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One-step Multiplex RT-PCR Assay for the Detection of Peste des petits ruminants Virus in Clinical Samples

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A single-tube one-step multiplex RT-PCR was standardized to amplify both 337 bp and 191 bp fragments of N and M genes of peste des petits ruminants virus (PPRV), respectively, and only a 337 bp fragment of N gene of Rinderpest virus (RPV). The RT-PCR using purified viral RNA was easily adopted for direct detection of PPRV in clinical field samples and its differentiation from RPV. The amplified N and M gene products were confirmed to be PPRV- and RPV-specific by their size in 1.5% agarose gel and restriction analysis. In the assay, the Qiagen one-step RT-PCR kit containing the Ominiscript and Sensiscript reverse transcriptases and Hot star Taq DNA polymerase was utilized. The sensitivity of the assay was found to be 100 fg of PPRV RNA. Compared with a two-step assay, the one-step assay is easier and time-saving as it requires just a single buffer for both reactions, reverse transcription (RT) and PCR. In experimentally infected goats, PPRV was detectable by the one-step RT-PCR in nasal and ocular swabs 7–17 days post infection (p.i.). and in oral swabs 7–15 days p.i. Out of 32 clinical field samples tested, 18 were positive by sandwich ELISA (S-ELISA), while 22 were positive by the one-step RT-PCR.

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agar gel immunodiffusion




horseradish peroxidase


Moloney murine leukaemia virus




post infection


peste des petits ruminants


PPR virus




rinderpest virus


reverse transcription


sandwich ELISA

TCID50 :

50% tissue culture infective dose


  1. Abu-Elzein, E.M.E., Hassanien, M.M., Al-Afaleq AI, Abd-Elhadi, M.A. and Housain, F.M.I., 1990. Isolation of peste des petits ruminants virus from goats in Saudi Arabia. The Veterinary Record, 127, 309–310

  2. Aleyas, A.G., 2002. Comparative evaluation of different gene targets for PCR diagnosis of peste des petits ruminants, (MVSc thesis, Indian Veterinary Research Insititute, Deemed University, Izatnagar)

  3. Andreoletti, L., Hober, D., Belaich, S., Lobert, P.E., Dewilde, A. and Wattre, P., 1996. Rapid detection of entero virus in the clinical specimens using PCR and microwell capture hybridization assay. Journal of Virological Methods, 62, 1–10

  4. Barlic-Maganja, D. and Grom, S., 2001. Highly sensitive one tube RT-PCR and microplate hybridization assay for the detection and for the discrimination of classical swine fever virus from other pestiviruses. Journal of Virological Methods, 95, 101–110

  5. Brindha, K., Raj, G.D., Ganesan, P.I., Thiagarajan, V., Nainar, A.M. and Nachimuthu, K., 2001. Comparison of virus isolation and polymerase chain reaction for diagnosis of peste des petits ruminants. Acta Virologica, 45, 169–172

  6. Callens, M. and Clercq, K., 1999. Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection. Journal of Virological Methods, 77, 87–99

  7. Cattaneo, R., Rebmann, G., Baczko, K., Ter Meulen, V. and Billeter, M.A., 1987. Altered ratios of measles virus transcripts in diseased human brains. Virology, 160, 523–526

  8. Chomczynski, P. and Sacchi, N., 1987. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162, 56–159

  9. Couacy-Hymann, E., Roger, F., Hurard, C., Guillou, J.P., Libeau, G. and Diallo, A., 2002. Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay. Journal of Virological Methods, 100, 17–25

  10. Devireddy, L.R., Raghavan, R., Ramachandran, S. and Shaila, M.S., 1998. Protection of rabbits against lapinised rinderpest virus with purified envelope glyproteins of peste des petits ruminants viruses. Acta Virologica, 42, 299–306

  11. Dhar, P., Sreenivasa, B.P., Barrett, T., Corteyn, M., Singh, R.P. and Bandyopadhyay, S.K., 2002. Recent epidemilogy of peste des petits ruminants virus (PPRV). Veterinary Microbiology, 88, 153–159

  12. Diallo, A., 1990. Morbillivirus group: genome organization and proteins. Veterinary Microbiology, 23, 155–163

  13. Diallo, A., Barrett, T., Barbron, M., Shaila, M.S. and Lefevre, P.C., 1989. Differentiation of rinderpest and peste des petits ruminants viruses using specific cDNA clones. Journal of Virological Methods, 23, 127–136

