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.
Molecular diagnostic methods can be used as complement existing tools to improve the diagnosis of Salmonella typhi. However, they require good laboratory infrastructure thereby restricting their use to some laboratories and studies. Therefore, it is very important to find some new simpler molecular platforms. The recently developed reverse transcription loop-mediated isothermal amplification （LAMP） method is relatively simple and it can be applied for detection of S. typhi. In this study, we designed 6 primers following STY3671 gcne of S. typhi strain CT-18, and set up a RT-LAMP method used for S. typhi test. Pure strain and blood simulation samples of CT-18 were used to determine the sensitivity and specificity of this system. We compared RT-LAMP to a real-time fluorescent quantitative reverse transcription polymerase chain reaction （rRT-PCR） method. Our results show that RT-LAMP reaction will be done within 60 minutes at 65 ℃. All of the samples from 134 S. typhi strains were tested positive by RT-LAMP. No amplification was observed with the 47 non-S, typhi species. The sensitivity ofRT-LAMP in detecting blood simulation samples was 7 cfu/mL and higher than that in rRT-PCR method for 10 times. In conclusion, this RT-LAMP method has great potential as a field usable molecular tool for diagnosis of S. typhi. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and disease control programs.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2013年第7期682-689,共8页
Chinese Journal of Biochemistry and Molecular Biology
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)~~
关键词
伤寒沙门菌
逆转录-环介导等温核酸扩增
检测
almoneUa typhi
reverse transcription loop-mediated isothermal amplification （RT-LAMP）
detection
