实时荧光环介导等温扩增技术检测熏肉中单增李斯特氏菌的研究  

Detection of Listeria monocytogenes by real-time fluorescence loop mediated isothermal amplification

作　　者：李森[1] 贾丽娜 王鑫 杨倩 马晓燕[1] 魏丹 袁耀武[1] 张伟[1,2] LI Sen;JIA Li-na;WANG Xin;YANG Qian;MA Xiao-yan;WEI Dan;YUAN Yao-wu;ZHANG Wei(College of Food Science and Technology,Hebei Agricultural University,Baoding 071001;College of Science and Technology,Hebei Agricultural University,Cangzhou 061100)

机构地区：[1]河北农业大学食品科技学院,保定071001 [2]河北农业大学理工学院,沧州061100

出　　处：《食品科技》2018年第8期319-324,共6页Food Science and Technology

基　　金：河北省自然科学基金面上项目(C2008000216);河北省高等学校科学技术研究项目(ZD2017237)

摘　　要：建立一种实时荧光环介导等温扩增方法(Real-Time Fluorescence Loop-mediated Isothermal Amplification,RTF-LAMP)来快速、灵敏地检测熏肉中单增李斯特氏菌。针对单增李斯特氏菌的特异性基因inl B设计4条LAMP引物,并建立一套最优的RTF-LAMP反应体系。对该方法进行特异性验证,同时对灵敏度和人工污染熏肉的检出限进行测定。3株单增李斯特氏菌被检测为阳性,17株非单增李斯特氏菌为阴性。表明引物具有良好的特异性。RTF-LAMP和传统LAMP检测单增李斯特氏菌的灵敏度分别为1.1×10~1、1.1×10~2 CFU/m L。通过对熏肉进行人工污染,得出RTF-LAMP的检出限为3.5×10~1 CFU/g。通过与国标方法GB 4789.30—2016进行对比并计算RTF-LAMP反应的敏感性,特异性和准确度,结果分别为100.00%、98.44%和98.46%。研究建立的RTF-LAMP能够实时、快速地检测熏肉中单增李斯特氏菌,具有良好的应用前景。To rapidly and sensitively detect Listeria monocytogenes （L. monocytogenes） in bacon, a real- time fluorescence loop-mediated isothermal amplification （RTF-LAMP） method was developed. Four LAMP primers were designed using the specific gene inlB of L. monocytogenes to establish an optimal RTF-LAMP reaction system. The performance of the assay was evaluated by specificity, sensitivity and the detection limit of artificially contaminated bacon. 3 L. monocytogenes were positive and other 17 non-L, monocytogenes strains were negative, which indicated the good specificity of primers. The sensitivity of RTF-LAMP and traditional LAMP for the detection of L. monocytogenes were 1.1×10^1 CFU/ mL and 1.1x102 CFU/mL, respectively. The detection limit of L. monocytogenes in artificially contaminated bacon was 3.5×10^1 CFU/g. Sensitivity, specificity and accuracy of RTF-LAMP system were calculated compared wirh the national standard GB 4789.30--2016, resulting in 100.00%, 98.44% and 98.46%, respectively. The RTF-LAMP established in this study can detect L. monocytogenes in bacon rapidly, sensitively and sDecificallv with a aood aDolication DrosDect.

关 键 词：实时荧光环介导等温扩增 单增李斯特氏菌 inlB 检测 熏肉 

分 类 号：O657.32[理学—分析化学]