荧光MIRA法快速检测食品中产气荚膜梭菌  

Fluorescent MIRA assay for the rapid detection of Clostridium perfringens in food

作　　者：张士财 丁卫平 伊廷存 张瑞 朱富强 崔俊凤 霍胜楠 ZHANG Shicai;DING Weiping;YI Tingcun;ZHANG Rui;ZHU Fuqiang;CUI Junfeng;HUO Shengnan(Binzhou Testing Center,Shandong Binzhou 256600,China;Shandong Institute of Food and Drug Control,Shandong Ji’nan 250101,China)

机构地区：[1]滨州市检验检测中心,山东滨州256600 [2]山东省食品药品检验研究院,山东济南250101

出　　处：《中国食品卫生杂志》2024年第2期139-146,共8页Chinese Journal of Food Hygiene

基　　金：2021年度山东省市场监督管局科研项目(202108)。

摘　　要：目的基于荧光多酶恒温快速扩增(MIRA)技术,建立一种快速准确检测食品中产气荚膜梭菌的方法。方法利用产气荚膜梭菌共有α毒素编码基因plc保守序列设计引物探针,并建立和优化荧光MIRA方法;提取17种非目标菌基因组DNA进行方法特异性验证;梯度稀释目标菌基因组DNA和菌液用于方法灵敏度评价;选取4种食品进行基质干扰试验,评估方法的稳定性;检测实际样品并与国家标准方法进行比较,以验证该方法的实用性。结果该方法特异性强,检测多种非目标致病菌均呈阴性反应;灵敏度高,以基因组DNA为模板和菌液为模板时最低检测限分别为1.05×10^(1) fg/μL和7.5×10^(0) CFU/mL;该方法不受基质影响,抗干扰能力强;耗时短,约20 min即可完成检测。结论本研究中的荧光MIRA方法快速准确、灵敏度高、特异性强,可用于食品中产气荚膜梭菌的快速检测。Objective To establish a rapid and accurate molecular method for detecting Clostridium perfringens(C.perfringens)in food using fluorescent multi-enzyme isothermal rapid amplification(MIRA).Methods Primers and probes based on the conserved sequence of the C.perfringens alpha toxin-coding gene plc were designed separately,and the fluorescence MIRA method was established and optimized.The genomic DNA of 17 non-C.perfringens strains were extracted for specificity verification,and the isolated DNA and bacterial solutions of different dilutions were used for sensitivity evaluation.Matrix interference testing with 4 types of food media was also performed to evaluate the stability of the method,and the actual sample detection results were compared with the standard method to verify its practicality.Results The fluorescent MIRA method for C.perfringens used in this study had high specificity.The results returned negative for other non-C.perfringens strains.MIRA also had high sensitivity,with detection limits of 1.05×10^(1) fg/μL for genomic DNA and 7.5×10^(0) CFU/mL for pure C.perfringens cultures.It also had a strong anti-interference ability;the detection results were not affected by the matrix,and the detection time was short(approximately 20 min).Conclusion This study developed a rapid,accurate,sensitive and specific fluorescent MIRA method for the molecular detection of C.perfringens in food.

关 键 词：多酶恒温快速扩增 产气荚膜梭菌 plc基因 分子检测 食源性致病菌 

分 类 号：R155[医药卫生—营养与食品卫生学]