猪四种呼吸道病原菌四重PCR检测方法的建立与应用

Establishment and application of multiplex PCR method for detection of four respiratory bacterial pathogens of swine

为建立可同时快速检测猪支气管败血波氏杆菌（Bb）、副猪嗜血杆菌（Hps）、胸膜肺炎放线杆菌（App）和多杀性巴氏杆菌（Pm）的四重PCR方法,根据Bb的fla基因、Hps的16S rRNA基因、App的apxⅣ基因和Pm的特异性基因KMT1设计4对特异性引物,基于TaKaRa公司的Premix Taq PCR预混酶,对四重PCR的引物浓度和退火温度进行优化,确定该四重PCR的反应体系和反应条件,通过对特异性及同一种病原菌不同血清型检测的通用性、敏感性和重复性评价,建立快速检测猪这4种呼吸道病原菌的四重PCR方法,同时应用该方法对81份临床样本进行检测,检测结果与这4种病原菌单项PCR检测以及4种病原菌的分离鉴定结果进行比较。结果表明,该方法能同时对这4种病原菌进行检测,特异性和通用性好、重复性好,对这4种病原菌DNA的最低检测质量浓度为20 pg/L,对这4种细菌的最低检出量分别为1×10^3CFU/mL的Pm,1×10^2CFU/mL的Hps、Bb和App,敏感性高。81份临床样本的四重PCR检测结果和单项PCR检测结果一致,PCR检出率明显高于细菌分离鉴定结果。本研究成功建立了可同时检出猪支气管败血波氏杆菌、副猪嗜血杆菌、胸膜肺炎放线杆菌和多杀性巴氏杆菌的四重PCR方法,该方法可用于临床样本的快速检测以及这4种病原菌的鉴别检测。

To establish a multiplex PCR method for rapid detection of Bordetella bronchiseptica（Bb）,Haemophilus parasuis（Hps）,Actinobacillus pleuropneumoniae（App） and Pasteurella multocida（Pm）,four pairs of specific primers were designed according to the fla gene of B.bronchiseptica,the 16S rRNA gene of H.parasuis,the apx Ⅳ gene of A.pleuropneumoniae and the specific KMT1 gene of P.multocida.Based on TaKaRa Premix Taq,with the optimization of multiplex PCR amplification primer concentrations and annealing temperature,the multiplex PCR method was established to detect the four kinds of swine respiratory bacterial pathogens,and the specificity,the versatility of different serotypes of the same pathogen,as well as sensitivity and repeatability of the multiplex PCR were evaluated.Total 81 clinical samples were detected with the multiplex PCR,single PCR for the 4 bacterial pathogens and bacteria isolation and identification.This method can detect the four kinds of pathogens in the sample at the same time,the specificity,the versatility and the reproducibility were good,and the sensitivity was high.The detection concentration of the four kinds of pathogens was 20 pg/L,and the minimum detection concentration of Pm was 1 ×10~3 CFU/mL,Hps,Bb and App was 1 ×10~2 CFU/mL.The multiplex PCR results of 81 clinical samples were consistent with the single PCR results.The multiplex PCR method was developed for the simultaneous detection of B.bronchiseptica,H.parasuis,A.pleuropneumoniae and P.multocida,which can be used for rapid detection of clinical samples and identification of pathogens.