基于反转录-环介导等温扩增技术检测沙门氏菌

Detection of Salmonella by reverse transcriptase loop-mediated isothermal amplification

摘要:

【背景】沙门氏菌是世界上最常见的食源性致病菌之一，全球每年沙门氏菌中毒事件居细菌性中毒事件首位。【目的】建立反转录-环介导等温扩增(reverse transcriptase loop-mediated isothermal amplification，RT-LAMP)技术特异性检测沙门氏菌。【方法】针对沙门氏菌特异保守invA基因设计多组引物，通过对引物筛选和反应条件优化，建立了检测沙门氏菌的实时荧光RT-LAMP方法。利用沙门氏菌及其人工污染脱脂乳样品，研究了方法的特异性和灵敏性，并与RT-PCR (reverse transcription polymerase chain)方法的灵敏度进行比较。【结果】等温65 °C条件下30 min内可完成RT-LAMP反应。利用该方法检测5株沙门氏菌均为阳性，其余21种检测菌株为阴性。在人工污染脱脂乳样品的检测中，灵敏度达到60 CFU/g，比RT-PCR方法高100倍。【结论】建立的实时荧光RT-LAMP方法将为沙门氏菌现场快速检测提供有力手段。

Abstract:

[Background] Salmonella is currently one of most common food-borne pathogens worldwide. Every year, food poisoning caused by Salmonella ranks the first among all bacterial poisoning incidents. [Objective] A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) method was developed to detect Salmonella. [Methods] A series of primers were designed according to the specific conservative invA gene of Salmonella, Real-time fluorescent RT-LAMP method was established to detect Salmonella by optimizing primer selection and reaction conditions. The specificity and sensitivity of the method were studied by using Salmonella and its artificial contaminated skim milk samples, and compared with the sensitivity of reverse transcription polymerase chain (RT-PCR) method. [Results] The RT-LAMP assay successfully detected invA gene of Salmonella within 30 minutes at 65 °C. Five strains of Salmonella were positive and the other 21 tested strains were negative. In the extracting nucleotide from artificial contaminated skim milk samples, the sensitivity was 60 CFU/g, which was 100-fold higher than that of RT-PCR. [Conclusion] The established real-time fluorescent RT-LAMP method can rapidly detect Salmonella on site.