羊源尸毒梭菌三重PCR检测方法的建立及应用

Establishment and application of a triplex PCR for detection of Clostridium cadaveris isolated from goat

为建立一种快速检测羊源尸毒梭菌的方法,本研究参考尸毒梭菌23S rRNA基因、高致病性毒力岛irp2基因和溶血素hlyA基因的特异序列,设计合成3对特异性引物,通过优化条件建立了检测羊源尸毒梭菌的三重PCR方法,并评估了其特异性、敏感性和重复性。将尸毒梭菌人工感染小鼠,利用建立的三重PCR方法检测死亡小鼠各组织中的尸毒梭菌,并与16S rDNA PCR方法的检测结果相比较。结果显示,该三重PCR方法优化后的条件为:3对基因上、下游引物均为1μL终浓度均为10μmol/L,退火温度59℃。该方法仅对尸毒梭菌扩增出23S rRNA、irp2和hlyA 3个目的基因,而对蜡样芽孢杆菌、类志贺邻单胞菌、乳酸片球菌、藤黄微球菌、霍氏肠杆菌、阴沟肠杆菌、大肠杆菌、柠檬酸杆菌及枯草芽孢杆菌均未扩增出目的基因;对尸毒梭菌DNA的最低检出量为2.76×101拷贝/μL,敏感性较高;该方法对同一稀释度及不同稀释度尸毒梭菌DNA的重复性检测结果均一致,重复性较好。人工感染1.5×109 cfu/mL的尸毒梭菌的小鼠12 h全部死亡,对照组小鼠未出现发病和死亡,且经本研究建立的三重PCR方法检测结果显示,每只死亡小鼠的肝脏、肾脏和肠道样品均为阳性结果,而其心脏、脾脏、肺脏检测结果均为阴性,与16S rDNA PCR方法的阳性检测结果均一致。本实验建立的致病性羊源尸毒梭菌三重PCR检测法特异性强、敏感性高、重复性好,为致病性羊源尸毒梭菌的检测和其感染的防治提供了技术支持。

A rapid assay was established to detect the highly pathogenic Clostridium cadaveris isolated from goat.Three pairs of specific primers were designed based on 23S rRNA gene of Clostridium cadaveris,irp2 gene of HPI(High pathogenicity island)and hlyA gene encoding hemolysin.The triplex PCR method for detecting Clostridium cadaveris was established by optimizing the conditions,and its specificity,sensitivity and repeatability were evaluated.The mice were artificially infected with Clostridium cadaveris,and the triplex PCR method was used to detect Clostridium cadaveris in the tissues of the dead mice.And the results were compared with that of 16S rDNA PCR method.The results showed that the optimized conditions of the triplex PCR method were:three pairs of sense and antisense primers were 1μL and the final concentration of each primer was 10μmol/L.The annealing temperature was 59℃.The triplex PCR detection method could amplify three target genes of 23S rRNA,irp2 and hlyA with Clostridium cadaveris,while Bacillus cereus,Plesimonas shigelloides,Pediococcus acidilactici,Micrococcus luteus,Enterobacter hormaechei,Enterobacter cloacae,Escherichia coli,Citrobacter amalonaticus and Bacillus subtilis did not yield any product.The minimum detectable amount of Clostridium cadaveris was 2.76×101 copies/μL.The results of repeatability detection of Clostridium cadaveris DNA with the same dilution and different dilutions were consistent and the repeatability was good.All the artificially infected mice died within 12 hours,while no morbidity or mortality occurred in the control group.The detection results of the triplex PCR method established in this study showed that the liver,kidney and intestinal samples of each dead mouse all tested positive,while the detection results of heart,spleen and lung all tested negative,which was consistent with the positive detection results of 16S rDNA PCR method.The triplex PCR method of the pathogenic Clostridium cadaveris isolated from goat was established with high specificity,sensitivity and repeatability,which provided technical support for the detection,prevention and treatment of it.