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

BACKGROUND: Peste 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 tra...

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Autores principales: Abera, Tsegalem, Thangavelu, Ardhanary, Joy Chandran, Navamani Daniel, Raja, Angamuthu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896792/
https://www.ncbi.nlm.nih.gov/pubmed/24423231
http://dx.doi.org/10.1186/1746-6148-10-22
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author Abera, Tsegalem
Thangavelu, Ardhanary
Joy Chandran, Navamani Daniel
Raja, Angamuthu
author_facet Abera, Tsegalem
Thangavelu, Ardhanary
Joy Chandran, Navamani Daniel
Raja, Angamuthu
author_sort Abera, Tsegalem
collection PubMed
description BACKGROUND: Peste 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. RESULTS: The 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 TCID(50)/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. CONCLUSIONS: The 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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spelling pubmed-38967922014-01-22 A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus Abera, Tsegalem Thangavelu, Ardhanary Joy Chandran, Navamani Daniel Raja, Angamuthu BMC Vet Res Research Article BACKGROUND: Peste 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. RESULTS: The 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 TCID(50)/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. CONCLUSIONS: The 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. BioMed Central 2014-01-14 /pmc/articles/PMC3896792/ /pubmed/24423231 http://dx.doi.org/10.1186/1746-6148-10-22 Text en Copyright © 2014 Abera et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Abera, Tsegalem
Thangavelu, Ardhanary
Joy Chandran, Navamani Daniel
Raja, Angamuthu
A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus
title A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus
title_full A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus
title_fullStr A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus
title_full_unstemmed A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus
title_short A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus
title_sort sybr green i based real time rt-pcr assay for specific detection and quantitation of peste des petits ruminants virus
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896792/
https://www.ncbi.nlm.nih.gov/pubmed/24423231
http://dx.doi.org/10.1186/1746-6148-10-22
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