基于样品检测的布鲁氏菌荧光定量PCR方法的建立

目的 本文建立一种基于样品检测的特异性好、灵敏性高的布鲁氏菌荧光定量PCR检测方法。方法 以哺乳动物beta-actin基因和外源重组质粒为内参,针对布鲁氏菌属特异性基因IS711,建立用于样品检测的布鲁氏菌荧光定量PCR方法,并验证其敏感性、特异性及稳定性,绘制标准曲线。将该方法用于哺乳动物样品检测,并与细菌分离培养检测结果比较。结果 荧光定量PCR方法对布鲁氏菌检测有良好的特异性,3对引物对阴性对照菌均无非特异性扩增;该方法用于样品检测的最低检测限为17拷贝;内外源内参同时存在条件下,布鲁氏菌荧光定量PCR的标准曲线相关系数为R²=0.997(Y=-3.12X+40.9)。将该方法直接用于181份动物样本的检测,并与分离培养结果相比,荧光定量PCR检测阳性率为11.4%,细菌分离培养检测阳性率为9.9%,且细菌分离培养阳性样品与荧光定量PCR阳性样品中编号基本一致。结论 本文建立的荧光定量PCR检测方法灵敏性高、特异性及稳定性好,可直接应用于样品的检测,检测结果受内参基因质控。

Development of real-time PCR method based on sample detection for Brucella

A reliable and efficient real-time PCR method directly used for sample detection targeting the Brucella genus-specific IS711 gen was developped. The method also includes a target to a conserved region of the beta-actin gene to assess suitable of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. We verified the sensitivity, specificity and stability of this method, and made the standard curve. The method was directly used for the detection of 181 ani-mal samples, and compared with the bacteria separating culture. The limit of simulated sample detection was 17 copies, The specificity of this real-time PCR method was no cross-reactivity with Escherichia coil O:086, Escherichia coil O:157, Yersinia enterocolitica O:9, Ochrobactrum anthro, Candida albicans stra, Salmonella typhimuri, Agrobacterium rhizogenes, Pseudomonas aeruginosa. Standard curve of the three groups were made: only to Brucella standard products as template DNA; to Brucella standard products and exogenous recombinant plasmid reference DNA, Brucella standard products, exogenous recombinant plasmid and endogenous host gene. The correlation coefficient were 0.999% (Y=-3.34X+42.85), 0.997% (Y=-3.13X+40.50) and 0.997%(Y=-3.12X+40.9), respectively. The positive detection rate of 181 animal samples was 11.4% for real-time PCR and 9.9% for bacteria separating culture, and bacteria isolated culture positive samples were basically consistent with the number of real-time PCR positive samples. Result showed that with the advantages of higher sensitivity and reliable, the multiple real-time PCR method could be used to rapid detecting the Brucella qualitily.