中国兽医杂志2026，Vol.62Issue(5)：45-50,6.DOI:10.20157/j.cnki.zgsyzz.2026.05.006
H10亚型禽流感病毒TaqMan探针实时荧光定量反转录PCR检测方法的建立和应用
Establishment and Application of a TaqMan Probe-Based Real-Time Fluorescent Quantitative Reverse Transcription PCR Method for Detection of H10 Subtype Avian Influenza Virus
摘要
Abstract
To establish a method capable of simultaneously detecting the North American and Eurasian lineages of H10 subtype avian influenza virus(AIV),primers and probes targeting the hemagglutinin(HA)gene of H10 subtype AIV were designed.Reaction conditions were optimized to develop a real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-qPCR)assay for simultaneous detection of the two lineages.Standard curves were constructed,and the specificity,sensitivity,and repeatability of the method were evaluated,followed by testing of clinical samples.The results showed that optimal performance was achieved with primer and probe concentrations of 0.40 and 0.20 μmol/L,respectively,and an annealing temperature of 59℃.The established RT-qPCR assay exhibited good linearity for detecting Eurasian-and North American-lineage H10 subtype AIV(R²=1.000 and R²=0.999,respectively).The method demonstrated high specificity,with no cross-reactivity with other AIV subtypes or other common avian viruses.The limits of detection for the Eurasian and North American lineages of H10 subtype AIV were 5.58×10² and 1.57×10² copies/μL,respectively.The coefficients of variation for both intra-assay and inter-assay repeatability tests were less than 2%,indicating good repeatability.Among 36 clinical samples of H10 subtype AIV,34 Eurasian-lineage and 2 North American-lineage samples tested positive.These results indicate that the TaqMan probe-based RT-qPCR assay established in this study has good specificity,sensitivity,and repeatability,enabling simultaneous detection of North American and Eurasian lineages of H10 subtype AIV.This method will help improve clinical detection and surveillance of H10 subtype AIV.关键词
H10亚型禽流感病毒(AIV)/北美谱系/欧亚谱系/TaqMan探针/实时荧光定量反转录PCR/HA基因Key words
H10 subtype avian influenza virus(AIV)/North American lineage/Eurasian lineage/TaqMan probe/real-time fluorescent quantitative reverse transcription PCR/HA gene分类
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周婉婷,张雅馨,彭程,刘朔,杨吉喆,李金平,侯广宇,刘华雷,蒋文明..H10亚型禽流感病毒TaqMan探针实时荧光定量反转录PCR检测方法的建立和应用[J].中国兽医杂志,2026,62(5):45-50,6.基金项目
"十四五"国家重点研发计划(2021YFD1800201) （2021YFD1800201）