  14. Diallo, A., Barrett, T., Barbron, M., Meyer, G. and Lefevre, P.C., 1994. Cloning of the nucleocapsid protein gene of peste des petits ruminants virus: relationship to other morbiliviruses. Journal of General Virology, 75, 233–237

  15. Diallo, A., Libeau, G., Couacy-Hymann, E. and Barbron, M., 1995. Recent developments in the diagnosis of rinderpest and peste des petits ruminants. Veterinary Microbiology, 44, 307–317

  16. Forsyth, M.A. and Barrett, T., 1995. Detection and differentiation of rinderpest and peste des petits ruminants viruses in diagnostic and experimental samples by polymerase chain reaction using P and F gene-specific primers. Virus Research, 39, 151–163

  17. Forsyth, M.A., Parida, S., Alexandersen, S., Belsham, G.J. and Barrett, T., 2003. Rinderpest virus lineage differentiation using RT-PCR and SNAP-ELISA. Journal of Virological Methods, 107, 29–36

  18. Ghosh, A., Joshi, V.P. and Shaila, M.S., 1995. Characterization of an in vitro transcription system from rinderpest virus. Veterinary Microbiology, 44, 165–173

  19. Haffar, A., Libeau, G., Moussa, A., Cecile, M. and Diallo, A., 1999. The matrix protein gene sequence analysis reveals close relationship between peste des petits ruminants virus (PPRV) and dolphin morbillivirus. Virus Research, 64, 69–75

  20. Harris, E., Roberts, T.G., Smith, L., Selle, J., Kramer, L.D., Valle, S., Sandoval, E. and Balmaseda, A., 1998. Typing of dengue virus in clinical specimens and mosquitoes by single tube multiplex reverse transcriptase PCR. Journal of Clinical Microbiology, 36, 2634–2639

  21. Ismail, T.M., Yamanaka, M.K., Saliki, J.T., EL-Kholy, A., Mebus, C. and Yilma, T., 1995. Cloning and expression of the nucleoprotein of peste des petits ruminants virus in baculovirus for use in serological diagnosis. Virology, 20, 776–778

  22. Jones, L., Giavedoni, L., Saliki, J.T., Brown, C., Mebus, C. and Yilma, T., 1993. Protection of goats against peste des petits ruminants with a vaccinia virus double recombinant expressing the F and H genes of rinderpest virus. Vaccine, 11, 961–964

  23. Kim, Y.H., Cho, K.W., Youn, H.Y., Yoo, H.S. and Han, H.R., 2001. Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR. Journal of Veterinary Science, 2, 59–63

  24. Lee, L.H., Ting, L.J., Shein, J.H. and Shein, H.K., 1994. Single tube, non-interrupted reverse transcriptase polymerase chain reaction for detection of infectious bursal disease virus. Journal of Clinical Microbiology, 32, 1268–1272

  25. Libeau, G., Diallo, A., Colas, F. and Guerre, L., 1994. Rapid differential diagnosis of rinderpest and peste des petits ruminants using an immunocapture ELISA. The Veterinary Record, 134, 300–304

  26. Limo, M. and Yilma, T., 1990. Molecular cloning of the rinderpest virus matrix gene: comparative sequence analysis with other paramyxoviruses. Virology, 175, 323–327

  27. Mallet, F., Oriol, G., Mary, C., Verrier, B. and Mandrand, B., 1995. Continuous RT-PCR using AMV-RT and Taq DNA polymerase: characterization and comparison to uncoupled procedures. Biotechniques, 18, 678–687

  28. Muthuchelvan, D., 2004. Molecular characterization of a candidate peste des petits ruminants vaccine virus, (PhD thesis, Indian Veterinary Research Institute, Deemed University, Izatnagar)

  29. Obi, T.U. and Patrick, D., 1984. The detection of peste des petits ruminants (PPR) virus antigen by agar gel precipitation test and counter-immunoelectrophoresis. Journal of Hygiene, 93, 579–586

  30. Obi, T.U., McCullough, K.C. and Taylor, W. P., 1990. The production of peste des petits ruminants hyperimmune sera in rabbits and their application in virus diagnosis. Journal of Veterinary Medicine, 37, 345–352

