TaqMan探针荧光定量PCR支原体快速检测方法的建立及应用

Establishment of rapid detection method of major Mycoplasma contaminants in cell cultures using real-time PCR with TaqMan probe

目的:在支原体16SrRNA的种属特异性区域设计一对引物及其相应的Taqman探针,建立荧光定量PCR方法,检测细胞培养物中支原体。方法:选择16srRNA支原体种属特异性区域作为扩增区,设计支原体通用引物,PCR扩增片段的预期大小为288bp。回收PCR扩增产物,连接pMD-18T载体,转化JM109大肠杆菌,涂布Amp+琼脂平板,挑取阳性克隆进行PCR鉴定后提取质粒。构建荧光定量PCR绝对定量的标准品,设计荧光定量引物、探针,采用矩阵法对反应体系进行优化,建立绝对定量的标准曲线。利用十倍稀释法检验灵敏度并与普通PCR法及培养法进行比较;对支原体及其他5种革兰氏阳性杆菌进行特异性检验;对培养法、普通PCR法及荧光定量PCR法检测样本的灵敏度进行比较。结果:支原体PCR产物大小约为288bp,质粒质量浓度为13.9mg·mL-1,A260/A280(Ratio)为1.780,质粒溶液浓度为4.25×109拷贝.μL-1。10μmol.L-1的引物浓度和10μmol.L-1探针浓度为最佳反应浓度,双温循环及60℃退火温度为最佳的循环条件。检测灵敏度为4.250×101拷贝。拷贝数与Ct值之间的线性关系表达式:Ct=-2.916×log拷贝数+40.02。特异性检验中5种革兰氏阳性杆菌样本检测均为阴性,支原体样本为阳性,表明特异性好。准确性检测中变异系数小,表明稳定性好。组内及组间相同浓度样本检测具有相同的Ct值,表明重现性好。样本检验中,荧光定量PCR检测出的阳性样本高于普通PCR及培养法。结论:支原体荧光定量PCR法检测细胞样本及临床样本中支原体快速、特异性强、灵敏度高、稳定性好。

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-1.A260/A280（Ratio）was 1.780.The plasmid concentration was 4.25 × 109copy· μL-1.10 μmol·L-1 primer concentration and 10 μmol·L-1 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.