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

Abstract

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.

Diagnosis and control of peste des petits ruminants: a comprehensive review

“…Therefore, specific diagnosis of PPRV infection can be achieved by cDNA hybridization [41,105,127,149], mobility of N protein [40,149], neutralization test [28,116,149] and MAb-based ELISA [81,82,135,136], RT-PCR [7,53] and Real time RT-PCR [12,13,18]. A battery of serological tests and molecular assays are available to detect and identify PPRV antigen/nucleic acid and antibodies.…”

“…Various primers sets derived from 'N', 'F' and 'P' genes were described to detect and differentiate between RP and PPR by various workers [21,35,53]. The PCR techniques have been developed targeting F gene [53], N gene [35,57], M gene [7,57] and H gene [14,69] and used for specific detection of PPRV from clinical samples. A PCR for PPRV/RPV using PPR virus-specific external protein F gene primer (F1 and F2) [53] has gained great importance for differential diagnosis and for epidemiological studies.…”

“…Further, PCR strategies targeting M and N gene have been developed for detection and differentiation of PPRV in sheep and goats. In a similar direction, a RT-PCR assay based on the M and N gene has been developed [7,57], which was employed successfully for the direct detection of PPRV in the clinical samples [7]. The one-step multiplex RT-PCR based on N and M genes for use in differential diagnosis of PPR from RP in a single reaction, which has the potential to replace the existing F gene based PCR for diagnosis of PPR was developed [7].…”

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“…The total RNA was extracted from clinical materials by using RNA easy kit (RNeasy®Minikit Qiagen Inc, Valencia, CA, USA), and RT-PCR was performed using Qiagen Revert Aid First Strand cDNA Synthesis Kit for first strand synthesis and subsequently PCR was performed using virus specific reported primer sets (Forsyth and Barrett, 1995;Couacy et al, 2002;Balamurugan et al, 2006) using PCR Master mix reagents (Qiagen Inc, Valencia, CA, USA). The serum samples were tested for the presence of PPRV-specific antibodies by using competitive enzyme-linked immunosorbent assay (c-ELISA) kit from Indian Veterinary Research Institute (Singh et al, 2004b).…”

Potential Effect ofAcacia arabica. onPeste des Petits Ruminants. Virus Replication

“…Extracted RNA was subjected to RT-PCR using one-step RT-PCR kit (Qiagen Inc, Valencia, CA, USA) as described earlier (Balamurugan et al, 2006) using 20 pmol PPRV-specific primers with prescribed PCR conditions. The sensitivity of this assay was 100 femtogram viral RNA for detection of PPRV.…”