应用环介导等温扩增检测阪崎肠杆菌

Application of loop-mediated isothermal amplification detection of nterobacter sakazakii

建立环介导等温扩增检测阪崎肠杆菌的方法,通过肉眼可见的白色沉淀,判断检测结果。将阪崎肠杆菌(ATCC29544)的16S-23SrRNA间区序列作为靶基因,设计2对特异引物,优化反应条件,进行LAMP扩增。对产物进行酶切分析,与理论上的预期结果相一致。通过测序比对,与GenBank上报道的同源性达到99%。用25株食源性致病菌证实该引物特异性强。LAMP扩增20min,其灵敏度为4.3×102 fg/tube,结果表明,LAMP方法检测阪崎肠杆菌,灵敏度高、特异性强、耗时短、方法简便。

Establish a loop-mediated isothermal amplification method for detection of the Enterobacter sakazakii,through white precipitates viewed with the naked eye to determine test results.The sequences of 16S-23Sr RNA of Enterobacter sakazakii（ATCC29544）was as target gene,2 pairs of specifical primers were designed.By optimization of reaction conditions,the sequences were amplificated by LAMP.Detection of Enterobacter sakazakii strains by LAMP was positive.The positive products were digested with restriction enzyme,which the theoretical expected results are consistent with.the result of sequencing of PCR with the outer primers of LAMP was compared with gene sequences in GenBank and reached the reported 99% homology.Specificity of the LAMP primers was confirmed using 25 strains of food borne pathogens.The sensitivity of the LAMP was 4.3×102 fg for 20 min.The results showed that,LAMP method for detection of Enterobacter sakazakii was high sensitivity and specificity,shorter time-consuming and simple.