Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR
The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-li...
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| Veröffentlicht in: | Journal of virological methods 2010-11, Vol.169 (2), p.391-396 |
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| container_title | Journal of virological methods |
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| creator | Sun, Yingjie Yue, Zhiqin Liu, Hong Zhao, Yuran Liang, Chengzhu Li, Ye Shi, Xiujie Wu, Bin Xu, Biao Deng, Mingjun Zhu, Laihua Wang, Zhe |
| description | The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10
8 copies of HRV RNA. Regression analysis demonstrated a significant correlation with an
R
2 value of 0.9963 and a slope of −3.18 between the mean
C
t values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37–1.72% and 1.37–3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples. |
| doi_str_mv | 10.1016/j.jviromet.2010.08.011 |
| format | Article |
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8 copies of HRV RNA. Regression analysis demonstrated a significant correlation with an
R
2 value of 0.9963 and a slope of −3.18 between the mean
C
t values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37–1.72% and 1.37–3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples.</description><identifier>ISSN: 0166-0934</identifier><identifier>EISSN: 1879-0984</identifier><identifier>DOI: 10.1016/j.jviromet.2010.08.011</identifier><identifier>PMID: 20723563</identifier><identifier>CODEN: JVMEDH</identifier><language>eng</language><publisher>Kidlington: Elsevier B.V</publisher><subject>Animals ; Biological and medical sciences ; Birnavirus ; Diagnostic assay ; Fish Diseases - diagnosis ; Fish Diseases - virology ; Fishes ; Freshwater ; Fundamental and applied biological sciences. Psychology ; Hirame rhabdovirus ; Infectious haematopoietic necrosis virus ; Infectious pancreatic necrosis virus ; Microbiology ; Novirhabdovirus - genetics ; Novirhabdovirus - isolation & purification ; Quantitative RT-PCR ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction - methods ; Rhabdoviridae Infections - veterinary ; Rhabdoviridae Infections - virology ; Sensitivity and Specificity ; TaqMan probe ; Techniques used in virology ; Viral hemorrhagic septicemia virus ; Viral Load - methods ; Virology</subject><ispartof>Journal of virological methods, 2010-11, Vol.169 (2), p.391-396</ispartof><rights>2010 Elsevier B.V.</rights><rights>2015 INIST-CNRS</rights><rights>Copyright © 2010 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c429t-327d01e4e4668fd71577045f983e23ad9bd04b405637aed5f39dbc47f28821dd3</citedby><cites>FETCH-LOGICAL-c429t-327d01e4e4668fd71577045f983e23ad9bd04b405637aed5f39dbc47f28821dd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jviromet.2010.08.011$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=23303166$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20723563$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sun, Yingjie</creatorcontrib><creatorcontrib>Yue, Zhiqin</creatorcontrib><creatorcontrib>Liu, Hong</creatorcontrib><creatorcontrib>Zhao, Yuran</creatorcontrib><creatorcontrib>Liang, Chengzhu</creatorcontrib><creatorcontrib>Li, Ye</creatorcontrib><creatorcontrib>Shi, Xiujie</creatorcontrib><creatorcontrib>Wu, Bin</creatorcontrib><creatorcontrib>Xu, Biao</creatorcontrib><creatorcontrib>Deng, Mingjun</creatorcontrib><creatorcontrib>Zhu, Laihua</creatorcontrib><creatorcontrib>Wang, Zhe</creatorcontrib><title>Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR</title><title>Journal of virological methods</title><addtitle>J Virol Methods</addtitle><description>The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10
8 copies of HRV RNA. Regression analysis demonstrated a significant correlation with an
R
2 value of 0.9963 and a slope of −3.18 between the mean
C
t values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37–1.72% and 1.37–3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Birnavirus</subject><subject>Diagnostic assay</subject><subject>Fish Diseases - diagnosis</subject><subject>Fish Diseases - virology</subject><subject>Fishes</subject><subject>Freshwater</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hirame rhabdovirus</subject><subject>Infectious haematopoietic necrosis virus</subject><subject>Infectious pancreatic necrosis virus</subject><subject>Microbiology</subject><subject>Novirhabdovirus - genetics</subject><subject>Novirhabdovirus - isolation & purification</subject><subject>Quantitative RT-PCR</subject><subject>Reproducibility of Results</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - methods</subject><subject>Rhabdoviridae Infections - veterinary</subject><subject>Rhabdoviridae Infections - virology</subject><subject>Sensitivity and Specificity</subject><subject>TaqMan probe</subject><subject>Techniques used in virology</subject><subject>Viral hemorrhagic septicemia virus</subject><subject>Viral Load - methods</subject><subject>Virology</subject><issn>0166-0934</issn><issn>1879-0984</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUFvFCEUx4nR2G31KzRcjKdZYYCBuWlWrSZNNE09EwYeKZuZYQvMJP32Uner8dQT5PH7Px78ELqkZEsJ7T7st_s1pDhB2bakFonaEkpfoA1Vsm9Ir_hLtKlgV_eMn6HznPeEECEZe43OWiJbJjq2QeUzrDDGwwRzwWZ2GFYzLqaEOOPoscEZ5hxKWOHP6f1i5hKKORZyNg_Yx4TvQjIT4HRnBhfrXEvGg8ngcO3yX-Tmtvm5u3mDXnkzZnh7Wi_Qr69fbnffmusfV993n64by9u-NKyVjlDgwLtOeSepkJJw4XvFoGXG9YMjfOCkPkQacMKz3g2WS98q1VLn2AV6f-x7SPF-gVz0FLKFcTQzxCVrJTopZI0_S0oh-k7xnlSyO5I2xZwTeH1IYTLpQVOiH9XovX5Sox_VaKJ0VVODl6crlmEC9zf25KIC706AydaMPpnZhvyPY4ywKrRyH48c1K9bAySdbYDZggsJbNEuhudm-Q3YSLFa</recordid><startdate>20101101</startdate><enddate>20101101</enddate><creator>Sun, Yingjie</creator><creator>Yue, Zhiqin</creator><creator>Liu, Hong</creator><creator>Zhao, Yuran</creator><creator>Liang, Chengzhu</creator><creator>Li, Ye</creator><creator>Shi, Xiujie</creator><creator>Wu, Bin</creator><creator>Xu, Biao</creator><creator>Deng, Mingjun</creator><creator>Zhu, Laihua</creator><creator>Wang, Zhe</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7U9</scope><scope>F1W</scope><scope>H94</scope><scope>H95</scope><scope>L.G</scope></search><sort><creationdate>20101101</creationdate><title>Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR</title><author>Sun, Yingjie ; Yue, Zhiqin ; Liu, Hong ; Zhao, Yuran ; Liang, Chengzhu ; Li, Ye ; Shi, Xiujie ; Wu, Bin ; Xu, Biao ; Deng, Mingjun ; Zhu, Laihua ; Wang, Zhe</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c429t-327d01e4e4668fd71577045f983e23ad9bd04b405637aed5f39dbc47f28821dd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Birnavirus</topic><topic>Diagnostic assay</topic><topic>Fish Diseases - diagnosis</topic><topic>Fish Diseases - virology</topic><topic>Fishes</topic><topic>Freshwater</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hirame rhabdovirus</topic><topic>Infectious haematopoietic necrosis virus</topic><topic>Infectious pancreatic necrosis virus</topic><topic>Microbiology</topic><topic>Novirhabdovirus - genetics</topic><topic>Novirhabdovirus - isolation & purification</topic><topic>Quantitative RT-PCR</topic><topic>Reproducibility of Results</topic><topic>Reverse Transcriptase Polymerase Chain Reaction - methods</topic><topic>Rhabdoviridae Infections - veterinary</topic><topic>Rhabdoviridae Infections - virology</topic><topic>Sensitivity and Specificity</topic><topic>TaqMan probe</topic><topic>Techniques used in virology</topic><topic>Viral hemorrhagic septicemia virus</topic><topic>Viral Load - methods</topic><topic>Virology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sun, Yingjie</creatorcontrib><creatorcontrib>Yue, Zhiqin</creatorcontrib><creatorcontrib>Liu, Hong</creatorcontrib><creatorcontrib>Zhao, Yuran</creatorcontrib><creatorcontrib>Liang, Chengzhu</creatorcontrib><creatorcontrib>Li, Ye</creatorcontrib><creatorcontrib>Shi, Xiujie</creatorcontrib><creatorcontrib>Wu, Bin</creatorcontrib><creatorcontrib>Xu, Biao</creatorcontrib><creatorcontrib>Deng, Mingjun</creatorcontrib><creatorcontrib>Zhu, Laihua</creatorcontrib><creatorcontrib>Wang, Zhe</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Virology and AIDS Abstracts</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><jtitle>Journal of virological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sun, Yingjie</au><au>Yue, Zhiqin</au><au>Liu, Hong</au><au>Zhao, Yuran</au><au>Liang, Chengzhu</au><au>Li, Ye</au><au>Shi, Xiujie</au><au>Wu, Bin</au><au>Xu, Biao</au><au>Deng, Mingjun</au><au>Zhu, Laihua</au><au>Wang, Zhe</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR</atitle><jtitle>Journal of virological methods</jtitle><addtitle>J Virol Methods</addtitle><date>2010-11-01</date><risdate>2010</risdate><volume>169</volume><issue>2</issue><spage>391</spage><epage>396</epage><pages>391-396</pages><issn>0166-0934</issn><eissn>1879-0984</eissn><coden>JVMEDH</coden><abstract>The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10
8 copies of HRV RNA. Regression analysis demonstrated a significant correlation with an
R
2 value of 0.9963 and a slope of −3.18 between the mean
C
t values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37–1.72% and 1.37–3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples.</abstract><cop>Kidlington</cop><pub>Elsevier B.V</pub><pmid>20723563</pmid><doi>10.1016/j.jviromet.2010.08.011</doi><tpages>6</tpages></addata></record> |
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| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Animals Biological and medical sciences Birnavirus Diagnostic assay Fish Diseases - diagnosis Fish Diseases - virology Fishes Freshwater Fundamental and applied biological sciences. Psychology Hirame rhabdovirus Infectious haematopoietic necrosis virus Infectious pancreatic necrosis virus Microbiology Novirhabdovirus - genetics Novirhabdovirus - isolation & purification Quantitative RT-PCR Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction - methods Rhabdoviridae Infections - veterinary Rhabdoviridae Infections - virology Sensitivity and Specificity TaqMan probe Techniques used in virology Viral hemorrhagic septicemia virus Viral Load - methods Virology |
| title | Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR |
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