通过多重核酸内切酶限制实时环介导的等温扩增技术快速灵敏地检测副溶血性弧菌和创伤弧菌。,Molecules

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通过多重核酸内切酶限制实时环介导的等温扩增技术快速灵敏地检测副溶血性弧菌和创伤弧菌。
Molecules ( IF 4.2 ) Pub Date : 2016-01-23 , DOI: 10.3390/molecules21010111
Yi Wang ₁ , Dongxun Li ₂ , Yan Wang ₁ , Kewei Li ₃ , Changyun Ye ₁

Affiliation

副溶血性弧菌和创伤弧菌是两种海洋海鲜传播的病原体，可在人类和水生动物中引起严重疾病。在这项研究中，成功开发并评估了一种新开发的新颖的多重内切核酸酶限制性实时环介导的等温扩增技术（MERT-LAMP），可在单个反应中同时检测副溶血性弧菌和创伤弧菌。设计了两个MERT-LAMP引物组，以特异性靶向溶血弧菌的toxR基因和创伤弧菌的rpoS基因。在62°C下进行MERT-LAMP反应，使用副溶血弧菌和创伤弧菌菌株提取的基因组DNA模板在短短19分钟内产生阳性结果。可以基于实时格式的不同荧光曲线同时检测并正确区分存在于同一样品中的两种目标病原体。使用副溶血弧菌和创伤弧菌菌株的基因组模板，每个反应的MERT-LAMP测定灵敏度为250 fg和125 fg DNA，这与常规LAMP检测相符。与基于PCR的技术相比，MERT-LAMP技术的灵敏度比PCR和qPCR方法高100倍和10倍。而且，在人工污染的牡蛎样品中，副溶血性弧菌和创伤弧菌分离株的MERT-LAMP方法检测限为每个反应92 CFU和83 CFU。总之，此处介绍的MERT-LAMP测定是一种快速，特异性和灵敏的工具，用于检测副溶血性弧菌和V.

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Rapid and Sensitive Detection of Vibrio parahaemolyticus and Vibrio vulnificus by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique.

Vibrio parahaemolyticus and Vibrio vulnificus are two marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. In this study, a recently developed novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully developed and evaluated for simultaneous detection of V. parahaemolyticus and V. vulnificus strains in only a single reaction. Two MERT-LAMP primer sets were designed to specifically target toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus. The MERT-LAMP reactions were conducted at 62 °C, and the positive results were produced in as short as 19 min with the genomic DNA templates extracted from the V. parahaemolyticus and V. vulnificus strains. The two target pathogens present in the same sample could be simultaneously detected and correctly differentiated based on distinct fluorescence curves in a real-time format. The sensitivity of MERT-LAMP assay was 250 fg and 125 fg DNA per reaction with genomic templates of V. parahaemolyticus and V. vulnificus strains, which was in conformity with conventional LAMP detection. Compared with PCR-based techniques, the MERT-LAMP technology was 100- and 10-fold more sensitive than that of PCR and qPCR methods. Moreover, the limit of detection of MERT-LAMP approach for V. parahaemolyticus isolates and V. vulnificus isolates detection in artificially-contaminated oyster samples was 92 CFU and 83 CFU per reaction. In conclusion, the MERT-LAMP assay presented here was a rapid, specific, and sensitive tool for the detection of V. parahaemolyticus and V. vulnificus, and could be adopted for simultaneous screening of V. parahaemolyticus and V. vulnificus in a wide variety of samples.

更新日期：2019-11-01

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