副猪嗜血杆菌和猪鼻支原体双重PCR检测方法的建立及应用

Establishment and application of duplex PCR assay for detection of Haemophilus parasuis and Mycoplasma hyorhinis

为建立一种特异性鉴别副猪嗜血杆菌(Hps)和猪鼻支原体(Mhr)的检测方法,对Hps的P2蛋白基因和Mhr的P37蛋白基因分别设计了1对引物,建立Hps和Mhr双重PCR检测方法,并进行特异性和灵敏度试验,同时应用所建立方法对临床样品进行检测。结果显示,该方法可特异性检测出Hps和Mhr,对其他细菌无非特异性扩增,检测灵敏度可达到100 copies;并可根据扩增产物的大小区分Hps P2蛋白基因的Ⅰ和Ⅱ两个基因型,初步判定Hps的致病性。对32份呼吸道疾病临床样品的检测结果显示,Hps和Mhr阳性率分别为59.4%和53.1%,双重感染的样品为34.4%。表明,成功建立了一种灵敏、特异和快速的双重PCR方法,实现了Hps和Mhr的同时、快速、准确诊断。

To establish a specific assay for simultaneous detection and differentiation of Haemophilus parasuis and Mycoplasma hyorhinis, a duplex PCR detection method for Hps and Mhr was established.Primers were designed based on Hps P2 protein gene and Mhr P37 protein gene, Clinical samples were detected by duplex PCR after specificity and sensitivity experiments. The resuls showed that duplex PCR could detecte Hps and Mhr specificly, without nonspecific amplification, the sensitivity of the assay was 100 copies/reaction. Besides, GenotypeⅠ and GenotypeⅡ of Hps P2 gene could be distinguished according to the size of amplification products, determining the pathogenicity of Hps.Detection of 32 clinical samples of respiratory disease showed that the Hps and Mhr positive rate was 59.4% and 53.1%,repectively, and the infection rate of samples infected by two pathogen was 34.4%. Conclusion： A sensitive, specific and rapid PCR method was successfully established for accurate diagnosis of Hps and Mhr,simultaneously and rapidly.