To establish a multiplex PCR for simultaneous detection of Escherichia coli (E. coli), Salmonella, Klebsiella pneumoniae (K. pneumoniae), and Staphylococcus aureus (S. aureus), four pairs of specific primers were designed according to the conservative regions of phoA gene for E. coli, invA gene for Salmonella, khe gene for K. pneumoniae, nuc gene for S. aureus. The quadruple PCR system was established through optimization of multiplex PCR and detection of specificity, sensitivity, and stability. The results showed that target gene bands of E. coli (622 bp), Salmonella (801 bp), K. pneumoniae (303 bp), and S. aureus (464 bp) could be amplified by this method specifically and simultaneously from the same sample containing the four pathogens, with a detection sensitivity of 100 pg/μL. Meanwhile, no bands of common clinical bacteria, including Clostridium perfringens, Pseudomonas aeruginosa, Pasteurella multocida, Streptococcus pneumoniae, Streptococcus pneumoniae, Proteus mirabilis, Staphylococcus sciuri, Staphylococcus pseudintermedius, Acinetobacter baumannii, Enterococcus faecalis, and Bacillus subtilis were amplified. In addition, 380 tissue samples were detected by multiplex and single PCR established in current study, respectively. Among the 368 carcass samples, positive detection rates of E. coli, K. pneumoniae, Salmonella, and S. aureus were 33.7, 12.0, 10.6, and 13.9%. Among the 12 visceral tissue samples, positive detection rates of E. coli, K. pneumoniae, Salmonella, and S. aureus were 41.7, 25.0, 16.7, and 8.3%, respectively. Positive detection rates of multiplex PCR were consistent with that of single PCR. Compared with single PCR, the multiplex PCR method had the advantages of time-saving, high specificity and high sensitivity. The results showed that the minks in these farms had mixed infection of these four pathogens, and the method established in this study could be applied to the rapid and accurate detection and identification of these four bacteria. In conclusion, the multiplex PCR method has stable detection results, good repeatability, and short detection time. It is suitable for the rapid and accurate detection of four kinds of bacteria above the carcass of fur animals, which could be suitable in microbial epidemiology investigation. It can provide a reliable technical reference for rapid clinical diagnosis and detection.

建立多重PCR以同时检测 大肠杆菌 （大肠杆菌）， 沙门氏菌，肺炎克雷伯菌 （肺炎克雷伯氏菌），以及 金黄色葡萄球菌 （金黄色葡萄球菌），根据保守区域设计了四对特异性引物 磷酸 基因 大肠杆菌，invA 基因 沙门氏菌 基因 肺炎克雷伯菌 基因 金黄色葡萄球菌。通过优化多重PCR并检测特异性，敏感性和稳定性，建立了四重PCR系统。结果显示靶基因带大肠杆菌 （622 bp）， 沙门氏菌 （801 bp）， 肺炎克雷伯氏菌 （303 bp），和 金黄色葡萄球菌通过这种方法，可以同时从含有四种病原体的同一样品中特异性扩增出约464 bp（464 bp）的片段，检测灵敏度为100 pg /μL。同时，没有常见的临床细菌带，包括产气荚膜梭菌，铜绿假单胞菌，多杀性巴斯德氏菌，肺炎链球菌，肺炎链球菌，奇异变形杆菌，sciphylococcus sciuri，pseudintermedius葡萄球菌，鲍曼不动杆菌，粪肠球菌和 枯草芽孢杆菌被放大了。另外，通过当前研究中建立的多重和单次PCR分别检测了380个组织样品。在368具car体样品中，阳性检出率为大肠杆菌，肺炎克雷伯菌，沙门氏菌和 金黄色葡萄球菌分别为33.7、12.0、10.6和13.9％。在12个内脏组织样本中，大肠杆菌，肺炎克雷伯菌，沙门氏菌和 金黄色葡萄球菌分别为41.7％，25.0％，16.7％和8.3％。多重PCR的阳性检出率与单一PCR一致。与单一PCR相比，多重PCR方法具有省时，高特异性，高灵敏度的优点。结果表明，这些养殖场的水貂混合感染了这四种病原体，本研究建立的方法可用于快速，准确地检测和鉴定这四种细菌。总之，多重PCR方法检测结果稳定，重复性好，检测时间短。它适用于快速，准确地检测毛皮动物above体上方的四种细菌，可能适用于微生物流行病学调查。