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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 biology diagnostic technique is an important assistant method for the detection of Salmonella typhi, however, it is very important to establish a simple detection platform because of the limited application and development of experimental equipment and conditions.Reverse transcription-mediated Currently, the method of reverse transcription loop-mediated isothermal amplification (RT-LAMP) is rapidly developed for the detection of pathogenic microorganisms, and the method is relatively simple and rapid.This study designed six specific primers for Salmonella typhi STY3671 gene , RT-LAMP method was established to detect the target gene by optimizing the reaction conditions.The specificity and sensitivity of this method were studied by using the standard strain of CT-18 and its blood simulant, (RT-LAMP) assay was performed within 30-60 minutes under isothermal temperature of 65 ° C. The results showed that 134 strains of S. typhi were all positive , And the remaining 47 kinds of test strains were negative for amplification.In the whole blood analog samples extracted nucleic acid as a template for detection, Up to 7 cfu / mL, 10-fold higher than the lower limit of detection by rRT-PCR.The results of this study indicate that the method we developed to detect Salmonella typhi in blood with high sensitivity and specificity was Salmonella typhi The rapid diagnosis provides a simple means for the early diagnosis of typhoid fever, prevention and control and clinical treatment.