利用RT-LAMP技术鉴别伤寒沙门菌

发布时间：2018-03-26 05:29

本文选题：伤寒沙门菌　切入点：逆转录-环介导等温核酸扩增　出处：《中国生物化学与分子生物学报》2013年07期

【摘要】：分子生物学诊断技术是用于检测伤寒沙门菌的重要辅助手段,但由于需要较好的实验设备和条件使其应用与发展受到限制,所以建立简捷的检测平台显得尤为重要.逆转录-环介导等温核酸扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)方法目前用于病原微生物的检测发展迅速,此方法相对简单、快速.本研究针对伤寒沙门菌STY3671基因设计6条特异性引物,通过优化反应条件,建立了检测该靶基因的RT-LAMP方法.利用伤寒标准菌株CT-18及其血模拟标本,研究了该方法的特异性及灵敏性,并与rRT-PCR(real-time fluorescent quantitative reverse transcription polymerase chain reaction)方法的敏感性进行比较.结果显示:等温65℃条件下,30～60 min内可完成RT-LAMP检测反应,利用该方法检测134株伤寒沙门菌均为阳性,其余47种检测菌株均扩增阴性.在以全血模拟样品提取核酸为模板的检测中,敏感性达7 cfu/mL,比rRT-PCR检测低限高10倍.本研究结果表明:我们建立的敏感性高、特异性好的RT-LAMP检测血液中伤寒沙门菌的方法,为伤寒沙门菌感染的快速诊断提供了简便的手段,可用于对伤寒的早期诊断、预防控制及临床治疗.
[Abstract]:Molecular biological diagnostic technique is an important auxiliary method for detection of Salmonella typhimurium, but its application and development are limited by the need of better experimental equipment and conditions. Therefore, it is very important to establish a simple detection platform. Reverse transcription loop-mediated isothermal amplification reverse transcription loop-mediated isothermal amplification RT-LAMPP has been developed rapidly in the detection of pathogenic microorganisms, and this method is relatively simple. In this study, six specific primers were designed for the STY3671 gene of Salmonella typhimurium. By optimizing the reaction conditions, a RT-LAMP method for detecting the target gene was established. The standard typhoid strain CT-18 and its blood mimic samples were used to detect the target gene of Salmonella typhimurium. The specificity and sensitivity of this method were studied and compared with that of rRT-PCR(real-time fluorescent quantitative reverse transcription polymerase chain reaction. 134 strains of Salmonella typhimurium were positive by this method, and all the other 47 strains were amplified negative. The sensitivity was 7 cfur / mL, 10 times higher than the lower limit of rRT-PCR. The results showed that the high sensitivity and high specificity RT-LAMP method for detection of Salmonella typhimurium in blood provided a simple and convenient method for the rapid diagnosis of Salmonella typhimurium infection. It can be used for early diagnosis, prevention, control and clinical treatment of typhoid fever.
【作者单位】：中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室;
【基金】：国家十二五重大专项-重大传染病应急处置检测技术平台(No.2011ZX10004-001) 国家十一五重大专项——病原体网络化监测技术研究(No.2008ZX10004-008)~~
【分类号】：R378

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