免提核酸的高通量DNA检测技术

目的介绍一种基于捕获的高通量DNA检测技术,无需核酸提取,适用于不同样品的各种应用,以提供遗传分析有力的技术手段。方法全血、唾液、干血斑和口腔拭子等样品裂解后释放DNA,经热变性后,通过与一系列特异探针杂交使靶DNA被捕获至96孔板;洗去未结合探针后,直接利用特异性引物进行荧光定量扩增(qPCR),或用酶连接连续排列的杂交探针,形成两端为特定序列的单链DNA模板,最后用通用引物进行qPCR分析;以全血中P16基因为靶标评估免提核酸技术的DNA检测效果,并将其应用于全血、血清及干血片样品中寄生虫DNA的检测,评估其灵敏度和特异性,同时评估了不同储藏方法下唾液及口腔拭子DNA检测的稳定性。结果成功应用于全血、干血斑、唾液、口腔拭子等多种样品的DNA检测;无需核酸提取,唾液及拭子样品在室温及4℃下保存15d后检测仍具有较高的稳定性;该方法对疟原虫的检测下限低至0.06个疟原虫/μL,较传统镜检具有更高的灵敏度,可准确检测到被感染样品中的寄生虫DNA。结论建立了无需核酸提取的高通量DNA检测技术,适用于不同样本类型及靶标检测,为大规模基因筛查分析提供一个灵敏、高效的工具。

High-throughput DNA detecting technology without nucleic acid extraction

Objective To introduce a high throughput DNA detection technique based on capture, which does not require nucleic acid extraction and suitable for various applications with different samples and so to provide a powerful tool for genetic analysis. Methods The whole blood, dry blood spots, saliva and oral swabs were lysed as samples. After washing off the unbound probe, DNA can be amplified by qPCR with specific primers, or enzymatic ligating the probes to form a single strand-template with specific sequence at both ends, and qPCR is carried out with universal primers. By using P16 gene as a target, the effect of this technique was evaluated, and the technique was applied to detect parasite DNA in whole blood, serum and dry blood spots, its sensitivity and specificity were evaluated. The stability of DNA detection was evaluated with saliva and oral swabs under different storage conditions. Results This method can be successfully applied to detect DNA in whole blood, dry blood spots, saliva and oral swabs. The results of saliva and swabs were still stable after being stored at room temperature and 4 ℃ for 15 days.The limit of detection of this method for Plasmodium was 0.06 parasites/μL, which was more sensitive than traditional microscopic examination and can accurately detect the parasite DNA in infected samples.Conclusions A high-throughput DNA detection technique without nucleic acid extraction is developed, which is suitable for different sample types and targets. It can provide a sensitive and efficient tool for large scale gene screening analysis.