B型禽偏肺病毒TaqMan实时荧光定量PCR方法的建立及应用  

Development and Application of a TaqMan Fluorescence Real-time Quantitative PCR Method for Avian Metapneumovirus Subtype B

作　　者：沈海强 车艳杰 宋新宇 高冉 付旭彬 金天明 SHEN Haiqiang;CHE Yanjie;SONG Xinyu;GAO Ran;FU Xubin;JIN Tianming(Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300392;Tianjin RingPu Bio-Technology Co.,Ltd.,Tianjin 300308)

机构地区：[1]天津农学院动物科学与动物医学学院,天津市农业动物繁育与健康养殖重点实验室,天津300392 [2]天津瑞普生物技术股份有限公司,天津300308

出　　处：《中国家禽》2024年第9期121-127,共7页China Poultry

摘　　要：试验旨在开发一种特异性检测B亚型禽偏肺病毒(aMPV-B)的方法。研究参照GenBank中的aMPV-G基因序列设计一对特异性引物,建立aMPV-B TaqMan荧光定量PCR方法,优化该方法的反应条件,建立标准曲线,进行特异性、重复性和敏感性试验,并将该方法用于临床样品的检测。结果显示:建立的TaqMan荧光定量PCR方法只能检测出B亚型aMPV,其敏感性能达到1.34×10^(1) copies/µL,斜率为-3.346,截距为40.01,相关系数(R2)为1.00,循环阈值(Ct)与模板拷贝数之间呈现良好的相关性;临床样品检测结果显示,该方法检测出18份样品阳性,而普通PCR仅检测出10份阳性。综上所述,建立的aMPV-B TaqMan荧光定量PCR方法在样品检测中表现出良好的特异性和较高的敏感性,适用于临床样品的aMPV-B检测。The aim of this study was to develop a method for the specific detection of avian metapneumovirus subtype B(aMPV-B).The study was conducted to design a pair of specific primers according to the aMPV-G gene sequence published in GenBank and to establish a TaqMan fluorescence quantitative PCR method for aMPV-B.The reaction conditions of the method were optimized,a standard curve was established,and specificity,reproducibility and sensitivity tests were performed.The method was used for the detection of clinical samples.The results showed that the established TaqMan real-time fluorescent quantitative PCR method could only detect the B subtype of aMPV,and its sensitivity could reach 1.34×10^(1) copies/µL,with a slope of-3.346,an intercept of 40.01,a correlation coefficient(R2)of 1.00,and a good correlation between the cycling threshold(Ct)value and the template copy number.The results of the clinical samples showed that 18 positive copies were detected the established method,while only 10 positive copies were detected by common PCR.In conclusion,the aMPV-B TaqMan fluorescent quantitative PCR method showed higher specificity and sensitivity in sample detection,which was suitable for the aMPV-B detection of clinical sample.

关 键 词：B型禽偏肺病毒 G基因 实时荧光定量PCR TapMan荧光探针 

分 类 号：S855.3[农业科学—临床兽医学]