利用M-RAPD技术绘制不同病原微生物基因指纹图  

Application of M-RAPD Technique to Obtain the Genomic Fingerprints of Various Pathogenic Microbials

作　　者：刘刚[1] 陶传敏[2] 翟朝阳[1] 

机构地区：[1]四川大学基础医学与法医学院生物化学与分子生物学实验室,成都610041 [2]四川大学华西医院实验医学科临床微生物室

出　　处：《四川大学学报（医学版）》2004年第4期563-567,共5页Journal of Sichuan University(Medical Sciences)

摘　　要：目的　探讨一种多重随机引物 PCR方法 (M- RAPD)对不同种株病原微生物进行鉴定的可能性。方法　将 1 0个碱基的随机引物进行随机组合 ,利用三条组合的随机引物在较高的退火温度下对不同种株病原微生物染色体 DNA进行扩增 ,通过 1 5 g/L 琼脂糖凝胶电泳获得不同种株病原微生物的扩增图谱 ,并用软件 Gelworks1 d Intermediate进行分析。结果　初步实验表明 ,M- RAPD技术可以得到稳定的种株特异的 DNA指纹图谱 ,其条带清晰、数目较多、片段大小分布均匀。三引物的扩增产物包含了其中任意两引物的绝大多数扩增产物 ,大、小片段均可出现增减 ,但小片段更易增加 ;对同一种病原微生物三引物所提供的基因信息要远多于两引物所提供的信息 ,增加的信息量约为原来的 5 0 % ;不同耐药菌株间及其与相应标准株间差异亦很明显 ,但同种不同株间相同的扩增产物要多于不同的扩增产物。软件 Gelworks1 d Interm ediate的分析数据亦支持我们的实验结果。结论　 M- RAPD技术对不同种株病原微生物染色体 DNA扩增时 ,能获取较丰富的遗传信息 ,可迅速、简便地鉴定不同种株的病原微生物。Objective To obtain genomic fingerprints of different pathogenic microbials and make certain whether their patterns can be used in the identification of microbials by means of multiplex random amplified polymorphic DNA analysis (M RAPD). Methods Arbitrary primers of 10 oligonucleotides were randomly grouped, and various microbials chromosomal DNA were amplified with three combinatorial primers at a special higher annealing temperature. The products were detected by 15 g/L agarose electrophoresis and the patterns were analyzed by the software of Gelworks 1d Intermediate. Results Specific and resistant DNA fingerprints for different pathogenic microbials with combinatorial primers were gained. The profiles were clear, well distributed and the number was great. The products of the three primers included most products of every two primers and would appear with no relation to their length, but small products had more opportunity; three primers could provide information contents half as many again as that two primers could provide for the same pathogenic microbials. There were obvious differences among different drug resistant strains and between the drug resistant strains and the corresponding reference strains, but different strains of the same microorganism had more similarity than discrepancy. The analytic data of the software of Gelworks 1d Intermediate also support our results. Conclusion M RAPD is a simple and rapid technique for the identification of different kinds of pathogenic microbials, and it can provide rich genetic information.

关 键 词：多重随机引物PCR方法 病原微生物 基因指纹图 

分 类 号：R37[医药卫生—病原生物学]