【作者】 刘刚；

【导师】 翟朝阳；

【作者基本信息】 四川大学 ， 遗传学， 2004， 硕士

【摘要】 目的:建立一种多重随机引物PCR方法即Multiplex RAPD技术(M-RAPD技术),探讨其对不同种株病原微生物进行有效、快速鉴定的可能性及应用前景。方法:将10个碱基的寡核苷酸RAPD引物进行随机组合,利用三条组合的RAPD引物在较高的退火温度(40℃)下对不同种株病原微生物染色体DNA进行扩增,通过15g/L琼脂糖凝胶电泳获得不同种株病原微生物的染色体DNA扩增指纹图谱,并用软件Gelworks 1d Intermediate对指纹图谱进行分析。结果:初步实验表明,利用特定的引物组合(S15、S18、S103,S14、S18、S66与S15、S18、S66),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;不同病原微生物间差异显示明显,不同种菌株对于同一RAPD引物组合未发现相同的扩增产物(包括亮度差异),有鉴定不同种类病原微生物的能力,具有临床意义;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50%;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。应用软件Gelworks 1d Intermediate对所得不同病原微生物的M-RAPD指更多还原

【Abstract】 Objective to obtain genomic fingerprints of different pathogenic microbials and analyze whether these patterns can be used to the quick and effective identification of the microbials, a technique called multiplex random amplified polymorphic DNA analysis (M-RAPD)was used. We also discussed the prospect of M-RAPD techniques. Methods arbitrary primers of 10 oligonucleotides were grouped randomly and various microbials chromosomal DNA were amplified with three combinatorial primers in a special higher annealing temperature(40℃). 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 combinational primers(S15, S18, S103; S14, S18, S66 and S15, S18, S66)were gained. The profiles were clear, well-distributed and the number was greater. The products of three primers included most products of every two primers and would appear with no relation to its length, but smallproducts had more opportunity; the discrepancy of different microbials was obvious, there was no identical product between different strains with the same combinational primers(including the difference of concentration), which proved the techniques had the ability to identify various bacterium; three primers could provide a half more information than two primers to the same pathogenic microbialsl; the difference was eye-catching between different drug-resistant strains and also the corresponding reference strains, but different strains of the same microorganism had more similarity than discrepancy. The analytic data of the software Gelworks Id Intermediate was also supporting our results. The value of Posn is the distance to the slot of different profiles in the same lane and the value of Peaks Intensity is the concentration of amplified profiles. The difference of various pathogenic microbials is obvious according to the value of Posn and Peaks Intensity, that is, the discrepancy of wave form. Conclusion The M-RAPD technique is a simple and rapid technique for the identification of different kinds of pathogenic microbials, and can provide rich genetic information, which makes it very suitable to the detection of pathogenic microorganism and large scale epidemiological studies.更多还原