实时荧光环介导等温扩增快速检测鸡肉中沙门氏菌

Development and Application of a Real-Time Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Salmonella enterica ser. Enteritis Retrieved from Chicken

根据沙门氏菌inv A基因序列设计特异性引物,利用Midori Green新型核酸荧光染料,建立了沙门氏菌实时荧光定量环介导等温扩增检测方法。结果表明,检测过程仅需45 min,灵敏度达6 CFU/管,而且细菌数量对数值(lg(CFU/管))与扩增荧光指数增加时间(Tp值)具有良好正相关线性关系,R2为0.985 1。通过模拟沙门氏菌人工污染25 g鸡肉样品,经37℃保温4 h,提取样品制备DNA模板用于实时荧光定量环介导等温扩增检测,结果表明灵敏度达450 CFU/g,共需时约7 h。随机从市场购买冷冻鸡肉样品21份,采用国标沙门氏菌检测方法和鸡肉样品实时荧光定量环介导等温扩增检测进行对比检测,结果两种方法均检测出同一份样品为沙门氏菌阳性,其余20份样品均为沙门氏菌阴性,表明本研究建立的鸡肉沙门氏菌实时荧光定量环介导等温扩增检测,其灵敏性和准确性与国标检测方法相当,但检测时间可缩短至7 h。

In this study,a real-time loop-mediated isothermal amplification（Rti-LAMP） assay system was developed for the rapid detection of Salmonella enterica in chicken.It showed that the lowest level of 6 CFU/reaction for pure Salmonellaculture could be detected in 45 minutes by the Rti-LAMP targeting the inv A gene using Midori Green as nucleic acid dye.Furthermore,25 g of chicken was artificially with various numbers（CFU） of Salmonella,followed by enrichment culture at 37 ℃ for 4 h and then filtration through Whirl-Pak bag and the bacterial cells were pelleted by centrifugation at 13 000 g.The resulting pellets were suspended in saline and processed for cell lysis and DNA purification.Finally,2 μL of purified DNA sample was incorporated into 25 μL of Rti-LAMP reactions at 65 ℃ for 60 min.The lowest level of 450 CFU/g of Salmonella was consistently detected by the Rti-LAMP assay.The entire assay could be completed in 7 h.A total of 21 chicken samples purchased from local market were detected by the Rti-LAMP assay using Salmonella culture as positive control.The same one sample was detected positive to Salmonella by the Rti-LAMP assay and the method described in the Chinese national standard.It suggested that the sensitivity and accuracy of the Rti-LAMP assay were comparable to those of the national standard method,and the former took only 7 h,which was highly time-saving compared with the latter.