食品中克罗诺杆菌属双重PCR检测试剂盒的评价

Evaluation of duplex PCR detection kit for detection of Cronobacter in food samples

【背景】在食品安全领域,克罗诺杆菌属于需要重点监测的致病菌,当前随着分子检测相关技术的不断发展,研制有关食品中克罗诺杆菌简便、高效的检测产品至关重要。【目的】研制克罗诺杆菌检测的双重PCR检测试剂盒并评价其用于食品中克罗诺杆菌检测的实效性。【方法】优化双重PCR反应体系,反应试剂采用冻干工艺,确立了试剂盒组成,并评价其特异性、灵敏度、重复性、保质期等性能指标。【结果】克罗诺杆菌标准菌株和分离菌株均在目标位置出现两条明显条带,非克罗诺杆菌标准菌株和分离菌株均检测为阴性,纯基因组DNA检测灵敏度为2.3×10-1 ng/μL,纯培养菌检验限为3.2×104CFU/mL;对65份食品样品进行克罗诺杆菌检测,该试剂盒检测结果与标准方法检测结果一致性较高;批内、批间检测重复率均为100%,可在42°C环境放置120 h且其检测效力不受影响,4°C保质期可长达12个月。【结论】该试剂盒性能好,检测结果稳定、可靠,适用于食品中克罗诺杆菌的快速检测。

[Background]Cronobacter spp.are major foodborne pathogens that need to be intensively monitored for food safety.At present,with continuous development of the molecular detection technology,it is very important to develop simple and efficient detection methods for Cronobacter detection in food.[Objective]Develop duplex PCR detection kit for detecting Cronobacter and evaluate its detecting efficacy in food samples.[Methods]Duplex PCR reaction system was optimized and the components of the kit were confirmed.The main reaction reagents were made into powder pattern by freeze drying.Then the specificity,sensitivity,repeatability,shelf life and other performance indexes of the kit were evaluated.[Results]All Cronobacter standard strains and isolated strains were detected positive with two distinct bands in the target size,while all non-Cronobacter standard strains and isolated strains were detected negative without target band observed.The detection sensitivity of Cronobacter purified genomic DNA and pure cultures was respectively 2.3×10-1 ng/μL and 3.2×104 CFU/mL.Cronobacter in 65 food samples was detected.The detection result obtained by the kit was highly consistent with that obtained by conventional microbial detection method.The intra-batch and inter-batch testing repetition rates were all 100%.The kit had no efficacy loss after 120 h storage at 42°C and 12 months at 4°C.[Conclusion]The kit is stable,reliable,and applicable for rapid detection of Cronobacter in food.