272 samples were undetermined by ELISA. Ninety-nine (66.8%) of these 148 undetermined samples were foundFMDV RNA-positive with mRT-PCR. Consequently, the diagnostic sensitivity and specificity of mRT-PCR was determined as 95% and84%, respectively. The study showed that mRT-PCR was more efficient than ELISA and concluded that the technique can be used as analternative to ELISA for molecular typing of FMDV samples. This is the first report providing the differentiation of FMDV serotypes Aand O in Turkey by mRT-PCR.

PDF

___

1. Clavijo A, Wright P, Kitching P. Developments in diagnostic techniques for differentiating infection from vaccination in foot-and-mouth disease. Vet J 2004; 167: 9-22.
2. Fry EE, Stuart DI, Rowlands DJ. The structure of foot-and- mouth disease virus. Curr Top Microbiol Immunol 2005; 288: 71-101.
3. Knowles NJ, Samuel AR. Molecular epidemiology of foot-and- mouth disease virus. Virus Res 2003; 91: 65-80.
4. Alexendersen S, Zhang Z, Donaldson A, Garland AJ. The pathogenesis and diagnosis of foot-and-mouth disease. J Comp Pathol 2003; 129: 1-36.
5. Carrillo C , Tulman ER, Delhon G, Lu Z, Carreno A, Vagnozzi A, Kutish GF. Comparative genomics of foot-and-mouth disease virus . J Virol 2005; 79: 6487-6450.
6. Domingo E, Escarmis C, Martinez MA, Martinez-Salas E, Mateu MG. Foot-and-mouth disease virus populations are quasispecies. Curr Top Microbiol Immunol 1992; 176: 33-47.
7. Ferris NP, Dawson M. Routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases. Vet Microbiol 1988; 16: 201-209.
8. Liu D. Foot-and-mouth disease virus. In: Liu D, editor. Molecular Detection of Animal Viral Pathogens. Boca Raton, FL, USA: CRC Press; 2016. p. 64.
9. Reid SM, Ferris N, Hutchings GH, Samuel AR, Knowles NJ. Primary diagnosis of FMD by reverse transcription PCR. J Virol Techniques 2000; 89: 167-176.
10. Hoffmann B, Beer M, Reid SM, Mertens P, Oura CA, Van Rijn PA, Slomka MJ, Banks J, Brown IH, Alexander DJ et al. A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health. Vet Microbiol 2009; 139: 1-23.
11. Rodriguez A, Martinez-Salas E, Dopazzo J, Davila M, Saiz JC, Sobrino F. Primer design for specific diagnosis by PCR of highly variable RNA viruses: typing of foot-and-mouth disease virus. Virology 1992; 189: 63-67.
12. Callens M, Clercg KD. Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction. J Virol Techniques 1997; 67: 35-44.
13. Reid SM, Hutchings GH, Ferris N, Clercq KD. Diagnosis of foot and mouth disease by RT-PCR: Evaluation of primers for serotypic characterisation of viral RNA in clinical samples. J Virol Techniques 1999; 83: 113-123.
14. Reid SM, Ferris N, Hutchings GH, Clercg KD, Newman BJ, Knowles NJ, Samuel AR. Diagnosis of FMD by RT-PCR: use of phylogenetic data to evaluate primers for the typing of viral RNA in clinical samples. Arch Virol 2001; 146: 2421-2434.
15. Saiz M, Morena DB, Blanco E, Nunez J, Fernandez R, Sanchez- Vizcaino JM. Detection of foot-and-mouth disease virus from culture and clinical samples by reverse transcription-PCR coupled to restriction enzyme and sequence analysis. Vet Res 2003; 34: 105-117.
16. Suryanarayana V, Madanamohan B, Bist P, Natarajan C, Tratschin, JD. Serotyping of FMDV by Ag-capture RT-PCR. J Virol Methods 1999; 80: 45-52.
17. Vangrysperre W, Clercq KD. Rapid and sensitive polymerase chain reaction based detection and typing of FMDV in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other genomically and/or symptomatically related virus. Arch Virol 1996; 141: 331-344.
18. Grindharan P, Hemadri D, Tosh C, Sanyan A, Bondyopadhyay SK. Development and evaluation of multiplex PCR for differentiation of foot-and-mouth disease virus strains native to India. J Virol Techniques 2005; 126: 1-11.
19. Bao HF, Li D, Guo ZJ, Lu ZJ, Chen YL, Liu TX, Xie QG. A highly sensitive and spesific mRT-PCR to detect foot-and- mouth disease virus in tissue and food samples. Arch Virol 2008; 153: 205-209.
20. Le VP, Lee K, Nguyen T, Kim S, Cho I, Quyen DV, Khang DD, Park J. Development of one-step multiplex RT-PCR method for simultaneous detection and differentiation of foot-and- mouth disease virus serotypes O, A, and Asia-1 circulating in Vietnam. J Virol Methods 2011; 175: 101-108.
21. Reid SM, Mioulet V, Knowles NJ, Shirazi N, Belsham GJ, King DP. Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot- and-mouth disease virus lineages circulating in the Middle East. J Virol Methods 2014; 207: 146-153.
22. Jamal SM, Belsham GJ. Development and characterization of probe-based real time quantitative RT-PCR assays for detection and serotyping of foot-and-mouth disease viruses circulating in West Eurasia PLoS One 2015; 10: e0135559.
23. Elnifro ME, Ashshi AM, Cooper RJ, Klapper P. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 1992; 13: 559-570.
24. Henagariu O. Multiplex PCR: Critical parameters and step by step protocol. Biotechniques 1995; 23: 504-511.
25. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate phenolchloroform extraction. Anal Chem 1987; 162: 156-159.
26. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press; 1989.
27. Peters IR, Helps CR, Hall EJ, Day MJ. Real time RT-PCR. Considerations for efficient and sensitive assay design. J Immunol Methods 2004; 286: 203-217.
28. Witwer C, Rauscher S, Hofacker LI, Stadler PF. Conserved RNA secondary structures in Picornaviridae genomes. Nuc Acid Res 2001; 29: 5079-5089.
29. Cheung A, Delamarter J, Weiss S, Küpper H. Comparison of the major antigenic determinants of different serotypes of foot- and-mouth disease virus. J Virol 1983; 48: 451-459.
30. Reid SM, Forsyth, MA, Hutchings, GH, Ferris, NP. Comparison of reverse transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation for the routine diagnosis of foot-and-mouth disease. J Virol Methods 1998; 70: 213-217.