Abstract:

Abstract:Objective To establish  a TaqMan PCR-based method for the detection of Mycoplasma in cell culture through designing  a pair of primers and Taq- Man  probe  according to conservative sequence of Mycoplasma 16s ribosomal RNA gene.Methods 16srRNA Mycoplasma species-specific region was selected as amplified regions,and the Mycoplasma universal primers were designed.The expected size of PCR amplified fragments was 288 bp.Recovery of PCR amplified products,connection of pMD-18T vector,transformation of  E.coli JM109,coating of Amp+ agar plate were made,and the positive clones were picked for  PCR identification,and then the  plasmids were  extracted.The  fluorescence quantitative PCR absolute quantification of standard was constructed, the fluorescence quantitative PCR primers and probe were designed,matrix method was used to optimize the reaction system,An absolute quantitative standard curve was established.Ten-fold dilution method was used to detect the sensitivity and compared with the general PCR method and culture method.The specificities of  5 kinds of gram-positive bacteria and Mycoplasma were  tested.The sensitivities of the samples were compared between  the culture method,general-PCR and fluorescence quantitative PCR assay.Results The size of PCR product  of Mycoplasma  was about 288 bp,the plasmid concentration was 13.9 mg/L.A260/A280 (Ratio) was 1.780. The plasmid concentration was 4.25 × 109copy/ μL.10 μmol/L primer concentration and 10 μmol/L probe concentration were the best response to the concentration.Two-temperature cycles and 60℃ annealing temperature were the best cycling conditions.The detection sensitivity was 4.25 ×101 copies.The expression of the linear relationship between copy number and Ct value: Ct =－2.916 × log copy number of +40.02.In  specificity  test,the   five kinds of gram-positive bacteria samples were negative,Mycoplasma sample was positive,indicating good specificity. CV of the accuracy of detection was small,indicating good stability.The same concentration samples got the same Ct value,indicating good reproducibility.The positive samples obtained by fluorescence PCR were  higher than those  ordinary PCR and culture method in sample test.Conclusion The detection system based on real-time RT-PCR is rapid,consistently sensitive and steady,which could be used to detect the laboratory samples and clinical samples.

Key words: TaqMan probe；Mycoplasma；real-time PCR