副猪嗜血杆菌荧光定量PCR的建立和临床应用

Development and Clinical Application of Quantitative Fluorescence PCR for Detection of Hamophilus parasuis

为了检测血液、鼻拭子、组织等样本中副猪嗜血杆菌的含量,本研究根据GenBank副猪嗜血杆菌不同血清型的保守区设计引物,建立了SYBR GreenⅡ荧光定量PCR方法,并对其敏感性、特异性、重复性进行了分析。结果显示:所建立的SYBR GreenⅡ荧光定量PCR的组间差异性小于4%,组内差异性小于2.5%;溶解曲线峰值单一,特异性良好;其敏感性是普通PCR的100倍,且明显优于细菌分离方法。该方法可用于副猪嗜血杆菌感染早期的诊断和流行性调查。

In the present study,primers specifi c for different serotypes of Haemophilus parasuis were designed based on the reference strains in GenBank for development of a SYBR GreenⅡfl uorescence quantitative PCR method.Subsequently,this PCR method was used to test H.parasuis in nasal swabs,blood and tissue samples collected from experimentally infected pigs.The sensitivity,specifi city and reproducibility of this method were analyzed for its optical performance.The intra-variability of this assay was less than 4%and the inter-variability was less than 2.5%.The assay was highly specifi c for its single dissolution curve and 100 times sensitive as compared with conventional PCR.Therefore,this PCR method performed signifi cantly better than classical bacterial isolation method and could be used for early diagnosis and epidemiological investigation of H.parasuis infection.