CAJ | 학술논문

目的建立一种检测副溶血性弧菌(Vp)快速且特异的环介导等温扩增(LAMP)检测方法.方法针对副溶血性弧菌小耐热溶血素(tlh)基因特异性序列的6个位点设计4条LAMP引物,65℃保温约60 min,完成对副溶血性弧菌的扩增,扩增产物经肉眼、SYBR Green I染色、电泳和酶切鉴定.利用I.AMP和普通PCR方法同时检测1株副溶血性弧菌和12株非副溶血性弧菌来验证LAMP方法的特异性;将副溶血性弧菌菌液作一系列lO倍稀释后用LAMP和PCR方法进行检测,比较两者敏感性.结果 1株副溶血性弧菌出现LAMP扩增反应:通过肉眼、SYBR Green I染色和电泳均能观察到LAMP扩增产物的出现,酶切证实了 LAMP产物的特异性;12株非副溶血性弧菌未出现LAMP扩增.LAMP检测tlh基因的特异性和敏感性与普通PCR相同,LAMP检测tlh基因的检测下限为15 cfu/mL.结论建立了一种快速、敏感、特异的副溶血性弧菌检测方法,适合日常监测及快速榆测的需要.
목적 건립일충검측부용혈성호균(Vp)쾌속차특이적배개도등온확증(LAMP)검측방법.방법 침대부용혈성호균소내열용혈소(tlh)기인특이성서렬적6개위점설계4조LAMP인물,65℃보온약60 min,완성대부용혈성호균적확증,확증산물경육안、SYBR Green I염색、전영화매절감정.이용I.AMP화보통PCR방법동시검측1주부용혈성호균화12주비부용혈성호균래험증LAMP방법적특이성;장부용혈성호균균액작일계렬lO배희석후용LAMP화PCR방법진행검측,비교량자민감성.결과 1주부용혈성호균출현LAMP확증반응:통과육안、SYBR Green I염색화전영균능관찰도LAMP확증산물적출현,매절증실료 LAMP산물적특이성;12주비부용혈성호균미출현LAMP확증.LAMP검측tlh기인적특이성화민감성여보통PCR상동,LAMP검측tlh기인적검측하한위15 cfu/mL.결론 건립료일충쾌속、민감、특이적부용혈성호균검측방법,괄합일상감측급쾌속유측적수요.
Objective To develop a loop-mediated isothermal amplification(LAMP) assay for the rapid and specific detection of vibrio parahaemolyticus(Vp). Methods Four primers which recog-nized 6 distinct regions on the thermolabite hemolysin(tlh) gene of Vp were designed and used for LAMP assay. Vp DNA was amplified under isothermal conditions(65 ℃) for 60 rain, then the ampli-fied product was judged by naked eye, SYBR Green I staining, electrophoretie analysis and restriction digestion. To evaluate the specificity of the assay, 1 strain of Vp and 12 strains of non-Vp were tested by LAMP and conventional PCR simultaneously. In addition, the detection limit of LAMP was com-pared with that of PCR by using the Vp strain, that were 10-fold serially diluted and was amplified by LAMP and PCR. Results With 1 strain of Vp, the naked eye, SYBR Green I staining and electro-phoretic analysis could detect the products in the LAMP assay. The specificity of LAMP products was confirmed by digestion of LAMP products using restriction enzymes. Amplification of LAMP was not observed when 12 strains of non-Vp were tested. The specificity and sensitivity of LAMP were similar to those of conventional PCR assay and the detection limit of LAMP assay was 15 cfu/mL. Conclusion These results indicate that the LAMP assay is a rapid, specific and sensitive detection method for Vp. The method is suitable fo: daily monitoring and rapid diagnosis of Vp.