粪便基因组DNA提取方法比较及在沙门菌定量PCR检测中的应用

Comparison of three protocols for extraction of total fecal DNA and identification of Salmonella with real time PCR

目的建立并优化1种可用于临床微生物检测的粪便微生物基因组DNA提取方法并应用于沙门菌定量PCR快速检测。方法分别利用土壤微生物基因组DNA提取试剂盒法、微生物基因组DNA提取试剂盒法和本实验室改进酚/氯仿抽提法提取腹泻患者粪便基因组DNA,根据沙门氏菌16srDNA、dT等基因或DNA功能区序列设计PCR引物,进行多基因定量PCR检测。结果3种方法提取的基因组DNA均可进行有效的定量PCR扩增,采用所建立的多重PCR方法可特异鉴别粪便沙门氏菌。结论本研究改进的粪便基因组DNA提取方法及临床微生物的定量PCR检测可在较短的时间内实现对大量临床样品快速诊断,有一定应用前景。

Objective To investigate the effects of different fecal DNA extraction methods on the molecular analysis of the diver sity of getting diarrhea. Methods Three protocols were used to extract total DNA from one fecal sample of an adult with diarrhea. The products of DNA were evaluated by agarose gel electrophoresis and then amplicons of 16S rRNA gene and dT fermentation of the three DNA isolation protocols were compared. Results The DNA products from the three protocols were all amplified hy the primer of S rRNA gene and dT fermentation with real time PCR. Conclusion Different DNA isolation methods have different effects on the amplification of DNA fragments with real time PCR for DNA extraction from fecal samples, the SYBR Green I real time PCR could probably be used to detect other pathogens in fecal samples for Salmonell.