| PCR is a simple and quick detection method with the characters of specificity and sensitivity. It has been widely used for the early diagnosis of mycoplasmosis and preliminary screening of mycoplasma contamination. The target sequences of detecting mycoplasma were usually designed in the highly conservative domain of mycoplasma genera (rRNA gene) or some functional gene domains. The exons of mycoplasma functional genes were specific but obvious in heterology, so PCR assay was low in sensitivity. The16S rRNA gene with high sensitivity was ideal target sequence in PCR detection. However, because of the cross reaction with other mycolasmas and the poor reproducibility, the16S rRNA gene application was subject to certain restrictions. So the PCR applied in detecting mycolasmas, The selection in target genes had become the experimental crux in the characters of specificity and sensitivity.In this study, Mycoplasma gallisepticum(MG) was incubated and collected, then the genomic DNA of MG was extracted and purified. According to the MG gene sequences published in GenBank, the highly conservative domain of16S rRNA gene, pvpA gene and TM-1gene were selected as the target genes. Specific primers were designed respectively, the PCR assay was established and the sensitivity and specificity were compared. The results showed that as low as2.75×10-8ng/μ L MG DNA could be detected by the PCR using the target sequence of16S rRNA,0.028ng/μL MG DNA could be detected using the target sequence of TM-1, and0.275ng/μL MG DNA could be detected using the target sequence of PvpA. In the specificity test, it indicated that only MG DNA was positive, the DNA or the cDNA of other species were all negative. Then the established PCR methods were applied to detect MG in the samples such as vaccines, chick embryo and eggs, and the results were effective. The broad-spectrum and precise PCR methods were established and it would make some basis for the clinical detection of MG. |
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