作　　者： ; ; ; ;

机构地区： 华南农业大学兽医学院广东省兽药研制与安全评价重点实验室

出　　处： 《中国兽医科技》 2005年第6期445-449,共5页

摘　　要： 采用珠磨法、冻融研磨法和酶法对粪便细菌进行裂解,提取粪便细菌DNA,并采用16SrRNAV3区引物(P3GCf、P2r)和V6V8区引物(U968GCf、L1401r)扩增细菌16SrRNA基因可变区,对变性梯度凝胶电泳(DGGE)方法用于肠道菌群多样性分析的各种条件进行了优化,建立了分析人和动物粪便菌群多样性的DGGE方法。2对引物的粪便DGGE结果都表明,珠磨法和冻融研磨法对细菌的裂解效果优于酶法;P3GCf、P2r引物的条带数明显多于U968GCf、L1401r引物;珠磨法PCRDGGE对普通拟杆菌、青春双歧杆菌和粪肠球菌的检测限分别为(6.4±0.1)×102、(6.1±0.7)×103和(7.2±0.2)×105个细菌,珠磨法对这3种细菌的检测限均低于酶法。结果表明,珠磨法和P3GCf、P2r引物更适宜用于粪便菌群多样性DGGE分析。 Effects of difgerent DNA isolation protocols and primers on fecal flora PCR-DGGE profiles were compared and a PCR-DGGE method was developed for the analysis of fecal microflora. Fecal bacterial DNA was extracted by glass bead beating method, freeze/thaw grinding method or enzymatic method. By using P3-GC-f,P2-r primers and U968-GC-f ,L1401-r, bacterial 16S rRNA V3 and V6-V8 regions were amplified separately. Then PCR products were analysed by DGGE. A higher diversity in fecal samples was observed with the bead beating method and freeze/thaw grinding methodthan with the enzymaticmethod by using two pairs of primers. Primers P3-GC-f,P2-r were superior to primers U968-GC-f ,L1401-r for ~fecal bacterial community structure and diversity analysis because the fecal DGGE profile has more bands by using primers P3-GC-f,P2-r . The PCR detection limit of 16S rDNA amplicons from B.vulgatus, B.thetaiotaomicron and E.faecalis were (6.4±0.1)×10~2 ,(6.1±0.7)×10~3 and (7.2±0.2)×10~5 cells by bead beating method. The results indicated that glass bead beating and primers for 16S rRNA V3 region were more efficient for the fecal bacterial community structure and diversity analysis.

关 键 词： 变性梯度凝胶电泳 肠道菌群 提取 菌群多样性

领　　域： [农业科学] [农业科学] [农业科学] [生物学]