环介导等温扩增技术结合乳胶微球试纸条检测副溶血性弧菌

Detection of Vibrio parahaemolyticus using loop-mediated isothermal amplification combined with latex microsphere test strips

目的建立环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术结合乳胶微球试纸条(latex microsphere test strips,LMTS)快速检测副溶血性弧菌(Vibrio parahaemolyticus,VP)的方法。方法以副溶血性弧菌的不耐热溶血素(TLH)基因作靶标设计引物,6-羧基荧光素(6-carboxyfluorescein,6-FAM)和生物素(Biotin)标记引物,对体系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、聚合酶链式反应(polymerase chain reaction,PCR)和实时荧光定量LAMP(quantitative LAMP,qLAMP)反应,比较三者的敏感度;对副溶血性弧菌、大肠埃希氏菌、福氏志贺氏菌、粪链(肠)球菌、单核细胞增生李斯特菌、鼠伤寒沙门氏菌、铜绿假单胞菌进行LAMP扩增,验证其特异性;虾样品用菌液进行模拟污染,分析LAMP-LMTS的可靠性。结果LAMP-LMTS方法的灵敏度可达4.16×10^(2)copies/μL,与qLAMP、LAMP-琼脂糖凝胶电泳(agarose gel electrophoresis,AGE)方法结果一致,检出限要比利用外引物建立的PCR方法低10倍的拷贝数,灵敏度更高。结论该方法检测副溶血性弧菌具有检测时间短、灵敏度高、特异性强、操作简便等特点,可应用于基层实验室、应急检测或现场监测,具有较高的推广价值。

Objective To establish a rapid detection method for Vibrio parahaemolyticus(VP)using loop-mediated isothermal amplification(LAMP)combined with latex microsphere test strips(LMTS).Methods Using thermolabile hemolysin gene(TLH)of Vibrio parahaemolyticus as target,primers were designed and labeled with 6-carboxyfluorescein(6-FAM)and Biotin,and the reaction parameters of the system were optimized.The fresh bacterial liquid was diluted by gradient of 10 times,and then LAMP,polymerase chain reaction(PCR)and quantitative LAMP(qLAMP)were carried out to compare their sensitivities.The specificity of Vibrio parahaemolyticus,Escherichia coli,Shigella flexneri,Enterococcus faecalis,Listeria monocytogenes,Salmonella typhimurium and Pseudomonas aeruginosa was verified by LAMP amplification.The shrimp samples were polluted by bacterial liquid,and the reliability of LAMP-LMTS was analyzed.Results The sensitivity of the LAMP-LMTS method could reach 4.16×10^(2)copies/μL,consistent with qLAMP and LAMP-agarose gel electrophoresis(AGE)results.The limit of detection was 10 times lower than qPCR method established by using primers,indicating higher sensitivity.Conclusion This Vibrio parahaemolyticus detection method exhibits characteristics of short detection time,high sensitivity,strong specificity,and easy operation.It is applicable to grassroots laboratories,emergency testing,or on-site monitoring,demonstrating significant potential for widespread use.