兔源多杀性巴氏杆菌、金黄色葡萄球菌和肺炎克雷伯菌多重PCR检测方法的建立

Establishment of a Multiplex PCR Assay for Simultaneous Detection Pasteurella Multocida、Staphyloccocus Aureus and Klebsiella Pneumoniae from Rabbit

目的建立兔源多杀性巴氏杆菌(Pm)、金黄色葡萄球菌(Sa)和肺炎克雷伯菌(Kp)的多重PCR方法。方法本研究参考Pm kmt1基因、Sa nuc基因和Kp kh1基因的保守区域,设计3对特异性引物,以温度梯度PCR法确定最适退火温度(Tm),采用控制单一变量法优化引物浓度,构建kmt1、nuc和kh1基因的重组质粒标准品pMDPm、pMD-Sa和pMD-Kp,确定最小检测量;以兔源支气管败血波氏杆菌和产气荚膜梭菌等9种病原体核酸样本确定多重PCR法的特异性;通过批间和批内实验验证其重复性;经检测疑似感染的临床样本与已报道的检测方法进行比较,评估其临床应用效果。结果最适退火温度为54.5℃;扩增kmt1和nuc基因的引物最适浓度均为1μL(10μmol/L),扩增kh1基因的引物最适浓度为0.5μL(10μmol/L);pMD-Pm最低检测限为20.6 copies/μL,pMDSa最低检测限为18.2 copies/μL,pMD-Kp最低检测限为23.2 copies/μL,表明其灵敏性高;该方法仅对Pm、Sa和Kp有特异性扩增条带,对其他病原体均无扩增条带,表明特异性较强;批间和批内检测结果一致,表明重复性较好;经临床样本检测Pm、Sa和Kp单独的阳性率分别为62.92%、25.84%和49.43%,与已报道的检测方法结果一致。结论本研究成功建立了一种能够同时快速检测兔源多杀性巴氏杆菌、金黄色葡萄球菌和肺炎克雷伯菌的多重PCR方法。

Objective This study aimed to establish a multiplex PCR method for detecting three pathogens in rabbits:Pasteurella multocida(Pm),Staphylococcus aureus(Sa),and Klebsiella pneumoniae(Kp).Method In this study,three pairs of specific primers were designed,targeting the conserved regions of the kmt1 gene in Pm,the nuc gene in Sa,and the kh1 gene in Kp.The optimal annealing temperature(Tm)was determined using gradient PCR.The primer concentrations were optimized using the controlled single-variable method.Recombinant plasmid standards(pMD-Pm,pMDSa,and pMD-Kp)were constructed,and the minimum detection limit was established.The specificity of the multiplex PCR was confirmed using nucleic acid samples of nine pathogens,including rabbit bronchial septicemic Pasteurella and gas-producing clostridia.The repeatability was validated through inter-batch and intra-batch experiments.Clinical samples suspected of infection were tested using the established method and compared with previously reported detection methods to evaluate its clinical application.Result The optimal annealing temperature was determined to be 54.5℃.The optimal primer concentrations for amplifying kmt1 and nuc genes were 1μL(10μmol/L),while for the kh1 gene,it was 0.5μL(10μmol/L).The lowest detection limits were 20.6 copies/μL for pMD-Pm,18.2 copies/μL for pMD-Sa,and 23.2 copies/μL for pMD-Kp,indicating high sensitivity.The multiplex PCR method showed specific amplification bands only for Pm,Sa,and Kp,with no amplification bands observed for other pathogens,demonstrating strong specificity.The results of inter-batch and intra-batch tests were consistent,indicating good repeatability.Clinical sample testing revealed individual positivity rates of 62.92%for Pm,25.84% for Sa,and 49.43% for Kp,which were consistent with the results obtained using previously reported detection methods.Conclusion This study successfully established a multiplex PCR method capable of simultaneously and rapidly detecting Pm,Sa,and Kp in rabbits.