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Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane

Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germ...

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Autores principales: Sun, Sheng-Ren, Ahmad, Kashif, Wu, Xiao-Bin, Chen, Jian-Sheng, Fu, Hua-Ying, Huang, Mei-Ting, Gao, San-Ji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6106854/
https://www.ncbi.nlm.nih.gov/pubmed/30175148
http://dx.doi.org/10.1155/2018/8678242
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author Sun, Sheng-Ren
Ahmad, Kashif
Wu, Xiao-Bin
Chen, Jian-Sheng
Fu, Hua-Ying
Huang, Mei-Ting
Gao, San-Ji
author_facet Sun, Sheng-Ren
Ahmad, Kashif
Wu, Xiao-Bin
Chen, Jian-Sheng
Fu, Hua-Ying
Huang, Mei-Ting
Gao, San-Ji
author_sort Sun, Sheng-Ren
collection PubMed
description Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germplasm. In this study, we develop two quick, sensitive, and reliable protocols for real-time quantitative PCR (qPCR) of Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV) using two sets of TaqMan probes and primers targeting the reverse transcriptase/ribonuclease H (RT/RNase H) region. The two assays had a detection limit of 100 copies of plasmid DNA and were 100 times more sensitive than conventional PCR. High specificity of the two assays was observed with respect to SCBIMV and SCBMOV. A total of 176 sugarcane leaf tissue samples from Fujian and Yunnan provinces were collected and analyzed in parallel by conventional PCR, SCBIMV-qPCR, and SCBMOV-qPCR. The SCBIMV-qPCR and SCBMOV-qPCR assays indicated that 50% (88/176) and 47% (83/176) samples tested positive, respectively, whereas only 29% (51/176) tested positive with conventional PCR with the primer pairs SCBV-F and SCBV-R. We demonstrate for the first time that SCBIMV and SCBMOV occur in China and reveal coinfection of both Badnavirus species in 29% (51/176) of tested leaf samples. Our findings supply sensitive and reliable qPCR assays for the detection and quantitation of SCBV in sugarcane quarantine programs.
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spelling pubmed-61068542018-09-02 Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane Sun, Sheng-Ren Ahmad, Kashif Wu, Xiao-Bin Chen, Jian-Sheng Fu, Hua-Ying Huang, Mei-Ting Gao, San-Ji Biomed Res Int Research Article Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germplasm. In this study, we develop two quick, sensitive, and reliable protocols for real-time quantitative PCR (qPCR) of Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV) using two sets of TaqMan probes and primers targeting the reverse transcriptase/ribonuclease H (RT/RNase H) region. The two assays had a detection limit of 100 copies of plasmid DNA and were 100 times more sensitive than conventional PCR. High specificity of the two assays was observed with respect to SCBIMV and SCBMOV. A total of 176 sugarcane leaf tissue samples from Fujian and Yunnan provinces were collected and analyzed in parallel by conventional PCR, SCBIMV-qPCR, and SCBMOV-qPCR. The SCBIMV-qPCR and SCBMOV-qPCR assays indicated that 50% (88/176) and 47% (83/176) samples tested positive, respectively, whereas only 29% (51/176) tested positive with conventional PCR with the primer pairs SCBV-F and SCBV-R. We demonstrate for the first time that SCBIMV and SCBMOV occur in China and reveal coinfection of both Badnavirus species in 29% (51/176) of tested leaf samples. Our findings supply sensitive and reliable qPCR assays for the detection and quantitation of SCBV in sugarcane quarantine programs. Hindawi 2018-08-06 /pmc/articles/PMC6106854/ /pubmed/30175148 http://dx.doi.org/10.1155/2018/8678242 Text en Copyright © 2018 Sheng-Ren Sun et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Sun, Sheng-Ren
Ahmad, Kashif
Wu, Xiao-Bin
Chen, Jian-Sheng
Fu, Hua-Ying
Huang, Mei-Ting
Gao, San-Ji
Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane
title Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane
title_full Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane
title_fullStr Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane
title_full_unstemmed Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane
title_short Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane
title_sort development of quantitative real-time pcr assays for rapid and sensitive detection of two badnavirus species in sugarcane
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6106854/
https://www.ncbi.nlm.nih.gov/pubmed/30175148
http://dx.doi.org/10.1155/2018/8678242
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