Development of a TaqMan probe real-time fluorescence quantitative PCR method for detection of H10 subtype avian influenza virus
This study aims to establish a faster,specific,and sensitive real-time fluorescence quanti-tative PCR detection method to strengthen the monitoring and prevention and control of H10 sub-type avian influenza virus.The HA gene sequences of H10 subtype avian influenza viruses were downloaded from NCBI and GISAID databases,and then compared,analyzed,and screened with the sequences of the strains stored in our laboratory.The fluorescent primers and a probe were de-signed by selecting the common conservative regions of these sequences.The reaction system and conditions were optimized,and the specificity,sensitivity and repeatability of the method were veri-fied.The results showed that this method had high specificity and no cross reaction with other avi-an viruses.The sensitivity of real-time fluorescence quantitative PCR was 1.7× 102 copies/μL,which was 10 times more sensitive than ordinary RT-PCR.The coefficients of variation(Cv)of in-tra-and inter-assay replicates were less than 1.4％,indicating good stability.The swabs and tissue samples of chicken infection test were simultaneously detected by virus isolation,TaqMan probe real-time fluorescent quantitative PCR and ordinary RT-PCR,the results showed that the total co-incidence rate of virus isolation method with the real-time fluorescent quantitative PCR method es-tablished in this study was 94.44％,while the total coincidence rate with the ordinary RT-PCR method was 90.74％;the positive detection rate of real-time fluorescent quantitative PCR was higher than that of ordinary RT-PCR.The results indicated that the established TaqMan probe re-al-time fluorescence quantitative PCR detection method has good specificity,sensitivity,and stabil-ity,which is helpful for the clinical detection and monitoring of H10 subtype avian influenza viru-ses and has certain significance for its prevention and control.
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