实时荧光环介导等温扩增技术检测土壤中的类鼻疽伯克霍尔德菌

目的 结合荧光染料EvaGreen与环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)探索快速检测类鼻疽伯克霍尔德菌的可行性。方法 根据类鼻疽伯克霍尔德菌filc基因片段设计了LAMP引物,利用荧光染料EvaGreen建立实时荧光环介导等温扩增技术(real-time fluorescence loop-mediated isothermal amplification,RealAmp),优化反应条件,扩增产物经电泳鉴定。通过类鼻疽伯克霍尔德菌和其他致病菌验证特异性,比较RealAmp与普通LAMP技术的敏感度,并测定人工污染土壤模拟样本的检出限。结果 恒温63 ℃条件下,20~60 min即可完成RealAmp检测;利用该方法检测类鼻疽伯克霍尔德菌为阳性,其余致病菌如鼠疫杆菌、布鲁氏杆菌、大肠杆菌等13株参考菌株及蜱虫的DNA(含大量假单胞菌属细菌)呈扩增阴性;RealAmp技术检测类鼻疽伯克霍尔德菌核酸的灵敏度为10² CFU/mL,检测克隆株质粒的灵敏度可达10¹ copies/μL,比普通LAMP技术的灵敏度高10倍;人工污染土壤模拟样本RealAmp的检出限为4.4×10¹ CFU/g,且20 min左右即可判定结果;直接检测24份浓度为4.4×10¹ CFU/g~4.4×10⁸ CFU/g的类鼻疽伯克霍尔德菌人工污染土壤样本,在检出限以上,检出率同荧光定量PCR一致。结论 该方法特异性强、灵敏度高,且可实时监测类鼻疽伯克霍尔德菌,有望成为快速检测类鼻疽伯克霍尔德菌的有效方法,增强抵御生物恐怖战争的能力。

Detection of Burkholderia pseudomallei in soil by real-time fluorescence loop-mediated isothermal amplification

The purpose of this study is using fluorescent dye EvaGreen and loop-mediated isothermal amplification technology to explore the feasibility of the rapid detection of Burkholderia pseudomallei. The specific LAMP primers designed were based on the filc gene of Burkholderia pseudomallei, and the best reaction conditions with EvaGreen inside for the real-time fluorescence loop-mediated isothermal amplification were optimized,and the products of amplification were detected by electrophoresis. Comparison of the sensitivity between RealAmp and general LAMP technology was conducted, and the detection limits of artificial contaminated soil was evaluated. The results showed that the RealAmp detection could be completed in 20-60 min under the condition of isothermality of 63 ℃; RealAmp is positive to detect Burkholderia pseudomallei but negative for others pathogenic bacteria such as Yersinia pestis, Brucella, Escherichia coli and other 13 strains of standard strains and the DNA from tick (including a large number of pseudomonas bacteria); the detection limita of Burkholderia pseudomallei with RealAmp technology is 10² CFU/mL for nucleic acid, and 10¹ copies/μL for cloning plasmid, which is 10 times higher than those of general LAMP technology. The detection limit by RealAmp in the artificial contaminated soil is 44 CFU/g, and can be determined by the reation in 20 min. The efficiency by RealAmp is in accord with that by the fluorescent quantitative PCR based on the detection of 24 samples of soil contaminated by Burkholderia pseudomallei with concentration gradient 4.4×10¹ CFU/g to 4.4×10⁸ CFU/g. It that due to the high specificity and sensibility on Burkholderia pseudomallei, RealAmp can be a potential method for the fast detection of Burkholderia Pseudomallei, and decreasing the risk of biological terror.