基于环介导等温扩增技术的产呕吐毒素蜡样芽胞杆菌特异性检测

Specific Detection of Emetic Bacillus cereus in Foods by Loop Mediated Isothermal Amplification (LAMP) Method

摘要:

本研究旨在建立食品中产呕吐毒素蜡样芽胞杆菌的快速检测方法。基于产呕吐毒素蜡样芽胞杆菌合成酶基因cesB靶基因，设计4条特异性引物（2条内引物、2条外引物），建立环介导等温扩增技术（LAMP）。然后采用2株产呕吐毒素蜡样芽胞杆菌、19株蜡样芽胞杆菌和41株非蜡样芽胞杆菌验证了该LAMP具有很好的特异性。LAMP检测的灵敏度在DNA水平上达到1.49 pg/μL，纯菌的灵敏度为5×103 cfu/mL。人工污染米饭样品，当起始污染量为2 cfu/g时，37 ℃增菌培养6 h，用试剂盒法和煮沸法提取的DNA，都可以检出产呕吐毒素蜡样芽胞杆菌。采用此LAMP方法进行了72份实际样品的检测，检出2份产呕吐毒素蜡样芽胞杆菌阳性样品，与传统方法一致。因此，本研究建立的LAMP检测方法可应用于食品中产呕吐毒素蜡样芽胞杆菌的快速特异检测。

Abstract:

The loop-mediated isothermal amplification (LAMP) assay for the detection of emetic Bacillus cereus was developed by designing four primers including two inner primers and two outer primers that specifically amplify segments of the cesB gene. Analysis of two emetic B. cereus strains, nineteen B. cereus strains and forty-one non-B. cereus strains demonstrated that this LAMP assay was specific. The detection limit of the LAMP method was 1.49 pg/μL with genomic DNA, 5×103 cfu/mL in pure culture. The detection limit of the LAMP method was 2 cfu/g in arti?cially contaminated rice for either boiling or kit-based DNA isolation method after enrichment at 37 ℃ for 6 h. Among seventy-two food samples, two was identified to be positive by this LAMP method, which was consistent with the international standard method. The result demonstrated that the specific LAMP assay can be used for rapid detection of emetic B. cereus strains in food.