含内参基因的高灵敏度生殖支原体双重荧光PCR方法的建立

Establishment of a high-sensitivity duplex fluorescence PCR technique with internal reference genes to detect genital mycoplasma

目的建立一种含有人源性内参基因的高灵敏度双重荧光PCR方法,以期用于生殖支原体感染的快速检测。方法根据生殖支原体MgPar重复序列中保守区域设计并合成特异性扩增引物和探针,添加人β-globin基因检测引物探针作为体系检测内参,建立并优化荧光PCR方法,使用标准浓度核酸进行扩增效率、灵敏度及特异度评价;用该方法检测62份临床标本,并与Mg-Pa荧光PCR结果相比较。结果建立的基于重复序列的荧光PCR方法对生殖支原体的检测限为0.5拷贝,Mg-Pa荧光PCR的检测限提高了一个数量级。该方法扩增16种其他支原体和常见生殖道致病菌均为阴性,特异度为100%。62份临床标本β-globin基因扩增均阳性,生殖支原体检测阳性率为3.2%(2/62)。结论建立的带内参检测生殖支原体的双重荧光PCR方法快速、灵敏、特异,并通过对标本进行质控,减少了假阴性结果的产生,提升了检测的准确性,具有良好的应用前景。

Objective To establish a high-sensitivity duplex fluorescence PCR assay with human-derived internal reference genes in order to facilitate the rapid and accurate detection of mycoplasma in clinical specimens. Methods Conserved regions in repeat sequences of Mycoplasma genitalium were selected to design and synthesize primers and probes.The human beta-globin gene was selected as a reference of this novel system of detection.A real-time PCR detection system was established and optimized,and its efficiency,sensitivity,and specificity were measured using standard concentrations of nucleic acids.Sixty-two clinical specimens were tested with this technique,and results were compared to the results of Mg-Pa fluorescence PCR. Results The limit of detection of the novel PCR technique established in this study was 0.5 copies,which was an order of magnitude higher than the detection limit of the existing real time PCR technique.This technique did not amplify 16 other mycoplasma strains or common genital tract pathogens,representing a specificity of 100%.All 62 clinical specimens were positive for amplification of theβ-globin gene.M.genitalium was detected at rate of 3.2%（2/62）. Conclusion A novel PCR technique with internal reference genes as established here can rapidly,sensitively,and specifically detect M.genitalium in specimens.Quality control in specimens can be performed with this technique,effectively reducing false negatives and improving detection accuracy.