A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ∼0.0001 cell culture infectious dose 50% units (TCID50). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.
Afzal M A, Osterhaus A D, Cosby S L, et al. 2003. Comparative evaluation of measles virus-specific RT-PCR method through an international collaborative study. J Med Virol, 70: 171–176.
Balamurugan V, Sen A, Saravanan P, et al. 2006. One-step multiplex RT-PCR assay for the detection of PPR virus in clinical samples. Vet Res Commun, 30: 566–666.
Balamurugan V, Sen A, Venkatesan G, et al. 2010. Isolation and Identification of virulent peste des petits ruminants viruses from PPR outbreaks in India. Trop Anim Health Prod, 42: 1043–1046.
Balamurugan V, Sen A, Venkatesan G, et al. Application of semi-quantitative M gene-based hydrolysis probe (TaqMan) real-time RT-PCR assay for the detection of peste des petitis ruminants virus in the clinical samples for investigation into clinical prevalence of disease (2010). Transbound Emerg Dis, 57(6): 383–395.
Bao J, Li L, Wang Z, et al. 2008. Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus. J Virol Methods, 148: 232–236.
Barlic-Maganja D, 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. J Virol Methods, 95: 101–110.
Couacy-Hymann E, Bodjo S C, Danho T, et al. 2007. Early detection of viral excretion from experimental infected goats with peste des petitis ruminants virus. Prev Vet Med, 78: 85–88.
Couacy-Hymann E, Bodjo S C, Koffi M Y, et al. 2009. The early detection of peste-des-petits-ruminants (PPR) virus antigens and nucleic acid from experimentally infected goats using RT-PCR and immunocapture ELISA techniques. Res Vet Sci, 87:332–335.
Couacy-Hymann E, Roger F, Hurard C, et al. 2002. Rapid and sensitive detection of Peste-des-petits-ruminants virus by a polymerase chain reaction assay. J Virol Methods, 100: 17–25.
Diallo A, Libeau G, Couacy-Hymann E, et al. 1995. Recent developments in the diagnosis of rinderpest and peste des petits ruminants. Vet Microbiol, 44: 307–317.
Diallo A, Minet C, Le Goff C, et al. 2007. The threat of peste des petits ruminants: progress in vaccine development for disease control. Vaccine, 25(30): 5591–5597.
El Mubarak H S, De Swart R L, Osterhaus A D, et al. 2005. Development of a semi-quantitative real-time RT-PCR for the detection of measles virus. J Clin Virol, 32: 313–317.
Elia G, Decaro N, Martella V, et al. 2006. Detection of canine distemper virus in dogs by real-time RT-PCR. J Virol Methods, 136: 171–176.
Forsyth M A, 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 Res, 39: 151–163.
Forsyth M A, Parida S, Alexandersen S, et al. 2003. Rinderpest virus lineage differentiation using RT-PCR and SNAP-ELISA. J Virol Methods, 107: 29–36.
George A, Dhar P, Sreenivasa B P, et al. 2006. The M and N genes-based simplex and multiplex PCRs are better than the F or H gene-based simplex PCR for Peste-des-petits-ruminants virus. Acta Virol, 50: 217–222.
Harris E, Roberts T G, Smith L, et al. 1998. Typing of dengue virus in clinical specimens and mosquitoes by single tube multiplex reverse transcriptase PCR. J Clin Microbiol, 36: 2634–2639.
Kim Y H, Cho K W, Youn H Y, et al. 2001. Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR. J Vet Sci, 2: 59–63.
Libeau G, Diallo A, Colas F, et al. 1994. Rapid differential diagnosis of rinderpest and Peste-des-petits-ruminants using an immunocapture ELISA. Vet Rec, 134: 300–304.
Mallet F, Oriol G, Mary C, et al. 1995. Continuous RT-PCR using AMV-RT and Taq DNA polymerase: characterization and comparison to uncoupled procedures. Biotechniques, 18: 678–687.
Shaila M S, Purushothaman V, Bhavasar D, et al. 1989. Peste des petits ruminants of sheep in India. Vet Rec, 125: 602.
Singh R P, Sreenivasa B P, Dhar P, et al. 2004. A sandwich-ELISA for the diagnosis of Peste des petits ruminants (PPR) infection in small ruminants using anti-nucleocapsid protein monoclonal antibody. Arch Virol, 149: 2155–2170.
Singh R P, Sreenivasa B P, Dhar P, et al. 2004. Development of monoclonal antibody based competitive-ELISA for detection and titration of antibodies to peste des petits ruminants virus. Vet Microbiol, 98: 3–15.
van Regenmortel M H V, Fauquet C M, Bishop D H L. 2000. Virus taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, San Diego: Academic Press.
Foundation items: Indian Council of Agricultural Research, New Delhi, India under Niche Area of Excellence: Production and Quality control of Veterinary Immunodiganostics and immunoprophylactics (F.No.10(11)2005-EP&D.dated 15.12.2005)
Rights and permissions
About this article
Cite this article
Balamurugan, V., Sen, A., Venkatesan, G. et al. A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of peste des petits ruminants virus in the clinical samples. Virol. Sin. 27, 1–9 (2012). https://doi.org/10.1007/s12250-012-3219-z
- M gene
- SYBR green RT-PCR
- Early diagnosis
- Clinical samples