摘要
建立一种检测沙门菌属快速敏感的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法。针对沙门菌属侵袭蛋白(invA)基因序列的六个区域设计四条LAMP引物,65℃保温约60min,完成对沙门菌属的扩增,扩增产物经电泳和酶切鉴定。利用LAMP和普通PCR方法同时检测4株沙门菌和9株非沙门菌(对照组)来验证LAMP方法的特异性;将肠炎沙门菌菌液做一系列10倍稀释后用LAMP和PCR方法进行检测来比较两者敏感性。4株沙门菌扩增出LAMP特征性梯状条带,9株非沙门菌没有出现LAMP扩增,LAMP产物的特异性通过限制性内切酶得到了证实;LAMP检测沙门菌属的特异性与普通PCR相同,但其敏感性比普通PCR高10倍,LAMP检测沙门菌属的检测下限为10cfu/ml,PCR检测下限为100cfu/ml。LAMP检测速度相比PCR更快速,在60min内即可完成扩增反应。建立了一个快速、特异、敏感的沙门菌属LAMP检测方法。
To develop a loop- mediated isothermal amplification （LAMP） assay for the rapid and sensitive detection of Salmonella. 4 primers which recognized 6 distinct regions on the invA gene of Salmonella were designed and used for LAMP assay. Salmonella DNA was amplified under isothermal conditions （65~0） for 60 min, then, LAMP results were judged by electrophoretic analysis and restriction digestion. To evaluate the specificity of the LAMP assay, 4 strains of Salmonella and 9 strains of non - Salmonella were tested by LAMP and conventional PCR. To compare the sensitivity of the LAMP to that of conventional PCR, S. Enteritidis were 10- fold serially diluted and was amplified by LAMP and PCR. After LAMP reaction, ladder patterns unique to the LAMP assay were observed with 4 strains of Salmonella, amplification was not observed when 9 strains of non - Salmonella were tested. The specificity of LAMP products was confirmed by restriction enzymes. The specificity of LAMP assay was similar to that of a PCR assay, but the sensitivity of LAMP was 10 times higher than that of conventional PCR assay. The detection limit of LAMP assay for invA gene was 10 cfu/ml of S. Enteritidis and that of PCR was 100 cfu/ml. LAMP method is superior to conventional PCR for its rapid detection of S. Enteritidis within 60 min. These results indicate that a rapid, specific, and sensitive LAMP method has been developed for detection of Salmonella.
出处
《实用预防医学》
CAS
2008年第6期1945-1947,共3页
Practical Preventive Medicine
