目的 建立反转录-环介导等温核酸扩增(RT-LAMP)方法特异检测伤寒沙门菌。 方法 针对伤寒沙门菌 fliC-d 基因设计6条特异性引物,通过优化反应条件,建立检测该靶基因的RT-LAMP方法,利用48个血清型的纯培养伤寒和非伤寒沙门菌菌株、常见非沙门致腹泻病原菌以及发热为主要症状的8种常见病原菌RNA评价该方法的特异性及敏感性,同时对伤寒沙门菌全血模拟标本进行敏感性检测,并与rRT-PCR方法的敏感性进行比较。 结果 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44株伤寒沙门菌均阳性,除4种少见的非伤寒沙门菌血清型扩增阳性外,其余30种非伤寒沙门菌血清型、致腹泻的其他5种肠道致病菌以及发热为主要症状的8种常见非沙门菌也均扩增阴性。在对纯菌总RNA检测中,RT-LAMP的最低检测限为0.5 pg/反应,即97个拷贝/反应。在以全血模拟样品提取核酸为模板的检测中,敏感性达1 cfu/ml,比rRT-PCR检测低限高100倍。 结论 建立了敏感性高、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及不明原因发热症状的病原初步鉴别提供了简便的手段,可用于对伤寒的早期诊断、预防控制及临床治疗。
Objective To establish reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect Salmonella Typhi in blood. Methods A set of six specific primers recognizing distinct regions of fliC-d gene of S. Typhi were designed and modified to establish RT-LAMP assay and the specificity and sensitivity of RT-LAMP with RNA as template were evaluated and verified by detecting cultured Salmonella and non-Salmonella strains in 48 serotypes, the non-Salmonella strains causing diarrhea and the strains of 8 bacteria inducing bacteremia with fever as main symptom. The simulated blood specimens supplemented with S. Typhi were tested with RT-LAMP assay, and the detection limit of RT-LAMP was compared with that of real-time PCR. Results The RT-LAMP assay successfully detected fliC-d gene of S. Typhi within 30 to 60 minutes at 65 ℃. Totally 44 strains of S. Typhi were detected to be positive. Except 4 rare serotypes of non-salmonella were positive in amplification, the amplification of the strains of non-Salmonella in 30 serotypes, the strains of 5 pathogens causing diarrhea and the strains of 8 bacteria causing bacteremia with fever as main symptom were negative. In the detection of total RNA from pure cultured isolates, the detection limit of RT-LAMP was 0.5 pg per reaction (97copies per reaction). In the extracting nucleotide from simulated blood, the sensitivity was 1 cfu /ml, which was 100 fold higher than that of real time PCR. Conclusion The RT-LAMP assay for detecting S. Typhi with high sensitivity and specificity was established, which would be suitable for rapid diagnosis of S. Typhi infection and identifying pathogen of fever with unknown origin , and can be used in early diagnosis, prevention/control and treatment of typhoid fever.