沙门氏菌LAMP检测方法的建立

Establishment and use of a loop-mediated isothermal amplification technique to detect Salmonella

目的建立检测沙门氏菌的环介导等温扩增技术。方法针对沙门氏菌侵袭蛋白基因(invA),用LAMP引物在线设计软件设计4条特异性引物(内、外引物各2条),建立了快速检测食品中沙门氏菌的环介导等温扩增方法。对dNTP浓度、Mg2+浓度、内引物浓度和外引物浓度等反应条件进行优化,并测定方法的特异性和灵敏度,然后用该方法对猪肉模拟样品和猪肉实物样品进行检测。结果优化的LAMP反应体系为:0.8 mmol/L dNTP,6.0 mmol/L MgCl2,0.2μmol/L外引物F3和B3,1.6μmol/L内引物FIP和BIP。对6种相关肠道细菌进行LAMP扩增,仅含invA基因的沙门氏菌扩增阳性。对细菌纯培养物检测的灵敏度为101 cfu/ml,对猪肉模拟样品检测的灵敏度为102 cfu/g。采用环介导等温扩增方法检测57份猪肉实物样品,invA基因阳性5份。LAMP方法检测(包括DNA提取、LAMP反应和电泳分析)可在2h内完成。结论检测沙门氏菌的环介导等温扩增方法快速、灵敏、特异,适合在基层食源性疾病监测和应急检测中应用。

Objective To establish a loop-mediated isothermal amplification（LAMP）technique for rapid detection of Salmonella. Methods Four primers（two outer and two inner）were designed specifically in accordance with the invA gene of Salmonellausing Primer Explorer software.A LAMP technique for rapid detection of Salmonellain food samples was established.The LAMP reaction conditions were optimized,including the concentration of dNTP,Mg2＋,and primer.The specificity and sensitivity of this system were evaluated.Salmonella was detected in artificially contaminated pork and pork samples. Results The optimal LAMP system consisted of 0.8mmol/L of dNTP,6.0mmol/L of MgCl2,0.2μmol/L of the outer primers F3 and B3,and 1.6μmol/L of the inner primers FIP and BIP.LAMP accurately identified Salmonellaisolates carrying the invAgene but failed to detect 6other bacteria.The sensitivity of the LAMP technique was 101 cfu/ml for bacterial samples and 102 cfu/g for Salmonellain artificially contaminated pork.The assay identified 5samples with Salmonella with invAprimers from 57 pork samples.The LAMP assay allowed Salmonellato be detected in less than 2h（including DNA extraction,LAMP,and electrophoresis）. Conclusion The LAMP technique for detection of Salmonellais rapid,specific,and sensitive,and this technique is suitable for monitoring and promptly detecting foodborne diseases in basic laboratories.