LAMP法检测乳粉中的阪崎肠杆菌

Rapid Detection of Enterobacter sakazakii in Infant Formula by Loop-mediated Isothermal Amplification

据WHO最新报告显示,乳粉中的阪崎肠杆菌是导致婴幼儿感染和死亡的主要原因之一。FDA于2002年公布了婴幼儿乳粉中阪崎肠杆菌检测的推荐方法,但检测周期长达1周左右,而且操作复杂,难以满足控制该疾病暴发和传播的需要。DNA环介导等恒扩增(loop-mediated isothermal amplification,LAMP)技术是一种特异性、灵敏度高、检测时间短的新型基因扩增技术。本文以阪崎肠杆菌为研究对象,建立了一套应用于婴幼儿乳粉中阪崎肠杆菌快速检测方法。针对阪崎肠杆菌16s RNA基因设计4条引物特异性识别靶基因的六个特殊区域,并在63℃进行恒温扩增。实验结果表明,使用该方法能够在5h之内检测出复原乳样品中101CFU/mL的阪崎肠杆菌。灵敏度可以达到100fg阪崎肠杆菌基因组DNA。应用环介导等温扩增法,克服了传统检测周期长、操作复杂的缺点,不仅能够用于婴幼儿乳粉中阪崎肠杆菌的检测,而且满足了对于婴幼儿乳粉中阪崎肠杆菌快速检测的迫切需要。

According to a report from WHO, Enterobacter sakazala＇i is one of the most causes of infant infection and death. There are many deficiencies in the general method of isolation and identification ofEnterobacter sakazalai. Loop-mediated isotheimal amplification （LAMP） is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. In this study, the LAMP method was employed in rapid detection ofEnterobacter sakazakii in infant formula. The species-specific primers were designed by targeting the 16s RNA gene. The results showed that Enterobacter sakazakii was rapidly and specifically detected by LAMP method among several common isolates within 5 h at constant temperature of 63 ℃. The method is very sensitive; the 101 CFU/mL of Enterobacter sakazakii or 100 fg genome DNA can be detected within 5 hours. This new method might facilitate genetic analysis applied in Enterobacter sakazakii in infant formula.