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目的建立反转录-环介导等温核酸扩增(RT-LAMP)方法特异检测甲型副伤寒沙门菌。方法针对甲型副伤寒沙门菌特异保守hsdM基因设计引物,通过优化反应条件,建立检测该靶基因的RT-LAMP体系。利用多种血清型沙门菌及非沙门菌菌株DNA评价该方法的特异性及敏感性。同时对甲型副伤寒沙门菌全血模拟标本及粪便模拟标本进行敏感性检测,并利用临床样本进行初步验证。结果等温65℃条件下,30~60 min内完成RTLAMP反应,利用该方法检测130株甲型副伤寒沙门菌均为阳性,其他非甲型副伤寒沙门菌均扩增阴性。对纯菌RNA检测中检测下限为50 pg/反应,约为19 000个拷贝/反应。在以全血模拟样本的检测中,敏感性达80 cfu/ml,略高于PCR检测低限。对粪便模拟标本的检测中,对原始样品检测下限为500 cfu/g;增菌后样品检测下限为0.8 cfu/g,比PCR检测低限高2~10倍。临床样本中甲型副伤寒患者血液标本检测均阳性,而伤寒样本检测均阴性。结论建立了敏感性高、特异性好的检测甲型副伤寒沙门菌的RT-LAMP方法,为甲型副伤寒沙门菌感染的快速诊断提供了简易手段,可试用于甲型副伤寒的早期诊断。
Objective To establish a reverse transcription-loop-mediated isothermal nucleic acid amplification (RT-LAMP) method for the specific detection of Salmonella paratyphi A. Methods Targeting Salmonella paratyphi-specific conservative hsdM gene, RT-LAMP system was established by optimizing the reaction conditions. The specificity and sensitivity of this method were evaluated using various serotypes of Salmonella and non-Salmonella strains. At the same time, Salmonella paratyphi A whole blood samples and stool samples were tested for sensitivity and clinical samples were used for preliminary validation. Results The RTLAMP reaction was completed within 30-60 minutes under the isothermal temperature of 65 ° C. All 130 strains of Salmonella paratyphi A were positive by this method, and all of the other non-Salmonella paratyphi strains were negative. The limit of detection for pure RNA detection is 50 pg / reaction, about 19 000 copies / reaction. In whole blood samples tested, the sensitivity was 80 cfu / ml, slightly above the detection limit of PCR. For fecal samples, the detection limit of the original sample was 500 cfu / g. The detection limit of the sample after enrichment was 0.8 cfu / g, which was 2 to 10 times higher than the detection limit of PCR. Clinical samples of Paratyphus A blood samples were tested positive, and typhoid samples were tested negative. Conclusion The RT-LAMP method for the detection of Salmonella paratyphi A with high sensitivity and specificity has been established. It provides a simple means for rapid diagnosis of Salmonella paratyphi A infection and can be used for the early diagnosis of Paratyphorin A .