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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

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.

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Correspondence to Vinayagamurthy Balamurugan.

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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)

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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

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Key words

  • PPR
  • M gene
  • SYBR green RT-PCR
  • Early diagnosis
  • Clinical samples