Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome

Abstract

A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/μl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection.

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

“…Some of earlier described RT-PCR assays are delineated in the OIEs Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [103]. A duplex real-time qRT-PCR assay was also developed for a simple and rapid diagnosis of PPR [107]. In the recent past, as a field application diagnostic tool, a simple, rapid, specific and highly sensitive novel approach called as LAMP have also been developed targeting N gene [38,79] and evaluated using field clinical samples.…”

Current Advances in Genome Detection of Peste des Petits Ruminants Virus

“…9.4, 9.5). Particularly, for the detection of PPRV genome, the TaqMan technology approach has been used by various authors (Bao et al 2008;Wang et al 2009;Kwiatek et al 2010;Batten et al 2011;Polci et al 2013). …”

“…Since these controls are amplified using the assay target-specific primers, a benefit of this type of control is that it directly monitors the performance of diagnostic primer sets, ensuring that the correct PCR components have been added. However, in contrast to the previously described exogenous ICs, these targetspecific control systems cannot be transferred to other assays, although a cassette approach can be designed to incorporate primer sequence of multiple diagnostic test (Hoffmann et al 2009;Polci et al 2013).…”

“…Both internal control systems have been used to perform PCR technique for the specific detection of PPRV genome (Polci et al 2013). …”

“…Different authors developed various multiplex systems for the detection of PPRV genome such as a duplex real-time PCR (Polci et al 2013) and multiplex RT-PCR (George et al 2006;Yeh et al 2011). Mainly, N, M and F gene-based multiplex PCR is in use in the laboratory for the specific diagnosis of PPR.…”