添加扩增内标的沙门氏菌PCR检测方法

Development of an Internal Amplification Control in the PCR Detection for Salmonella

通过构建人工扩增内标,建立可以有效指示沙门氏菌检测过程可能出现假阴性情况的PCR检测方法。本研究基于沙门氏菌invA基因设计特异性引物,复合法构建扩增内标,建立PCR检测体系。特异性引物LW,对33株沙门氏菌和6株非沙门氏菌标准株进行检测,结果显示,所有沙门氏菌均扩增出385 bp的目标片段,非沙门氏菌则只能扩增出484 bp的扩增内标片段,特异性良好。灵敏度实验表明,该检测体系的灵敏度可达6.35 fg/μL。人工污染实验表明,起始染菌量为3.2 CFU/25 mL时,仅需8h增菌培养便可检出。大量食品样品检测证明,该检测体系确实可以有效的避免PCR检测过程出现的假阴性,提高检测准确性。

By constructing an internal amplification control（IAC）, this study developed a PCR system for the detection of Salmonella, which could effectively indicate false-negative results. The specific primers were designed according to the conserved gene invA in Salmonella spp.. An IAC was constructed by the compound primer technology, and finally the PCR detection system was developed. The experiment indicated that the specific 385 bp DNA fragment was amplified against 33 reference strains of Salmonella spp., while 6 strains of non-Salmonella only showed the 484 bp amplified band of IAC. The detection limit of this PCR system forpurified genomic DNA was 6.35 fg/μL. The artificial contamination assays showed that Salmonella could be detected after eight hours enrichment when the original bacterial concentration was 3.2 CFU/25 mL. A large number of food samples were also tested, and the results demonstrated that the detection system could effectively avoid the false-negatives and improve the detection accuracy.