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. 2012 Jan 22;27(1):1–9. doi: 10.1007/s12250-012-3219-z

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

Vinayagamurthy Balamurugan 1,2,✉, Arnab Sen 1,3, Gnanavel Venkatesan 1, Vinita Yadav 1, Vandna Bhanot 1, Veerakyathappa Bhanuprakash 1,4, Raj Kumar Singh 1,5
PMCID: PMC8218091  PMID: 22270801

Abstract

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.

Key words: PPR, M gene, SYBR green RT-PCR, Early diagnosis, Clinical samples

Footnotes

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)

References

  • 1.Afzal M. A., Osterhaus A. D., Cosby S. L., et al. Comparative evaluation of measles virus-specific RT-PCR method through an international collaborative study. J Med Virol. 2003;70:171–176. doi: 10.1002/jmv.10371. [DOI] [PubMed] [Google Scholar]
  • 2.Balamurugan V., Sen A., Saravanan P., et al. One-step multiplex RT-PCR assay for the detection of PPR virus in clinical samples. Vet Res Commun. 2006;30:566–666. doi: 10.1007/s11259-006-3331-3. [DOI] [PubMed] [Google Scholar]
  • 3.Balamurugan V., Sen A., Venkatesan G., et al. Isolation and Identification of virulent peste des petits ruminants viruses from PPR outbreaks in India. Trop Anim Health Prod. 2010;42:1043–1046. doi: 10.1007/s11250-010-9527-0. [DOI] [PubMed] [Google Scholar]
  • 4.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. [DOI] [PubMed]
  • 5.Bao J., Li L., Wang Z., et al. Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus. J Virol Methods. 2008;148:232–236. doi: 10.1016/j.jviromet.2007.12.003. [DOI] [PubMed] [Google Scholar]
  • 6.Barlic-Maganja D., Grom S. 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. 2001;95:101–110. doi: 10.1016/S0166-0934(01)00302-0. [DOI] [PubMed] [Google Scholar]
  • 7.Couacy-Hymann E., Bodjo S. C., Danho T., et al. Early detection of viral excretion from experimental infected goats with peste des petitis ruminants virus. Prev Vet Med. 2007;78:85–88. doi: 10.1016/j.prevetmed.2006.09.003. [DOI] [PubMed] [Google Scholar]
  • 8.Couacy-Hymann E., Bodjo S. C., Koffi M. Y., et al. 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. 2009;87:332–335. doi: 10.1016/j.rvsc.2009.03.002. [DOI] [PubMed] [Google Scholar]
  • 9.Couacy-Hymann E., Roger F., Hurard C., et al. Rapid and sensitive detection of Peste-des-petits-ruminants virus by a polymerase chain reaction assay. J Virol Methods. 2002;100:17–25. doi: 10.1016/S0166-0934(01)00386-X. [DOI] [PubMed] [Google Scholar]
  • 10.Diallo A., Libeau G., Couacy-Hymann E., et al. Recent developments in the diagnosis of rinderpest and peste des petits ruminants. Vet Microbiol. 1995;44:307–317. doi: 10.1016/0378-1135(95)00025-6. [DOI] [PubMed] [Google Scholar]
  • 11.Diallo A., Minet C., Le Goff C., et al. The threat of peste des petits ruminants: progress in vaccine development for disease control. Vaccine. 2007;25(30):5591–5597. doi: 10.1016/j.vaccine.2007.02.013. [DOI] [PubMed] [Google Scholar]
  • 12.El Mubarak H. S., De Swart R. L., Osterhaus A. D., et al. Development of a semi-quantitative real-time RT-PCR for the detection of measles virus. J Clin Virol. 2005;32:313–317. doi: 10.1016/j.jcv.2004.08.017. [DOI] [PubMed] [Google Scholar]
  • 13.Elia G., Decaro N., Martella V., et al. Detection of canine distemper virus in dogs by real-time RT-PCR. J Virol Methods. 2006;136:171–176. doi: 10.1016/j.jviromet.2006.05.004. [DOI] [PubMed] [Google Scholar]
  • 14.Forsyth M. A., Barrett T. 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. 1995;39:151–163. doi: 10.1016/0168-1702(95)00076-3. [DOI] [PubMed] [Google Scholar]
  • 15.Forsyth M. A., Parida S., Alexandersen S., et al. Rinderpest virus lineage differentiation using RT-PCR and SNAP-ELISA. J Virol Methods. 2003;107:29–36. doi: 10.1016/S0166-0934(02)00186-6. [DOI] [PubMed] [Google Scholar]
  • 16.George A., Dhar P., Sreenivasa B. P., et al. 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. 2006;50:217–222. [PubMed] [Google Scholar]
  • 17.Harris E., Roberts T. G., Smith L., et al. Typing of dengue virus in clinical specimens and mosquitoes by single tube multiplex reverse transcriptase PCR. J Clin Microbiol. 1998;36:2634–2639. doi: 10.1128/jcm.36.9.2634-2639.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kim Y. H., Cho K. W., Youn H. Y., et al. Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR. J Vet Sci. 2001;2:59–63. [PubMed] [Google Scholar]
  • 19.Libeau G., Diallo A., Colas F., et al. Rapid differential diagnosis of rinderpest and Peste-des-petits-ruminants using an immunocapture ELISA. Vet Rec. 1994;134:300–304. doi: 10.1136/vr.134.12.300. [DOI] [PubMed] [Google Scholar]
  • 20.Mallet F., Oriol G., Mary C., et al. Continuous RT-PCR using AMV-RT and Taq DNA polymerase: characterization and comparison to uncoupled procedures. Biotechniques. 1995;18:678–687. [PubMed] [Google Scholar]
  • 21.Shaila M. S., Purushothaman V., Bhavasar D., et al. Peste des petits ruminants of sheep in India. Vet Rec. 1989;125:602. [PubMed] [Google Scholar]
  • 22.Singh R. P., Sreenivasa B. P., Dhar P., et al. A sandwich-ELISA for the diagnosis of Peste des petits ruminants (PPR) infection in small ruminants using anti-nucleocapsid protein monoclonal antibody. Arch Virol. 2004;149:2155–2170. doi: 10.1007/s00705-004-0366-z. [DOI] [PubMed] [Google Scholar]
  • 23.Singh R. P., Sreenivasa B. P., Dhar P., et al. Development of monoclonal antibody based competitive-ELISA for detection and titration of antibodies to peste des petits ruminants virus. Vet Microbiol. 2004;98:3–15. doi: 10.1016/j.vetmic.2003.07.007. [DOI] [PubMed] [Google Scholar]
  • 24.van Regenmortel M. H. V., Fauquet C. M., Bishop D. H. L. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press; 2000. Virus taxonomy. [Google Scholar]

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