支原体检测多重定量PCR方法的建立及应用

Establishment and Application of Multiplex Quantitative PCR Method for Detection of Mycoplasma

目的:建立一种快速、准确的方法检测支原体,这不仅可以有效地减少和预防支原体污染,还能为科研工作者提供一定的指导价值。方法:利用荧光定量PCR(TaqMan探针法)检测支原体,反应体系中同时存在标记两种颜色TaqMan探针及相关引物,分别检测支原体DNA和参考基因模板。根据支原体16S核糖体RNA保守区和参考基因TOP3A保守区设计引物和探针。通过对引物浓度、探针浓度和退火温度等反应条件的优化,建立了TaqMan探针多重定量PCR方法,并对该方法的特异性、敏感性和重复性进行了验证。结果:建立的双色荧光探针定量PCR方法的标准曲线相关系数r2和扩增效率分别为0.995和113.36%;该方法最低检测限为10 copies/μL;组内及组间变异系数均小于1%,证明该检测方法高效。利用该方法对随机挑选90例细胞抽提DNA样本进行检测,结果有60例为支原体阳性样本,阳性率67%,阳性率与相关研究报道一致。检测3个细胞培养上清样本,结果 1例支原体阳性,2例支原体阴性。从检测的样本中随机选择3个阳性样本及2个阴性样本使用普通PCR支原体检测试剂盒检测,结果一致;将其测序,测序结果比对正确。结论:本研究建立的多重定量PCR支原体检测方法能够应用于细胞抽提DNA及细胞培养上清的支原体检测,可以实现高效、快速检测支原体污染。

Objective: To establish a rapid and accurate method to detect mycoplasma, which can not only effectively reduce and prevent mycoplasma pollution, but also provide some guidance value for researchers. Methods: Mycoplasma was detected by real-time PCR(TaqMan probe method), and two color TaqMan probes and related primers were simultaneously labeled in the reaction system,which aims to detect mycoplasma DNA and reference gene template, respectively. Primers and probes were designed based on the conserved region of the 16 S ribosomal RNA of the mycoplasma and the conserved region of the reference gene TOP3 A. The TaqMan probe multiplex quantitative PCR method was established by optimizing the reaction conditions such as primer concentration, probe concentration and annealing temperature, and the specificity, sensitivity and repeatability of the method were verified. Results: The standard curve correlation coefficient r2 was 0.995 and amplification efficiency was 113.36%;the minimum detection limit of this method was 10 copies/μL;the intra-group and inter-group coefficient of variation was less than 1%, which suggesting that this method has good specificity. Furthermore, detected 90 samples of cell extract DNA samples using this method. The results showed that 60 cases were positive for mycoplasma, and the positive rate was 67%, which was in agreement with the related research reports. Moreover, three cell culture supernatant samples were detected, and one case was positive for mycoplasma and two cases were negative for mycoplasma. Randomly select 3 positive samples and 2 negative samples from the tested samples using the common PCR Mycoplasma test kit. The results were consistent;the sequencing results were correct. Conclusions: The multiplex quantitative PCR mycoplasma detection method established in this study can be applied to the detection of mycoplasma in cell extraction DNA and cell culture supernatant, thereby achieving efficient and rapid detection of mycoplasma.