兔泰泽病原体实时荧光PCR检测方法的建立

Establishment of Real-time PCR Method for Diagnosis of Tyzzer's Organism in Rabbits

目的 建立一种适用于兔泰泽病原体检测的实时荧光PCR检测方法。方法 根据泰泽病原体的16S rRNA序列设计了特异性的引物和探针,对引物和探针浓度进行了优化,并评价其敏感性、特异性和重复性。同时,使用该方法对临床样品进行了检测,并与已报道的套式PCR方法进行比较。结果 建立了能够特异检测泰泽病原体的实时荧光PCR检测方法,该方法检测质粒标准品的最低检测限为8 copies/μL,不与SPF级实验动物必须排除的多种细菌发生交叉反应,批内和批间重复变异系数均小于3%,重复性良好。对70份临床样品的检测结果显示该方法的阳性样品检出率高于套式PCR检测方法。结论 建立的实时荧光PCR方法特异性好、灵敏度高、重复性好,适用于兔粪球样本中泰泽病原体的检测。

Objective To establish a real-time PCR method for diagnosis of Tyzzer's organism in rabbits.Method One sets of primers and probes were designed according to the 16S rRNA sequence of Tyzzer's organism.The concentration of primers and probes was optimized,and the sensitivity,specificity and repeatability of this real-time PCR method were evaluated.At the same time,clinical samples were tested using this method and compared with nested PCR method reported before.Result A real-time PCR was developed to specifically detect Tyzzer's Organism,detection limit of the positive plasmids were 8 copies/μL.This method did not cross-react with various pathogens that should not be detected in laboratory animals.The coefficient of variation of intra-and inter-batch repetition was less than 3%,indicating that the repeatability of this method was good.According to the testing result of 70 clinical samples,it was found that the positive sample detection rate of this method was higher than that of the nested PCR method.Conclusion The real-time PCR method established in this study has good specificity,high sensitivity and good reproducibility,and was suitable for the detection of Tyzzer's organism in rabbit fecal samples.