致牛腹泻4种细菌多重荧光定量PCR检测方法的建立及应用

Establishment and Application of Multiplex Fluorescent Quantitative PCR for Detection of Four Bacteria Causing Calf Diarrhea

随着规模化和集约化养牛业的不断发展,多种致病菌混合感染造成牛腹泻严重制约了养牛业的快速发展,对腹泻病原进行快速准确鉴别诊断对疫病防控至关重要。根据沙门氏菌的ivnA基因、大肠埃希氏菌的23S rRNA基因、产气荚膜梭菌的plc基因以及志贺氏菌的ipaH基因的保守区域建立了可同时检测这4种细菌的多重荧光定量PCR检测方法,并对其特异性、灵敏性和重复性进行了研究。结果显示,建立的标准曲线线性关系良好;优化后的检测方法仅可特异性扩增出4种目标细菌;对大肠埃希氏菌、沙门氏菌、志贺氏菌和产气荚膜梭菌的最低检出限分别为9.4×10~1copies/μL、1.2×10~2copies/μL、9.1×10~1copies/μL和1.4×10~3copies/μL,该方法的灵敏性高于普通单项PCR检测方法10倍;批内、批间差异均小于5%。用此方法检测人工感染病料,每份病料均检测出对应攻毒细菌的荧光信号。以上结果表明,所建立的多重荧光定量PCR方法特异性强、灵敏度高、重复性好,可用于致牛腹泻细菌的实验室检测。

With the continuous strengthening of large-scale and intensive breeding,the mixed infections of different pathogens caused calf diarrhea,which seriously threatened the health of calves and restricted the rapid development of breeding industry.Rapid and accurate identification of pathogens is crucial for disease diagnosis and prevention and control.In this experiment,based on the conserved regions of the ivnA gene of Salmonella,the 23S rRNA gene of Escherichia coli,the PLC gene of Clostridium perfringens and the IPAH gene of Shigella,a multiplex fluorescence quantitative PCR method for detecting these four bacteria at the same time was established,and its specificity,sensitivity and reproducibility were studied.The results show that the linear relationship of standard curves established in this study is good.The optimized detection method could only amplify four target bacteria.The minimum detection limits of Escherichia coli,Salmonella,Shigella and Clostridium perfringens were 9.4×101 copies/μL,1.2×102 copies/μL,9.1×101 copies/μL and 1.4×103 copies/μL.The sensitivity of this method was 10 times higher than that of common single PCR.The intra-batch and inter-batch differences were less than 5%.Artificial infection materials were detected by this method,and the fluorescence signal of the corresponding challeng bacteria was detected in each material.The above results indicated that the method established in this study has strong specificity,high sensitivity and good repeatability,and can be used for laboratory detection of bacteria causing diarrhea in cattle.