Establishment of a PCR method with an internal amplication control for detection of Cronobacter sakazakii in foods
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摘要: 为了解决常规PCR方法检测阪崎克罗诺杆菌时因检测过程中环境和食品理化因素的影响而导致的假阴性结果的产生,本研究以细菌16S rRNA基因为扩增内标对照,以阪崎克罗诺杆菌特异性基因grx B为靶基因设计了一对特异性引物,并通过优化PCR反应条件,最终建立了一种添加扩增内标的阪崎克罗诺杆菌PCR检测方法,可以指示PCR过程中因存在DNA聚合酶抑制剂而导致的假阴性结果。通过对20种细菌进行PCR检测显示,该方法对阪崎克罗诺杆菌具有良好的特异性。灵敏度实验结果表明,该检测方法对阪崎克罗诺菌纯DNA模板的检测灵敏度为2.15×102fg/μL,对阪崎克罗诺杆菌纯培养物的检测灵敏度为9.4×103CFU/m L。对人工污染婴幼儿奶粉的检测结果显示,阪崎克罗诺杆菌接种量为0.94 CFU/g的婴幼儿奶粉样品经过8 h增菌培养后,即可检出。食品样品检测结果表明,不添加扩增内标的PCR检测方法中出现的假阴性结果可被本方法检出。该检测方法特异性强、灵敏度较高,能消除阪崎克罗诺杆菌常规PCR检测方法中可能出现的假阴性结果,适用于婴幼儿配方乳粉、乳制品等食品中阪崎克罗诺杆菌的快速检测。Abstract: The traditional detection method of Cronobacter sakazakii was time- consuming,operation- complicated,low sensitivity and was often influenced by the environmental factors,and the physical and chemical properties of food,which might lead to false negative results. In this study,we used 16 S rRNA gene as an internal amplification control,designed primers targeting grx B of C. sakazakii,optimized the reaction conditions and finally,established a PCR method with an internal amplication control for C.sakazakii detection.The results of detection of 20 strains including Cronobacter and non- Cronobacter by this method showed that these primers were specific for C. sakazakii detection. The sensitivity of established detection assay for C.sakazakii purified genomic DNA and pure cultures were 2.15 × 102 fg / μL and 9.4 × 103 CFU / m L,respectively. The detection for artificially contaminated infant milk powder showed that C.sakazakii could be detected after 8 h enrichment culture when the C.sakazakii inoculation was 0.94 CFU / g. This detection method demonstrated a good specificity and sensitivity,and could eliminate false- negative results caused by inhibitor of DNA polymerase in the PCR reaction reagents,and suitable for rapid detection of foodborne C.sakazakii.
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