利用双重PCR同时检测婴幼儿配方奶粉中克罗诺杆菌和沙门氏菌方法的建立
Detection of Cronobacter spp.and Samonella spp.in powder infant formula by duplex PCR
-
摘要: 建立能够同时检测婴幼儿配方奶粉中克罗诺杆菌和沙门氏菌的双重聚合酶链式反应(Polymerse chain reaction,PCR)的方法。分别根据沙门氏菌和克罗诺杆菌16S rDNA序列设计特异性引物Cro和InvA。对待检奶粉样本进行预增菌后,提取菌液基因组DNA作为模板,进行双重PCR扩增,对其特异性、灵敏度和抗杂菌干扰能力进行评估。结果表明:两对引物分别可特异性扩增出140、630bp的目的条带。双重PCR同时检测婴幼儿配方奶粉样本中两种食源性致病菌的方法具有较高灵敏度,在经过4h预增菌后可检测到初始浓度为100CFU/g的克罗诺杆菌和初始浓度为100CFU/g的沙门氏菌,且在高浓度杂菌干扰下,灵敏度无下降。在人工污染奶粉样本检测中,两种致病菌的检出准确率均达95%以上。初步建立了能同步、快速、准确、灵敏地检测婴幼儿配方奶粉中克罗诺杆菌和沙门氏菌的双重PCR方法。Abstract: A duplex PCR assay was developed to detect Cronobacter spp. and Samonella spp. in powder infant formula ( PIF) .Two sets of specific primers were designed according to the conservative region of 16 S rDNA gene sequences of Cronobacter and Samonella.The genomic DNA was extracted and used as the templates for duplex PCR after the 4- hour pre- enrichment.As the results showed that, two DNA fragments of 140 bp and 630 bp were amplified by two pairs of specific primers. The detection limit of this protocol could be 100 CFU /g for both Cronobacter spp. and Samonella spp., and bacteria interference didn't affect the detection sensitivity. A great sensitivity and specificity ( ≥95%) was observed in the detection of artificially contaminated PIF samples.Thus, the duplex PCR assay for joint detection of Cronobacter spp. and Samonella spp. in powder infant formula was sensitive, accurate, and also applicable to investigate and monitor these two pathogens in dairy products.
下载: