FQ-PCR检测肠黏膜结核杆菌DNA对肠结核的诊断价值

目的建立一种实时定量检测结核分枝杆菌（MTB）插入序列IS6110 DNA的方法,探讨其在肠结核（ITB）诊断中的价值。方法采用FQ-PCR技术对36例内镜活检ITB标本（30例石蜡、6例新鲜组织）、36例Crohn’s disease标本（16例石蜡手术标本,15例石蜡内镜活检标本,5例新鲜内镜活检组织）及34例阴性对照标本进行IS6110 DNA的实时定量检测,并比较上述标本抗酸染色结果。结果13例ITB抗酸染色阳性,CD无1例阳性,抗酸染色对ITB诊断的敏感性36.11%。MTBIS6110DNA检测结果：23例ITB（63.89%）阳性,6例CD（16.67%）阳性,另有1例阳性为升结肠腺癌癌旁正常组织。MTBIS6110 DNA在ITB与CD中的阳性率差异有统计学意义（P〈0.05）。FQ-PCR对ITB诊断的敏感性为63.89%,特异性83.33%。FQ-PCR敏感性显著高于抗酸染色（P〈0.05）。MTBIS6110 DNA阳性的ITB标本其定量结果范围为2.44×10^-4-2.26×10^1拷贝/细胞。结论FQ-PCR检测MTBIS6110 DNA是一种快速有效的ITB诊断方法,其定量范围广,检测灵敏度高。对活检组织少、病理改变不典型、抗酸染色阴性组织的诊断和鉴别诊断尤有意义。

The value of fluorescent quantitative polymerase chain reaction detecting Mycobacterium tuberculosis for the diagnosis of intestinal tuberculosis

Objective Use fluorescent quantitative polymerase chain reaction （FQ-PCR） for the detection of Mycobacterium tuberculosis （MTB） DNA to set up a real-time quantitative assay and identify its value for diagnosing intestinal tuberculosis（ITB）. Methods FQ-PCR was performed on 36 endoscopic biopsy specimens from patients with ITB（30 paraffin blocks,6 fresh biopsies）, 36 Crohn＇ s disease （CD） specimens （including 15 paraffin blocks which were surgically resected specimens, 16 paraffin blocks which were endoscopic biopsy specimens and 5 fresh biopsies） from those CD patients and 34 controlled specimens. DNA was amplified by FQ-PCR with the IS6110 primers. In an analysis of 36 specimens from patients with 1TB, results of FQ-PCR were compared with Ziehl- Neelsen （ZN） staining for acid-fast bacilli （AFB）. The sensitivity and＇specificity of FQ-PCR were analyzed. Results MTB IS6110 DNA was positive in 23 1TB and 6 CD patients. The positive rate of MTB IS6110 DNA in ITB is greater than in CD（ P 〈 0. 05 ）. The sensitivity of FQ-PCR detecting MTB IS6110 DNA for ITB was 63.89%, significantly higher than ZN staining （36.11% ）, and the specificity was 83.33 %. The positive specimens comprised MTB with the range from 2.44× 10^-4 copies/cell to 2.26×10^1 copies/ cell. Conclusion FQ-PCR is a rapid and reliable assay for the diagnosis of ITB, especially when the ZN staining is negative.