分子信标荧光定量PCR技术检测结核分枝杆菌方法建立及其临床应用

目的 建立结核分枝杆菌的分子信标荧光定量PCR检测方法 ,探讨该方法 检测结核分枝杆菌的临床应用价值.方法 针对结核分枝杆菌Ag85B DNA特异保守序列设计引物和分子信标探针,并构建标准品.采用分子信标荧光定量PCR法、抗酸染色法、集菌培养法检测158例肺结核患者痰液标本,比较3种方法 的检出率.结果 所建立方法 最低检测限度为2个基因拷贝/反应,在2×10~2～2×10~8基因拷贝/mL范围内,Ct值同DNA量的对数呈线性关系.该方法 检测5株非分枝杆菌和6株非结核分枝杆菌结果 均为阴性,检测结核分枝杆菌H37Rv出现75 bp特异条带.分子信标荧光定量PCR法、抗酸染色法、集菌培养法阳性检出率分别为60.13%、16.46%、31.01%,荧光定量PCR法检出率明显高于抗酸染色法和集菌培养法.结论 分子信标荧光定量PCR法具有较高的灵敏度和特异度,对结核病的诊断有一定的临床意义.

Establishment and Application of Molecular Beacon Real-time PCR in the Detection of Mycobacterium tuberculosis

Objective To establish a method for detection of M.tuberculosis by molecular beacon real-time PCR and evaluate its application in the clinical detection of M.tuberculosis.Methods The primers and molecular probe were designed to M.tuberculosis specific DNA sequence for AgS5B,and constitute a standards.158 sputum specimens from patients with pulmonary tuberculosis were detected using molecular beacon real-time PCR.acid-fast stain and cultivation,and the detection rates of the three methods were compared.Results The lowest detection limit of the detected method is 2 genome copies per reaction,the linear standard curve was obtained between 2×10~2 and 2×10~8 DNA copies/mL.The method detected 5 strains of non-mycobacterium,6 strains of non-tuberculosis mycobacterium and M.tuberculosis H37Rv,only M.tuberculosis H37Rv had 75 bp specific band.The positive rates of detection using molecular beacon real-time PCR,acid-fast stain and cultivation were 60.13%,16.46%,31.01%respectively,the positive rates of detection of real-time PCR was significantly higher than that of acid-fast stain and cultivation.Conclusion Molecular beacon real-time PCR has high sensitivity and specificity in the detection of M.tuberculosis,which is clinical significance for the diagnosis of tuberculosis.