H10亚型禽流感病毒TaqMan探针实时荧光定量PCR检测方法的建立
Development of a TaqMan probe real-time fluorescence quantitative PCR method for detection of H10 subtype avian influenza virus
张雅馨 1毛秋艳 2刘朔 3周婉婷 2周淑宁 4彭程 3李金平 3刘华雷 3蒋文明 3格日勒图1
作者信息
- 1. 内蒙古农业大学兽医学院,内蒙古呼和浩特 010018
- 2. 浙江农林大学动物科技学院/动物医学院,浙江杭州 311300
- 3. 中国动物卫生与流行病学中心,山东青岛 266032
- 4. 青岛农业大学动物医学院,山东青岛 266109
- 折叠
摘要
从NCBI和GISAID数据库中下载H10亚型禽流感病毒的HA基因序列信息,与本实验室保存毒株的序列进行比对、分析、筛选,选择其共同保守区域设计荧光引物和探针,优化反应体系和条件,并验证该方法的特异性、敏感性和重复性.结果显示,该方法具有高度特异性,与其他几种禽源性病毒均无交叉反应;敏感性可达到1.7 ×102拷贝/μL,比普通RT-PCR敏感10倍;批内和批间重复的变异系数(Cv)均小于1.4％,稳定性较好;分别使用鸡胚接种病毒分离法、本研究建立的TaqMan探针实时荧光定量PCR和普通RT-PCR检测鸡感染试验的拭子和组织样品,结果显示病毒分离法与本研究建立的实时荧光定量PCR方法的总符合率为94.44％,而与普通RT-PCR方法的总符合率为90.74％,实时荧光定量PCR的阳性检出率比RT-PCR更高.结果表明,建立的TaqMan探针实时荧光定量PCR检测方法具有较好的特异性、敏感性和稳定性,有助于H10亚型禽流感病毒的临床检测和监测,对其防控具有一定意义.
Abstract
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.
关键词
H10亚型禽流感病毒/TaqMan探针/实时荧光定量PCR/HA基因Key words
H10 subtype avian influenza virus/TaqMan probe/real-time fluorescence quantitative PCR/HA gene引用本文复制引用
基金项目
山东省重点研发计划(2022CXGC010606)
出版年
2024
NETL
NSTL
万方数据