病原微生物DNA的超快速提取方法建立与应用

Development and application of an ultra-fast DNA extraction method of pathogenic microorganism

目的:为明显提高基于实时荧光定量PCR的微生物检测速度,应用于突发公共卫生事件发生时传染性疾病的早期诊断。方法:在煮沸裂解法的基础上对提取剂与提取时间进行改进与优化,并与优质柱式核酸提取试剂盒法、煮沸裂解法进行提取时间与提取效果比对。结果:超快速法可在5 min内完成病原菌DNA提取,煮沸裂解法与柱式核酸提取试剂盒法分别需要30 min和50 min。3种提取方法对不同病原体的提取效果通过对扩增曲线形态、灵敏度、重复性与ct值4项指标的比较均呈现高度一致性,ct值误差均<±1.5S、cv值均<3.9%,3种方法的ct值数据经F与t检验比较差异均不显著。结论:超快速法对病原微生物DNA的提取效果良好.提取速度明显快于柱式核酸提取试验盒法及煮沸裂解法,与实时荧光PCR配套可明显缩短检测所用时间,多次用于突发公共卫生事件应急检测取得良好效果。

Objective：Drastically speed up the Real -time fluorescence quantitative PCR based pathogenic microorganism detection, and applied to early diagnosis of infectious disease in public health emergency. Methods ： The method was developed based on the boiling - lysis process, with improvement and optimization on extraction reagents and time. Timing and extraction ef- ficiency of our ultra - fast method were compared with column nuclear extraction kit and traditional boiling - lysis method. Results ： DNA extraction from pathogenic microbes could be done within five minutes using ultra - fast extraction method, while the column nuclear extraction kit and traditional boiling - lysis method need 30 to 50 minutes for the whole process. We applied these 3 different extraction methods for extracting DNA from various pathogenic microorganisms, then use the DNA as the templates for Real - time fluorescence quantitative PCR, the amplification curve, sensitivity, repeatability and CV are highly consistent： CT error 〈 ± 1.5S, all the cv value 〈3.9%, and shown no statistical significance according F and t test. Conclusion： Using the Ultra - fast DNA extraction method for pathogenic microorganism detection Our method needs much less time for DNA extraction comparing with column nuclear extraction kit and traditional boiling - lysis method, Combined with Real - time fluorescence quantitative PCR, it drastically cut the time required for the whole detection process. This method has been applied many times is the public health emergencies and returned good effects.