粪便DNA抽提方法的比较研究  

A comparison of four methods for extraction of human fecal DNA by using real time PCR

作　　者：吴仲文[1] 韩樱[1] 鲁海峰[1] 李兰娟[1] 盛吉芳[1] 左健[1] 

机构地区：[1]浙江大学附属第一医院传染科卫生部传染病重点实验室,杭州310003

出　　处：《中华检验医学杂志》2007年第1期67-71,共5页Chinese Journal of Laboratory Medicine

基　　金：科技部"973"资助项目(2003CB515506);浙江省医药卫生优秀青年科技人才专项科研基金(2003 QN005);浙江省科技厅重点资助项目(2006C23017)

摘　　要：目的比较国际主要微生态实验室4种常用的核酸抽提方法获得的粪便 DNA 的质量。方法采用针对目的细菌(总细菌、类杆菌.普氏杆菌属组、双歧杆菌、肠杆菌科细菌及肠球菌)16s rDNA 的特异性引物及实时定量 PCR 方法,分析 QIAamp粪便 DNA 试剂盒法(QIA 法)、FastDNAKit 试剂盒法(Fast 法)、酚-氯仿-玻璃珠击打法(酚-氯仿-玻珠法)及 QIAampDNA 粪便试剂盒+玻璃珠击打法(QIA+玻珠法)4种方法抽提获得的10份人粪便 DNA 的质与量。结果通用引物特异性扩增粪便总 DNA,发现4/10份由酚-氯仿-玻珠法获得的粪便 DNA 中存在 PCR 抑制剂,过柱纯化后 PCR 抑制现象消失。实时定量 PCR 检测粪便总细菌量,以 QIA 法与 QIA+玻珠法获得的量最高(分别为12.16±0.18,12.05±0.48),而 Fast 法与酚-氯仿-玻珠法获得的粪便细菌量(分别为11.26±0.40,11.21±0.16)与 QIA 法相比显著低下(均 P<0.05)。采用 QIA 法(11.63±O.23,8.68±0.51)及 QIA+玻珠法(11.67±0.56,8.77±0.56)抽提获得的粪便类杆菌-普氏杆菌及肠杆菌科细菌的 DNA 量显著高于 Fast 法(10.67±0.47,7.39±0.51)及酚-氯仿-玻珠法(10.65±0.33,7.40±0.18)(均 P<0.05)。对于双歧杆菌,酚-氯仿-玻珠法获得的量显著低下。但对肠球菌而言,以 Fast法获得的量最高。结论实时定量 PCR 检测结果表明,QIA 法及 QIA+玻珠法获得的粪便 DNA 质与量总体上优于 Fast 法及酚-氯仿-玻珠法,但根据不同的目的细菌群对象可以选择不同的方法。Objective To compare the relative efficacy and quality of extraction of human fecal DNA using four methods. Methods Real-time PCR were utilized for analysis both quantification and quality of the fecal targeted bacteria （ including gut all eubacterium, Bacter/odes-Ptevotella group, Bifidobacterium spp Enterebacteriaceae and Enterococcus spp） by using 16s rRNA gene- targeted genus or group- specific primer sets. Results The negative rat of PCR product from method 3 （ phenol-chloroform pins bead-boating） was about 40% （4/10） by using universal primers, the PCR inhibition disappeared after fecal DNA purified with column. The total fecal 16s rRNA gene copy numbers （ per gram of wet weight of feces） as well as the nnmhers of Bacteriodes-Prevotella group from method 1 （ QIAamp DNA stool mini kit） and 4 （ QIAamp DNA stool mini kit combined with bead-boating） was higher significantly than that from method 2 （ FastDNA Kit, Bio101 ） and 3 （ P 〈 0. 05, respectively）. Reductions in fecal Bifidobacteria by method 3 were significant compared with that by other three methods （ P 〈 0. 05 ）. The counts of Enterobacteriaceae by methods 2 and 3 were lowered markedly compared that by methods 1 and 4 （ P 〈 0. 05, respectively）. On the contrary, the results of Enterococcus spp from method 2 were higher significantly than that from other three methods （P〈0.05）. Conclusions Our data suggest that the efficacy and quality of extracted human fecal DNA by using QIAamp DNA stool mini kit combined with glass bead beating and QIAamp DNA stool mini kit method were superior to that by Fast DNA Kit and phenol-chloroform plus bead-boating methods, DNA extraction method should be carefully selected with particular regard to targeted bacterium.

关 键 词：粪便 DNA 聚合酶链反应 

分 类 号：R450[医药卫生—治疗学]