PRRSV、SS和Pm多重TaqMan荧光定量PCR检测方法的建立与初步应用
Establishment and preliminary application of PRRSV,SS and Pm multiplex Taq-Man real-time PCR detection methods
詹存林 1王庚 2孙秀秀 1冯贺龙 1林正丹 1胡薛英 1谷长勤 1张万坡 1陶攀 1陈品 1钱平 1曹胜波 1张伟超 2程国富1
作者信息
- 1. 华中农业大学 动物医学院,湖北 武汉 430000
- 2. 广西扬翔股份有限公司,广西贵港 537000
- 折叠
摘要
为了建立一种可同时检测并鉴别猪繁殖和呼吸综合征病毒(PRRSV)、猪链球菌(SS)和猪多杀性巴氏杆菌(Pm)多重TaqMan荧光定量PCR检测方法,本试验通过MEGA7.0分析NCBI上已登录的3种病原体基因序列,分别根据PRRSV的ORF6基因、SS的GDH基因和Pm的KMT1基因设计出特异性引物和探针,并对反应条件进行优化.结果显示,该方法能够特异性地检测PRRSV、SS和Pm,对其他病原无交叉反应;对PRRSV、SS和Pm的最低检测浓度分别为9.93×102、1.10×102和1.07× 102拷贝/μL;组内和组间变异系数均小于1.85％.结果表明,该方法特异性强、灵敏性高、稳定性好,可应用于临床检测.
Abstract
In order to establish a multiplex TaqMan PCR detection method that can simultaneously detect and identify porcine reproductive and respiratory syndrome virus(PRRSV),Streptococcus suis(SS)and Pasteurella multocida(Pm),the gene sequences of three pathogens registered on NCBI were analyzed by MEGA7.0,and specific primers and probes were designed according to the ORF6 gene of PRRSV,GDH gene of SS and KMT1 gene of Pm,respectively,and the reaction con-ditions were optimized.This method can specifically detect PRRSV,SS and Pm,and has no cross-reactivity against other pathogens.The minimum detection copy numbers for PRRSV,SS and Pm were 9.93×102 copies/μL,1.10 × 102 copies/μL and 1.07×102 copies/μL,respectively.The coeffi-cient of variation was less than 1.85％both within and between groups.The results show that the method has strong specificity,high sensitivity and good stability,and can be applied to clinical de-tection.
关键词
猪繁殖和呼吸综合征病毒/猪链球菌/猪多杀性巴氏杆菌/多重TaqMan荧光定量PCRKey words
PRRSV/SS/Pm/multiplex TaqMan real-time PCR引用本文复制引用
基金项目
中央高校基本科研业务费专项(140422008)
扬翔股份有限公司重大创新变革基金(YXNM-R&D2022-02)
出版年
2024
NETL
NSTL
万方数据