多重PCR检测婴幼儿配方奶粉中3种食源性致病菌

Multiplex PCR for the Detection of Three Foodborne Pathogens in Powdered Infant Formula

为建立一种能够同时检测婴幼儿配方奶粉(Powdered Infant Formula,PIF)中克罗诺杆菌、沙门氏菌和金黄色葡萄球菌3种食源性致病菌的多重PCR检测方法。通过单重PCR验证了9对引物的特异性,筛选出其中3对特异性好的引物建立了多重PCR体系,对其特异性和灵敏度进行评价,并将其应用于人工污染PIF中3种食源性致病菌的检测。结果表明,针对克罗诺杆菌、沙门氏菌和金黄色葡萄球菌等3种食源性致病菌所筛选的3对引物分别能扩增出469、638和796 bp的目的条带,具有高度特异性。多重PCR检测体系的最佳退火温度为55℃,最佳Mg^(2+)浓度为2.00 mmol/L,最佳引物浓度为400 nmol/L。在此条件下3种目的菌在10~2CFU/m L均可同时扩增出较清晰条带。将建立的多重PCR检测体系应用于人工污染PIF中3种食源性致病菌的检测,其检出限达到10~3CFU/g。本研究初步建立了一种能准确、快速、特异性的检测PIF中克罗诺杆菌、沙门氏菌和金黄色葡萄球菌的多重PCR方法,适用于PIF中3种常见的食源性致病菌的快速检测。

To establish a multiple PCR assay for the effective and simultaneous detection of Cronobacter spp.,Salmonella spp.and Staphylococcus aureus in powdered infant formula（ PIF）. Three pairs of primers（ Omp A,inv AF1 and clf A） with high specificity were screened from nine previously reported pairs of primers. The specificity and sensitivity of the established multiple PCR assay was evaluated,and then the multiplex PCR assay was applied to artificially contaminated PIF for the detection of the three foodborne pathogens. The results indicated that the amplified fragment sizes of Cronobacter spp.,Salmonella spp.and Staphylococcus aureus were 469,638 and 796 bp,respectively which had highly specificity. The optimal annealing temperature of the multiplex PCR assay for was 55 ℃,the Mg^2＋ concentration of 2.00 mmol/L,and the concentration of primers of 400 nmol/L for each specific primer for the simultaneous detection of Cronobacter spp.,Salmonella spp. and Staphylococcus aureus in a single PCR tube.The specificity of the multiplex PCR assay was high,and the sensitivity of multiplex PCR assay was 10^2 CFU/mL for pure cultures under the optimized conditions. When the multiplex PCR assay was applied to artificially contaminated PIF,the detection limit of the multiplex PCR was 10^3 CFU/g. The multiplex PCR assay was thus an accurate and specific method for the rapid detection of Cronobacter spp.,Salmonella spp.and Staphylococcus aureus in PIF.