Simultaneous detection of Rift Valley Fever, bluetongue, rinderpest, and Peste des petits ruminants viruses by a single-tube multiplex reverse transcriptase-PCR assay using a dual-priming oligonucleotide system

Abstract

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions.

Fig. 1.

Multiplex RT-PCR amplification for Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV) in 10% oral mucosal homogenate. M, 50-bp DNA molecular weight marker (50-bp DNA ladder; Gene Direx); lane Mix, four viruses in a single-tube; lane Btv, BTV strain IND2003/01(serotype 2); lane Pprv, PPRV strain Nigeria 75/1; lane Rvfv, RVFV strain Smithburn; lane Rpv, RPV strain LATC; lane Neg, 10% oral mucosal homogenate collected from asymptomatic cattle.