多重PCR方法快速检测猪的5种呼吸道病原菌  

Development of a Rapid Multiplex PCR for Simultaneous Detection of Five Major Pathogenic Bacteria of Respiratory Tract Diseases in Swine

作　　者：薛云[1,2,1] 赵战勤[1,2,1] 陈杨[1] 邹浩勇[1] 何启盖[1] 李文洁[1] 熊金凤[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院农业微生物学国家重点实验室,武汉430070 [2]河南科技大学动物科技学院动物病原微生物学实验室,洛阳471003

出　　处：《畜牧兽医学报》2009年第8期1222-1228,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技支撑计划项目(2006BAD06A12)

摘　　要：猪胸膜肺炎放线杆菌(App)、副猪嗜血杆菌(Hps)、支气管败血波氏杆菌(Bb)、产毒性多杀巴氏杆菌(T+Pm)和猪肺炎支原体(Mhp)是最常见的几种猪呼吸道病原菌。笔者参照文献报道的基因序列设计针对App、Hps、Bb、T+Pm和Mhp的特异性引物,对单一PCR条件进行系统优化后建立5重PCR方法。该多重PCR方法能对同一样品中5种病原菌的DNA模板进行扩增,且在5种病原菌之间没有交叉反应;对大肠杆菌等其它6种常见猪细菌性病原的检测结果均为阴性;对5种病原菌DNA模板检出的最小量均低于160 pg,且能从攻毒小鼠(App、Hps、Bb和T+Pm)肺脏组织8 h增菌的培养物中快速检测出相应的病原菌。对中国华中地区269份临床样本的检测结果表明,5种呼吸道病原菌在华中地区猪群中普遍存在,且混合感染情况十分严重。这些试验结果表明该多重PCR方法可用于猪呼吸道病原菌App、Hps、Bb、T+Pm和Mhp的单一或混合感染的鉴别诊断及病原流行病学调查。Actinobacillus pleuropneumoniae （App）, Haernophilus parasuis （Hps）, Bordetella bronchiseptica （Bb）, Toxigenic Pasteurella rnultocida （T^＋ Pro） and Mycoplasrna hyopneumoniae （Mhp） are five kinds of the most common pathogenic bacteria which can cause a series of respiratory tract diseases in swine. Five pairs of specific oligonucleotide primers were designed according to the DNA sequences of App apxIV, Hps 16S rRNA, Bb fla, toxigenic T＋Pm toxA and Mhp ldh reported in NCBI website. A multiple PCR detection method was established according to improved systems and programmes on the basis of five single PCR methods. No cross reaction was detected by the multiplex PCR method between these five bacteria templates and other common bacteria templates. The sensitivity of multiple PCR was lower than 160 pg genome template.Using this multiple PCR method, bacteria could be detected from lung tissues of challenged mice with App, Hps, Bb and T^＋Pm respectively after being cultured for 8 h in Tryptic Soy Broth （TSB） supplemented with 5% calf serum and NAD^＋ factor. Simultaneous detection of pathogens in 269 clinical samples from the central area of China revealed the universal existence of App, Hps, Bb, T^＋Pm and Mhp. In conclusion, the multiple PCR assay showed excellent specificity, sensitivity and reduplication and could detect five pathogenic bacteria of respiratory tract in swine. We expected that this multiple PCR assay could be useful extensively for epidemiological survey and clinical differential diagnosis of five common respiratory diseases in swine.

关 键 词：猪 呼吸道病原 多重PCR 检测 

分 类 号：S858.285.1[农业科学—临床兽医学] S852.61[农业科学—兽医学]