  31. Pandey, K.D., Baron, M.D. and Barrett, T., 1992. Differential diagnosis of rinderpest and PPR using biotinylated cDNA probes. The Veterinary Record, 131, 199–200

  32. Plowright, W. and Ferris, R.D., 1962. Studies with rinderpest virus in tissue culture. The use of attenuated culture virus as a vaccine for cattle. Research in Veterinary Science, 3, 172–182

  33. Saravanan, P., Singh, R.P., Balamurugan, V., Dhar, P., Sreenivasa, B.P., Muthuchevan, D., Sen, A., Aleyas, A.G., Singh, R.K. and Bandhyopadhyay, S.K., 2004. Development of a N gene based PCR ELISA for detection of PPR virus in clinical samples. Acta Virologica, 48, 249–255

  34. Shaila, M.S., Purushothaman, V., Bhavasar, D., Venugopal, K. and Venkatesan, R.A., 1989. Peste des petits ruminants of sheep in India. The Veterinary Record, 125, 602

  35. Shaila, M.S., Shamaki, D., Forsyth, M.A., Diallo, A., Groatley, L., Kitching, R.P. and Barrett, T., 1996. Geographic distribution and epidemiology of peste des petits ruminants viruses. Virus Research, 43, 149–153

  36. Sharma, B., Norrby, E., Blixenkrone-Moller, M. and Kovamees, J., 1992. The nucleotide and deduced amino acid sequence of the M gene of phocid distemper virus (PDV). The most conserved protein of morbilliviruses shows a uniquely close relationship between PDV and canine distemper virus. Virus Research, 13–25

  37. Singh, R.P., Sreenivasa, B.P., Dhar, P., Shah, L.C. and Bandyopadhyay, S.K., 2004a. Development of a monoclonal antibody based competitive ELISA for detection and titration of antibodies to peste des petits ruminants (PPR) virus. Veterinary Microbiology, 98, 3–15

  38. Singh, R.P., Sreenivasa, B.P., Dhar, P. and Bandyopadhyay, S.K., 2004b. A sandwich-ELISA for the diagnosis of peste des petits ruminants (PPR) infection in small ruminants using anti-nucleocapsid protein monoclonal antibody. Archives of Virology, 149 2155–2170

  39. Sinha, N.K., 1998. Rinderpest in India: the delegate declares the peninsular zone ‘provisionally free’ from the disease. Disease Information, 11, 61

  40. Sreenivasa, B.P., Dhar, P., Singh, R.P. and Bandyopadhyay, S.K., 2000. Evaluation of an indigenously developed homologous live attenuated cell culture vaccine against peste des petits ruminants infection of small ruminants. Proceedings of the XX Annual Conference of Indian Association of Veterinary Microbiologists, Immunologists and Specialists in Infectious Diseases (IAVMI), Pantnagar, Uttaranchal, India, 84

  41. Steinhauer, D.A., De La Torre, J.C. and Holland, J.J., 1989. High nucleotide substitution error frequencies in clonal pools of vesicular stomatitis virus. Journal of Virology, 63, 2063–2071

  42. Tosh, C., Hemadri, D., Sanyal, A., Pattnaik, B. and Venkataramanan, R., 1997. One tube and one—buffer system of RT-PCR amplification of ID gene of foot-and-mouth disease virus field isolates. Acta Virologica, 41, 153–155

  43. Van Regenmortel, M.H.V., Fauquet, C. M. and Bishop, D.H.L., 2000. Virus taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, (Academic Press, San Diego)

  44. Zientara, S., Saileau, C., Moulay, S. and Cruciere, C., 1994. Diagnosis of the African horse sickness virus serotype-4 by a one-tube, one-manipulation RT-PCR reaction from infected organs. Journal of Virological Methods, 46, 179–188

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Correspondence to V. Balamurugan.

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Balamurugan, V., Sen, A., Saravanan, P. et al. One-step Multiplex RT-PCR Assay for the Detection of Peste des petits ruminants Virus in Clinical Samples. Vet Res Commun 30, 655–666 (2006). https://doi.org/10.1007/s11259-006-3331-3

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  • one-step multiplex RT-PCR
  • peste des petits ruminants virus
  • differentiation RPV
  • clinical samples