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OA

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.