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-  2017 

水产品中副溶血性弧菌LAMP检测方法的优化

DOI: 10.13982/j.mfst.1673-9078.2017.6.046

Keywords: 环介导等温扩增技术 副溶血性弧菌 tlh基因
loop-mediated isothermal amplification Vibrio parahaemolyticus tlh gene

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Abstract:

本文用副溶血性弧菌不耐热溶血素(tlh)基因作靶标,优化环介导等温扩增技术检测水产品副溶血性弧菌的方法。体系用Bst DNA聚合酶催化,恒温反应60 min,产物分别用2%琼脂糖凝胶电泳和SYBR Green I染色鉴定,对各项反应参数进行优化;将新鲜菌液作10倍梯度稀释后进行LAMP和PCR反应,比较二者的敏感度;对32株食源性病原菌(包括分离于水产品的25株副溶血弧菌,1株副溶血性弧菌ATCC17802标准株,大肠杆菌D5、金黄色葡萄球菌CMCC26003、志贺氏菌B4、蜡样芽胞杆菌63302、单核细胞增生李斯特菌54001和沙门氏菌50041各1株)进行LAMP扩增,验证其特异性;虾样品用菌液进行模拟污染,分析LAMP的可靠性。各参数最佳条件为:Mg2+浓度为3.6 mmol/L,dNTPs浓度为0.96 mmol/L,Bst DNA聚合酶用量为4.8 U,内外引物浓度比为8:1,最佳反应温度为63 ℃,时间为60 min;LAMP的检测限为1 CFU/mL,低于PCR方法的最低检测限;特异性试验检测26株副溶血性弧菌均为阳性,6株非副溶血性弧菌为阴性;人工污染试验中检测限达1 CFU/mL,无假阳性。建立的LAMP方法操作简单且灵敏度高,适用于水产食品中副溶血弧菌的现场快速检测。
The heat-sensitive hemolysin (tlh) gene was used as the target in this study, and loop-mediated isothermal amplification (LAMP) conditions were optimized to develop a method for the detection of Vibrio parahaemolyticus in aquatic products. The LAMP reaction system was catalyzed using Bst DNA polymerase and the reaction proceeded at a constant temperature for 60 min. The amplified products were identified by 2% agarose gel electrophoresis and SYBR Green I staining, and all reaction parameters were optimized. LAMP and polymerase chain reaction (PCR) amplification were performed after ten-fold serial dilution of the fresh bacterial culture, and the sensitivities of the two methods were compared. LAMP was performed on 32 foodborne pathogens (including 25 strains of Vibrio parahaemolyticus isolated from aquatic products, Vibrio parahaemolyticus strain ATCC 17802, Escherichia coli strain D5, Staphylococcus aureus strain CMCC26003, Shigella boydii strain B4, Bacillus cereus strain 63302, Listeria monocytogenes strain 54001, and Salmonella Enteritidis strain 50041), and the specificity of LAMP was verified. The reliability of LAMP was evaluated by measuring shrimp samples contaminated with V. parahaemolyticus culture. The optimum conditions were as follows: 3.6 mmol/L magnesium ion, 0.96 mmol/L dNTPs, 4.8 U Bst DNA polymerase, 8:1 ratio of inner and outer primers, reaction temperature of 63 ℃, and reaction time of 60 min. The detection limit of the LAMP assay was 1 CFU/mL, which was lower than that of the PCR method (1×105 CFU/mL). In specificity tests, all 26 strains of V. parahaemolyticus were positive, and the other six strains were negative. In the detection of artificially contaminated samples, the detection limits were 1 CFU/mL and there were no false positive results. In conclusion, the established LAMP assay is a simple, rapid, specific, and sensitive detection method that is suitable for the rapid on-site detection of V. parahaemolyticus in aquatic products.

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