兔源A型多杀性巴氏杆菌双重PCR检测方法的建立  

Development of duplex PCR assay for detecting Pasteurella multocida serogroup A strain in rabbits

作　　者：王锦祥[1,2] 付环茹 孙世坤 陈冬金[1,2] 高承芳 桑雷[1,2] 谢喜平 Wang Jinxiang;Fu Huanru;Sun Shikun;Chen Dongjin;Gao Chengfang;Sang Lei;Xie Xiping(Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agriculture Sciences,Fuzhou350013;Fujian Key Laboratory of Animal Genetics and Breeding,Fuzhou350013)

机构地区：[1]福建省农业科学院畜牧兽医研究所,福州350013 [2]福建省畜禽遗传重点实验室,福州350013

出　　处：《福建畜牧兽医》2023年第5期20-23,共4页Fujian Journal of Animal Husbandry and Veterinary medicine

基　　金：福建省科技计划公益类专项(2022R10260012);福建省农业科学院科技创新团队建设项目(CXTD2021007-2);福建省农业高质量发展超越“5511”协同创新工程项目(XTCXGC2021008);国家兔产业技术体系(CARS-43-G-5)资助。

摘　　要：为建立一种特异性强且敏感性高的兔源A型多杀性巴氏杆菌快速检测方法,本研究以兔源A型多杀性巴氏杆菌kmt1和hyaD基因为目的基因,设计2对特异性引物进行双重PCR扩增,经反应条件优化,建立检测兔源A型多杀性巴氏杆菌的双重PCR方法。该方法的最佳退火温度为57.8℃,最佳混合引物浓度为0.8μmol/L。该方法特异性强,对兔源A型多杀性巴氏杆菌为kmt1和hyaD基因双阳性,对兔源D型和F型多杀性巴氏杆菌为kmt1基因单阳性,对兔源其它细菌性病原和阴性对照则均为阴性。该方法敏感性高,对兔源A型多杀性巴氏杆菌kmt1和hyaD基因的最低检出限分别为1×10^(3)拷贝/μL和1×10^(4)拷贝/μL。该方法重复性好,且与已报道的多重PCR方法的符合率高达96.12%。本研究建立的双重PCR方法对兔源A型多杀性巴氏杆菌具有良好的特异性和敏感性,且重复性好、准确性高,为该病原的快速检测提供了有力的技术支持。To develop a specific and sensible assay for the rapid detection of Pasteurella multocida(P.multocida)serogroup A strain in rabbits.Two sets of specific primers targeting the genes kmt1 and hyaD of P.multocida serogroup A strain were designed,the reaction conditions were then optimized and a duplex PCR assay for the detection of P.multocida serogroup A strain in rabbits was developed.The results showed that the optimum reaction conditions including the annealing temperature and concentration of the primers were 57.8℃and 0.8μmol/L,respectively.The assay was specificity,which amplified both the genes kmt1 and hyaD from rabbit sourced P.multocida serogroup A strain and the gene kmt1 from rabbit sourced P.multocida serogroup D and F strains,and had no cross-reactions with the other bacterial pathogens and negative control.The assay was highly sensitivity,the detection limit for the genes kmt1 and hyaD were 1×10^(3) copies/μL and 1×10^(4) copies/μL,respectively.The assay was repeatable,and with the high accuracy of up to 96.12%compared to the reported multiplex PCR assay.The duplex PCR assay was specific and sensible for the genes kmt1 and hyaD of rabbit sourced P.multocida serogroup A strain.Moreover,the assay was repeatable and highly accuracy,which provides a potential technical support for the rapid detection of P.multocida serogroup A strain in rabbits.

关 键 词：兔 A型多杀性巴氏杆菌 kmt1基因 hyaD基因 双重PCR 

分 类 号：S852.61[农业科学—基础兽医学]