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Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3
BACKGROUND: The cocirculation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Ducklings with DHAV-1 or DHAV-3 infection show similar clinical signs and gross lesions; hence, it is important to identify the viral subtypes in infec...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675107/ https://www.ncbi.nlm.nih.gov/pubmed/31369566 http://dx.doi.org/10.1371/journal.pone.0219750 |
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author | Chen, Xueming Chen, Yuhuan Liu, Chungguo Li, Xiaojun Liu, Hongyu Yin, Xiuchen Bai, Xiaofei Ge, Ming Chen, Hongyan Liu, Ming Du, Yuanzhao Fan, Gencheng Zhang, Yun |
author_facet | Chen, Xueming Chen, Yuhuan Liu, Chungguo Li, Xiaojun Liu, Hongyu Yin, Xiuchen Bai, Xiaofei Ge, Ming Chen, Hongyan Liu, Ming Du, Yuanzhao Fan, Gencheng Zhang, Yun |
author_sort | Chen, Xueming |
collection | PubMed |
description | BACKGROUND: The cocirculation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Ducklings with DHAV-1 or DHAV-3 infection show similar clinical signs and gross lesions; hence, it is important to identify the viral subtypes in infected ducklings as early as possible for better clinical management. METHODS AND RESULTS: Based on multiple 5’ noncoding region (5’-NCR) sequences of DHAV-1 and DHAV-3 strain alignments, universal and type-specific primers were designed and synthesized. With three primers in one-tube reverse transcription-PCR (RT-PCR), reference DHAV-1 and DHAV-3 isolates ranging over 60 years and across many different countries were successfully amplified, indicating that the primer sequences were completely conserved. The sequence results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates are completely correlated with their subtypes. Moreover, with this one-tube RT-PCR system, amplicon sizes from liver samples of reference DHAV-1- or DHAV-3-infected birds fit closely with their subtypes, which was determined by virus isolation and neutralization testing. No other duck-origin RNA viruses were detected. The sensitivity of viral RNA detection was 10 pg. With this system, 20% subtype 1, 45% subtype 3, and 9% coinfection of two subtypes were detected in 55 clinical samples. CONCLUSIONS AND SIGNIFICANCE: This novel approach could be used for rapidly typing DHAV-1 or DHAV-3 infection in routine clinical surveillance or epidemiological screening. |
format | Online Article Text |
id | pubmed-6675107 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-66751072019-08-06 Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3 Chen, Xueming Chen, Yuhuan Liu, Chungguo Li, Xiaojun Liu, Hongyu Yin, Xiuchen Bai, Xiaofei Ge, Ming Chen, Hongyan Liu, Ming Du, Yuanzhao Fan, Gencheng Zhang, Yun PLoS One Research Article BACKGROUND: The cocirculation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Ducklings with DHAV-1 or DHAV-3 infection show similar clinical signs and gross lesions; hence, it is important to identify the viral subtypes in infected ducklings as early as possible for better clinical management. METHODS AND RESULTS: Based on multiple 5’ noncoding region (5’-NCR) sequences of DHAV-1 and DHAV-3 strain alignments, universal and type-specific primers were designed and synthesized. With three primers in one-tube reverse transcription-PCR (RT-PCR), reference DHAV-1 and DHAV-3 isolates ranging over 60 years and across many different countries were successfully amplified, indicating that the primer sequences were completely conserved. The sequence results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates are completely correlated with their subtypes. Moreover, with this one-tube RT-PCR system, amplicon sizes from liver samples of reference DHAV-1- or DHAV-3-infected birds fit closely with their subtypes, which was determined by virus isolation and neutralization testing. No other duck-origin RNA viruses were detected. The sensitivity of viral RNA detection was 10 pg. With this system, 20% subtype 1, 45% subtype 3, and 9% coinfection of two subtypes were detected in 55 clinical samples. CONCLUSIONS AND SIGNIFICANCE: This novel approach could be used for rapidly typing DHAV-1 or DHAV-3 infection in routine clinical surveillance or epidemiological screening. Public Library of Science 2019-08-01 /pmc/articles/PMC6675107/ /pubmed/31369566 http://dx.doi.org/10.1371/journal.pone.0219750 Text en © 2019 Chen et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Chen, Xueming Chen, Yuhuan Liu, Chungguo Li, Xiaojun Liu, Hongyu Yin, Xiuchen Bai, Xiaofei Ge, Ming Chen, Hongyan Liu, Ming Du, Yuanzhao Fan, Gencheng Zhang, Yun Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3 |
title | Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3 |
title_full | Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3 |
title_fullStr | Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3 |
title_full_unstemmed | Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3 |
title_short | Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3 |
title_sort | improved one-tube rt-pcr method for simultaneous detection and genotyping of duck hepatitis a virus subtypes 1 and 3 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675107/ https://www.ncbi.nlm.nih.gov/pubmed/31369566 http://dx.doi.org/10.1371/journal.pone.0219750 |
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