评估PCR-荧光探针法在临床标本诊断中的应用价值

Evaluation of PCR fluorescence probe technique in diagnosis of clinical samples

目的评估PCR-荧光探针法在临床标本中快速检测结核分枝杆菌(MTB)和非结核分枝杆菌(NTM)的临床价值。方法收集贵阳市肺科医院2012年4月-2013年2月住院及门诊的2 403例患者送检标本,包括痰液、纤维支气管镜刷检物、胸水标本、脓性分泌物,对标本涂片抗酸染色检测,同时采用PCR-荧光探针法,即用双重PCR技术和Taqman探针技术相结合,提取患者标本中的分枝杆菌核酸进行定性检测,比较两者结果。结果 2 403例患者临床标本经萋尼抗酸染色涂片镜检阳性696例,抗酸染色涂片镜检阴性1 707例,其中非肺结核患者539例,菌阴肺结核患者1 168例;在696例涂片阳性的标本中,应用PCR-荧光探针法,MTB核酸阳性标本635例和NTM核酸阳性9例;1 707例抗酸杆菌涂片阴性标本中MTB核酸阳性264例和NTM阳性6例,539例非肺结核患者标本中检测出MTB核酸假阳性4例;MTB感染患者标本经抗酸染色涂片与PCR-荧光探针法检测,阳性率分别为37.3%及48.8%,即PCR-荧光探针法检测灵敏度较高,差异有统计学意义(P<0.05)。结论利用PCR-荧光探针法可快速在临床标本中检测和区分MTB和NTM,具有较高的敏感性和特异性,值得在临床中推广应用。

OBJECTIVE To evaluate the diagnostic value of PCR‐fluorescence probe technique in rapid detection of clinical Mycobacterium tuberculosis （MTB）and nontuberculosis mycobacteria （NTM ） .METHODS Totally 2 403 samples（sputum ,analytes for fiberoptic bronchoscopy ,pleural fluid and purulent secretion ）from outpatients and inpatients during Apr .2012 to Feb .2013 were detected by PCR‐fluorescence probe technique and acid‐fast stain , i .e .,by combination of duplex PCR and Taqman probe .Nucleic acid （DNA） of Mycobacteria was extracted from patient samples for qualitative assay and the results by acid‐fast stain or PCR were compared .RESULTS In 2 403 samples ,696 cases were positive and 1707 cases were negative detected by acid‐fast stain .In the 1 707 acid‐fast negative cases ,539 cases were diagnosed as non‐tubertulosis and 1168 cases were pulmonary tuberculosis .In 696 acid‐fast positive samples ,635 were MTB positive and 9 were NTM positive detected by PCR‐fluorescence probe technique .In the 1707 acid‐fast negative samples ,264 cases were MTB DNA positive ,and 6 cases were NTM DNA positive .However ,in 539 samples from non‐pulmonary tubertulosis patients ,4 cases were MTB DNA positive .The positive rates detected by acid‐fast stain and PCR‐fluorescence probe technique were 37 .3% （696/1864）and 48 .8% （910/1864） respectively ,indicating that PCR‐fluorescence probe technique was more sensitive than acid‐fast stain （P〈0 .05） .CONCLUSION PCR‐fluorescence probe technique is a rapid method with high sen‐sitivity and specificity to determine and distinguish MTB and NTM in clinical samples ,which is worthy of clinical promotion .