鸡毒支原体PCR检测及特异扩增片断的克隆与测序

POLYMERASE CHAIN REACTION FOR DETECTION OF MYCOPLASMA GALLISEPTICUM AND CLONING AND SEQUENCING OF THE AMPLIFED DNA

根据ERNascimento等从鸡毒支原体(Mycoplasmagallisepricum,MG)基因文库分离的种特异性片段fMG2序列,设计合成一对长25bp的引物L1R1,经PCR反应条件优化选择,对5株MGDNA扩增均产生出预期的732bp特异扩增片断,而不能扩增滑液支原体(Mycoplasmasynoviae,MS)、Ecoli、PUC19质粒DNA,符合设计要求;DIG随机引物法标记扩增产物制成DNA探针,与上述菌株DNADotblot杂交,MG呈阳性,其它为阴性;将扩增产物克隆于pGEMT载体,经EcoRI和RsaI酶切,证实克隆片段与目的DNA大小及酶切位点相同;测序结果表明扩增片段与fMG-2靶序列同源性达972%,与已发表的DNA序列无明显同源性。说明所建PCR方法种特异性强,特异扩增片段克隆成功。

Based on the DNA sequence data of fMG 2 isolated from a MG DNA genomic library,a pair of 25bp primers (L 1,R 1) were synthesized and then used in PCR under optimum condition The result showed that,DNA of the 5 MG strains yielded an expected 732bp product while there was no amplified product in the DNA of non MG such as MS、E coli and PUC19 plasmid DNA The PCR product was used as a probe labelled with DIG 11 dUTP to hybridize with all the DNA above Dot blot assay suggested that the probe hybridized with the 5 MG strains but not with MS、E coli and PUC19 The amplified 732bp DNA was cloned into plasmid vector PGEM T,the positive recombinants were screened through PCR and restriction endonuclease analysis with EcoRI and RsaI One of them was chosen for sequencing It was proved that the PCR product was complete and correct in sequence It had no significant homology with other published DNA seuqences The MG PCR could detect as low as 1 pg MG DNA