Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3

PloS One
Xueming ChenYun Zhang

Abstract

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. 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 sys...Continue Reading

References

Feb 13, 2007·Virus Research·Chun-Hsien Tseng, Hsiang-Jung Tsai
May 10, 2007·Molecular Biology and Evolution·Koichiro TamuraSudhir Kumar
Jun 7, 2011·Veterinary Microbiology·Ming LiuYun Zhang
Feb 23, 2012·Journal of Virology·Ming LiuYun Zhang
Aug 3, 2012·Journal of Clinical Microbiology·Qiuxue HuangCheng Tang
Oct 2, 2013·Veterinary Microbiology·Chun-Guo LiuYun Zhang
Aug 12, 2014·Journal of Virological Methods·Tingting ZhangYun Zhang
Oct 4, 2016·Journal of Infection in Developing Countries·Huong Thi Thanh DoanThanh Hoa Le

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Methods Mentioned

BETA
genotyping
PCR
amplicon sequencing
electrophoresis
PCRs

Software Mentioned

TreeView
MegAlign
LASERGENE
MEGA
CLUSTAL W

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