Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples

Abera, Tsegalem; Thangavelu, A.

doi:10.1016/j.jviromet.2014.08.017

Cited by 7 publications

(3 citation statements)

References 13 publications

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“…M gene-based hydrolysis probe (TaqMan), SYBR Green I based real-time RT-PCR assays targeting the M gene of PPRV are in use (Abera and Thangavelu, 2014;Balamurugan et al, 2012c). Recently, a novel non-amplification technique was developed to detect nucleic acids of PPRV in which two probes complementary to the target sequences -one conjugated to magnetic microparticles and the second to gold nanoparticles labelled with horseradish peroxidase (Tao et al, 2013).…”

Section: Real-time Rt-pcr and Lateral Flow Assaysmentioning

confidence: 99%

Peste-Des-Petits-Ruminants: An Indian Perspective

Muthuchelvan¹

2015

Adv. Anim. Vet. Sci.

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Section: Real-time Rt-pcr and Lateral Flow Assaysmentioning

confidence: 99%

Peste-Des-Petits-Ruminants: An Indian Perspective

Muthuchelvan¹

2015

Adv. Anim. Vet. Sci.

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“…However, these conventional RT-PCR assays also have shortcomings, such as being labor intensive, time consuming and high risk of cross-contamination [ 16 ]. Real time RT-PCR assay has acquired extensive adoption over conventional RT-PCR and been developed for detection and quantitation of PPRV present in clinical samples [ 16 – 20 ]. But the real time RT-PCR assay still relies on specialized and expensive thermos cycling machine, as a result it is difficult to be used as a “pen-side” test and in endemic areas with low resources.…”

Section: Introductionmentioning

confidence: 99%

Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus

Yang

Qin

Song

et al. 2017

Virol J

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BackgroundPeste des petits ruminants (PPR) is an economically important, Office International des Epizooties (OIE) notifiable, transboundary viral disease of small ruminants such as sheep and goat. PPR virus (PPRV), a negative-sense single-stranded RNA virus, is the causal agent of PPR. Therefore, sensitive, specific and rapid diagnostic assay for the detection of PPRV are necessary to accurately and promptly diagnose suspected case of PPR.MethodsIn this study, reverse transcription recombinase polymerase amplification assays using real-time fluorescent detection (real-time RT-RPA assay) and lateral flow strip detection (LFS RT-RPA assay) were developed targeting the N gene of PPRV.ResultsThe sensitivity of the developed real-time RT-RPA assay was as low as 100 copies per reaction within 7 min at 40 °C with 95% reliability; while the sensitivity of the developed LFS RT-RPA assay was as low as 150 copies per reaction at 39 °C in less than 25 min. In both assays, there were no cross-reactions with sheep and goat pox viruses, foot-and-mouth disease virus and Orf virus.ConclusionsThese features make RPA assay promising candidates either in field use or as a point of care diagnostic technique.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0688-6) contains supplementary material, which is available to authorized users.

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“…Currently, various methods are used to detect PPRV, such as virus neutralization test (VNT)[ 17 ], monoclonal antibody-based complement enzyme-linked immunosorbent assay c-ELISA[ 18 , 19 ], agar diffusion test, hemagglutination tests[ 20 ], and virus isolation[ 21 , 22 ], as well as molecular biology-based methods [ 23 – 25 ]. The OIE recommends the VNT and c-ELISA as serologic diagnostic techniques for PPR.…”

Section: Introductionmentioning

confidence: 99%

Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule

et al. 2016

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From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field.

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Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples

Cited by 7 publications

References 13 publications

Peste-Des-Petits-Ruminants: An Indian Perspective

Peste-Des-Petits-Ruminants: An Indian Perspective

Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus

Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule

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