捕获和连接的环介导等温扩增技术

Capture and ligation-based loop-mediated isothermal amplification technology

目的建立捕获和连接的环介导等温扩增(CLIP-LAMP)技术,为医学诊断和病原体检测提供新的技术手段。方法根据靶标DNA或RNA序列设计特异性的捕获探针和具有茎环结构的检测探针。无需核酸提取,直接裂解全血和干血片等样本后,通过捕获探针将靶核酸固定于96孔板上。检测探针进行特异性的连接反应,形成扩增模板,进行荧光定量的环介导等温扩增反应。将此方法应用于疟疾的检测中,定量检测疟原虫的18S rRNA,评估方法的灵敏度、特异性和重复性,并将其应用于临床样本的检测。结果此法具有较高的灵敏度和特异性,可检测到低至0.01个/μL的疟原虫,较传统的镜检和RDT灵敏度高,可准确检测到疟原虫的感染样品。此方法可在4 h内完成96个样本的检测,快速高效。结论建立了一种捕获和连接的等温扩增技术,为疾病的分子诊断和基因筛查提供了新的灵敏、高效的方法。

Objective To establish a capture and ligation-based loop-mediated isothermal amplification technology(CLIP-LAMP)based on probe capture and ligation reaction to provide a new method for laboratory diagnosis and pathogen detection.Methods Capture probes and detection probes were designed with step-loop structure which can hybridize with the target DNA/RNA sequence.Whole blood or dry blood smears were lysed directly in the 96-well capture plate and target nucleic acid was fixed in the tube with capture probes without nucleic acid extraction.Detection probes which hybridized to target sequence were ligated to form a dumb-bell form DNA structure,which can serve as a template in the loop-mediated isothermal amplification.The specificity and sensitivity of this technique were evaluated by application to quantitative detection of 18S rRNA in plasmodium.Moreover,the technique was used to test some clinical samples.Results This method was successfully applied in plasmodium detection with high sensitivity and specificity.The method detected as low as 0.01 parasites/μL,which was more sensitive than RDT and traditional microscopy.Moreover,it accurately detected the 18S rRNA of parasite in infected samples.The method was able to test 96 samples in 4 hours.Conclusions A nucleic acid extraction-free,capture and ligation-based loop-mediated isothermal amplification is developed,which provide a more efficient and sensitive method formolecular diagnosis and screening analysis.