副猪嗜血杆菌荧光定量PCR方法的建立及初步应用

Establishment and Preliminary Application of Fluorescence Quantitative PCR Assay for Detection of Haemophilus parasuis

为建立一种快速、敏感、特异的副猪嗜血杆菌(Haemophilus parasuis,HPS)荧光定量PCR(Fluorescence Quantitative PCR,FQ-PCR)检测方法,根据Gen Bank登录的HPS 16S rRNA基因序列,设计1对特异性引物和1条特异性Taq Man-MGB探针,建立了HPS荧光定量PCR检测方法,并对方法的敏感性、特异性以及重复性和稳定性进行了验证。结果显示:本研究建立的HPS FQ-PCR方法在10~1~10~6拷贝/μL模板范围内有很好的线性关系,所得相关系数为0.998;检测灵敏度可达10拷贝/μL,是常规PCR的10倍;特异性好,对pGEM-T/HPS重组质粒扩增呈现阳性反应曲线,而对4个对照病原的扩增曲线均呈现阴性反应;对不同浓度的pGEM-T/HPS重组质粒分别重复扩增3次,重复结果良好。对30份临床疑似HPS感染的组织样品进行应用检测,检出19份阳性,比商品化的HPS FQ-PCR检测试剂盒及常规PCR方法的阳性检出率高。结果表明,本研究建立的HPS FQ-PCR方法具有敏感性高、特异性好、稳定性强的优点,可用于临床HPS感染猪的早期检测,对HPS的快速诊断、综合防控具有重要意义。

In order to develop a rapid,sensitive and specific fluorescence quantitative PCR method（FQ-PCR）for detection of Haemophilus parasuis（HPS）,based on the sequence of 16S rRNA of HPS published in GenBank,a pair of specific primers and a TaqMan-MGB probe were designed,and the FQ-PCR method was established. Then its sensitivity,specificity and repeatability were tested. The results indicated that the FQ-PCR assay was successfully established,and it showed a good linear relationship at the template range of 101 to106 copies/μL,with a coefficient correlation of 0.998; the lowest detection limit value was 10 copies/μL,which was 10 times than that of conventional PCR method; amplification of recombinant pGEM-T/HPS plasmid showed positive results,while amplification results of other four control pathogens were all negative. Using the established method to conduct repeated amplification towards different concentrations of pGEM-T/HPS plasmid for 3 times,the results were stable. Meanwhile,the method was applied in clinic to test 30 suspected tissue samples,and 19 samples were detected positive,the detection rate of the method was higher than that of commercial detection kits and conventional PCR. As a conclusion,the established HPS FQ-PCR assay was sensitive,specific and stable,it was suitable for early detection of HPS and had great significance in rapid diagnosis and comprehensive prevention and control of HPS.