关键词: 种特异性, pvpA, 鸡毒支原体, 实时荧光PCR, SYBR Green I

Abstract:

【Objective】The objective of this study is to develop a SYBR Green I Real-Time PCR assay with species-specific surface-exposed protein gene pvpA as the target for detection of Mycoplasma gallisepticum. 【Method】A pair of primers was designed within highly conservative region of M. gallisepticum pvpA gene following BLASTn all published pvpA sequences accessible to GenBank. Ninety bp fragment of pvpA was amplified from M. gallisepticum isolate, then cloned into PMD19-T vector. Confirmed with PCR or digestion with EcoRⅠand Hind Ⅲ, a recombinant plasmid rPvpA90 was obtained when competent E.coli DH5α was transformed with the PMD19-pvpA vector. The positive plasmid rPvpA90 was used as the quantitative template to generate standard curve and melt curve. Analytical specificity, sensitivity, and reproducibility were evaluated respectively and clinical sampiles were detected. 【Result】The results demonstrated standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration. The melt curve was specific with the correlation coefficient of 0.990. The test had a detection limit of 74 copies per 20 μL when tested with M. gallisepticum genomic DNA. The sensitivity of the assay was at least 100-fold higher than that of the conventional PCR. The assay was confirmed to be highly specific when amplified with either single or mixed DNA sample from microorganisms. The variation coefficient of Ct values of diluted standard DNA was less than 2%, which indicated a good reproducibility. The test result of clinical samples demonstrated that the detection rate of the assay was significantly higher than that of the conventional PCR.【Conclusion】The developed Real-Time PCR assay was highly specific, sensitive, and reproducible and could be a potential tool for diagnosis and monitoring of M.gallisepticum in poultry farm.

Key words: species-specific, pvpA gene, Mycoplasma gallisepticum, Real-Time PCR, SYBR Green I dye