肉牛呼吸道感染主要细菌性病原多重PCR检测方法的建立

doi:10.3969/j.issn.1004-1524.2020.02.04

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (2): 210-217.DOI: 10.3969/j.issn.1004-1524.2020.02.04

肉牛呼吸道感染主要细菌性病原多重PCR检测方法的建立

张玉龙, 马志宇, 崔耀成, 谭天宇, 姚彩霞, 樊利虹, 左之才^*, 才冬杰^*

四川农业大学动物医学院动物疫病与人类健康四川省重点实验室,四川雅安 625014

收稿日期:2019-09-16 出版日期:2020-02-25 发布日期:2020-03-13
通讯作者: *左之才,E-mail:zzcjl@126.com;才冬杰,E-mail:18811782766@163.com
作者简介:张玉龙(1991—),男,河南商丘人,硕士研究生,研究方向为中西兽医与临床。E-mail:YULO_ZH@163.com
基金资助:
国家重点研发计划(2018YFD0501800); 四川省科技计划(2018NZ0002); 国家现代农业产业技术体系四川肉牛创新团队项目(035Z2036)

Establishment of multiplex PCR assay for major bacterial pathogens in respiratory tract infection in beef cattle

ZHANG Yulong, MA Zhiyu, CUI Yaocheng, TAN Tianyu, YAO Caixia, FAN Lihong, ZUO Zhicai^*, CAI Dongjie^*

Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China

Received:2019-09-16 Online:2020-02-25 Published:2020-03-13

摘要/Abstract

摘要： 为了建立一种能同时检测肺炎克雷伯菌(Klebsiella pneumoniae)、约翰逊不动杆菌(Acinetobacter johnsonii)、多杀性巴氏杆菌(Pasteurella multocida)这3种肉牛呼吸道病原菌的多重PCR检测方法,采用肺炎克雷伯菌Khe基因、约翰逊不动杆菌Ptk基因、多杀性巴氏杆菌Abhd基因设计特异性引物,经过特异性、敏感性、引物浓度、退火温度试验,建立多重PCR检测方法的反应条件和体系。结果显示,该方法能同时扩增出肺炎克雷伯菌、约翰逊不动杆菌和多杀性巴氏杆菌,3种目的菌扩增测序与GenBank上的细菌基因序列相似性均高于99%,对其他10种肉牛呼吸道病原菌扩增结果均为阴性。对3种病原基因组DNA的检测下限分别为69.8×10^-5、208.9×10^-4、70.8×10^-3 ng·μL^-1,最佳引物浓度比例为1:1:1,最佳的退火温度为58 ℃。该方法特异性强、敏感性高,为上述3种病原的快速检测、鉴定和临床牛呼吸道疾病的诊断提供了一种方便、快捷和准确的工具。

关键词: 肉牛呼吸道, 肺炎克雷伯菌, 约翰逊不动杆菌, 多杀性巴氏杆菌, 多重PCR

Abstract: To establish a multiplex PCR assay for simultaneous detection of Klebsiella pneumoniae, Acinetobacter johnsonii, Pasteurella multocida of beef cattle, specific primers were designed based on Khe gene of Klebsiella pneumoniae, Ptk gene of Acinetobacter johnsonii, and Abhd gene of Pasteurella multocida. The reaction conditions and system of the multiplex PCR detection method were established by specificity test, sensitivity test, primer concentration test and annealing temperature test. The results showed that the method can simultaneously amplify Klebsiella pneumoniae, Acinetobacter johnsonii and Pasteurella multocida, and the similarity between bacterial sequences of three kinds of target bacteria amplification and sequencing on GenBank were more than 99%. Meanwhile, the amplification results of the other 10 kinds of beef respiratory pathogens were all negative. The detection limits of the above three pathogen genomic DNA were 69.8×10^-5, 208.9×10^-4, 70.8×10^-3 ng·μL^-1, and the optimal primer concentration ratio was 1:1:1. The optimum annealing temperature was 58 ℃. The method had a strong specificity and high sensitivity, and provided a convenient, rapid and accurate tool for rapid detection and identification of the above three pathogens and diagnosis of clinical bovine respiratory diseases.

Key words: beef cattle respiratory tract, Klebsiella pneumoniae, Acinetobacter johnsonii, Pasteurella multocida, multiplex PCR

中图分类号:

S858.23

张玉龙, 马志宇, 崔耀成, 谭天宇, 姚彩霞, 樊利虹, 左之才, 才冬杰. 肉牛呼吸道感染主要细菌性病原多重PCR检测方法的建立[J]. 浙江农业学报, 2020, 32(2): 210-217.

ZHANG Yulong, MA Zhiyu, CUI Yaocheng, TAN Tianyu, YAO Caixia, FAN Lihong, ZUO Zhicai, CAI Dongjie. Establishment of multiplex PCR assay for major bacterial pathogens in respiratory tract infection in beef cattle[J]. , 2020, 32(2): 210-217.

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肉牛呼吸道感染主要细菌性病原多重PCR检测方法的建立

Establishment of multiplex PCR assay for major bacterial pathogens in respiratory tract infection in beef cattle

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