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

Balamurugan, V.; Sen, Abhik; Venkatesan, Gnanavel; Yadav, Vinita; Bhanot, Vandna; Bhanuprakash, V.; Singh, Rajkumar

doi:10.1111/j.1865-1682.2010.01160.x

Cited by 25 publications

(19 citation statements)

References 32 publications

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“…Since 1995, several reverse transcription‐polymerase chain reaction (RT‐PCR) methods have been developed for the fast, specific detection of PPRV genome (Forsyth and Barrett, ; Couacy‐Hymann et al., ). However, these are now being replaced by more sensitive and robust qRT‐PCR assays targeting the matrix protein (M) gene (Balamurugan et al., ) or different sequences of the Np gene (Bao et al., ; Kwiatek et al., ; Batten et al., ). However, the reliability of molecular diagnostic assays could be limited by the lack of stable internal controls, leading to false‐negative results.…”

Section: Introductionmentioning

confidence: 99%

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

Polci

Cosseddu

Ancora

et al. 2013

Transbound Emerg Dis

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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.

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Section: Introductionmentioning

confidence: 99%

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

Polci

Cosseddu

Ancora

et al. 2013

Transbound Emerg Dis

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“…Earlier, N gene based one step TaqMan real-time RT-PCR has been developed for detection of PPRV nucleic acid in the clinical samples with high sensitivity [18]. Recently, M gene based two step TaqMan hydrolysis probe [12] and one step real-time RT-PCR based on SYBR Green chemistry [13] have been optimized for specific detection of PPRV in clinical samples. Some of earlier described RT-PCR assays are delineated in the OIEs Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [103].…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

“…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.…”

Section: Diagnosismentioning

confidence: 99%

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

Balamurugan¹,

Hemadri²,

Gajendragad³

et al. 2013

VirusDis.

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130

118

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Peste des petits ruminants (PPR) is an acute, highly contagious, world organization for animal health (OIE) notifiable and economically important transboundary viral disease of sheep and goats associated with high morbidity and mortality and caused by PPR virus. PPR is considered as one of the main constraints in augmenting the productivity of small ruminants in developing countries and particularly severely affects poor farmer's economy. The disease is clinically manifested by pyrexia, oculo-nasal discharges, necrotizing and erosive stomatitis, gastroenteritis, diarrhoea and bronchopneumonia. The disease can be diagnosed from its clinical signs, pathological lesions, and specific detection of virus antigen/antibodies/genome in the clinical samples by various serological tests and molecular assays. PPR is the one of the priority animal diseases whose control is considered important for poverty alleviation in enzootic countries. Availability of effective and safe live attenuated cell culture PPR vaccines and diagnostics have boosted the recently launched centrally sponsored control programme in India and also in other countries. This review article primarily focus on the current scenario of PPR diagnosis and its control programme with advancement of research areas that have taken place in the recent years with future perspectives.

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“…To check the performance of the newly developed assay, clinical samples (2 nasal swabs from goats) were spiked with known titre of PPR virus as described by Balamurugan et al [8]. Infected cell culture fluid containing 10 5 TCID 50 /ml viruses was diluted 1: 10 serially by using the two negative nasal swabs as diluent.…”

Section: Spikingmentioning

confidence: 99%

“…Real time RT-PCR has gained wider acceptance over conventional RT-PCR because it is more rapid, sensitive and reproducible. Few real time RT-PCR assays have been described for detection and quantification of PPRV in clinical samples using TaqMan chemistry [7][8][9][10]. In comparison with TaqMan-PCR, SYBR Green I based realtime RT-PCR assay has the advantages of being more cost-effective, easy to design, more precise and produced a more linear decay plot [11].…”

Section: Introductionmentioning

confidence: 99%

A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus

Abera

Thangavelu²,

Chandran³

et al. 2014

BMC Vet Res

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BackgroundPeste des petits ruminants (PPR) is an economically important disease of small ruminants such as sheep and goats. The disease is characterized by severe pyrexia, oculo-nasal discharge, pneumonia, necrosis and ulceration of the mucous membrane and inflammation of the gastro-intestinal tract leading to severe diarrhea. A SYBR Green I based real time RT-PCR targeting the N gene of PPRV has not been established for PPRV detection. Thus, the objective of present study was to develop highly sensitive N gene target SYBR Green I real time RT-PCR for specific detection and quantification of PPRV in clinical samples. A set of primers was designed to detect the nucleocapsid (N) gene of PPRV.ResultsThe assay exhibited high specificity as all the viruses which have clinical and structural similarities to PPRV including Canine distemper virus (CDV), Measles virus (MV), Bluetongue virus (BTV) and Newcastle disease virus (NDV) failed to show an amplification signal. The lower detection limit of the assay was 5.11 copies/μl (Ct value of 33.67 ± 0.5) and 0.001 TCID50/ml (Ct value of 34.7 ± 0.5) based on plasmid copy number and tissue culture infectivity titre. The assay was 3-log more sensitive than the conventional RT-PCR. The coefficient of variation (CV) values for intra- and inter-assay variability were low, ranging from 0.32% - 2.31%, and 0.71% - 5.32%, respectively. To evaluate the performance of the newly developed assay, a total of 36 clinical samples suspected of PPR were screened for the presence of PPRV in parallel with conventional RT-PCR. The real time RT-PCR assay detected PPRV in 30 (83.3%) of clinical samples compared to 16 (44.4%) by conventional RT-PCR.ConclusionsThe two-step SYBR Green I based real time RT-PCR assay reported here is highly sensitive, specific, reproducible and rapid for detection and quantification of PPRV nucleic acids.

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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

Cited by 25 publications

References 32 publications

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

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

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

A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus

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