Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, 97 Machang Road, Tongzhou District, Beijing, 101149, China.
BMC Infect Dis. 2020 Sep 7;20(1):657. doi: 10.1186/s12879-020-05375-y.
Tuberculosis is still a significant diagnostic and therapeutic challenge with high proportion of smear- and culture- negative incidences worldwide. The conventional diagnostic tests are time-consuming and have a low sensitivity. Digital PCR is a novel technology which can detect target sequences with relatively low abundance and obtain the absolute copy numbers of the targets.
We assessed the accuracy of dPCR in TB diagnosis using more than 250 specimens, and for the first time, we selected M.tuberculosis-specific IS1081 in addition to widely used IS6110 as the amplification targets for dPCR. The quantification of target DNA was calculated using QuantaSoft Version 1.7.4.0917 (BioRad), and SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.
IS6110-dPCR was more sensitive than IS1081, with the sensitivity and specificity accounting for 40.6 and 93.4% respectively. When we classified the TB patients by personal factors for high copy number of M.tuberculosis derived DNA in plasma: bilateral TB, extrapulmonary TB and disseminated TB, the sensitivity of both IS6110- and IS1081- dPCR was the highest in patients with disseminated TB (IS6110, 100%; IS1081, 68.8%), while their sensitivity was a bit higher in patients with extrapulmonary TB (IS6110, 50.0%; IS1081, 39.3%) than that in bilateral TB (IS6110, 43.3%; IS1081, 33.3%). Compared with traditional TB diagnostic tests, joint detection IS6110 & IS1081-dPCR was not as sensitive as smear microscope or mycobacterial culture, but it was higher than IS6110 qPCR (p < 0.05) and was able to detect 47.4% of smear-negative TB patients.
Our study suggested that plasma IS6110-dPCR is a rapid, moderate accurate and less-invasive method to detect M.tuberculosis DNA in plasma of TB patients and IS6110 & IS1081-dPCR has a potential to aid diagnosis of smear-negative TB.
结核病仍然是一个重大的诊断和治疗挑战，在全球范围内，涂片和培养阴性的比例都很高。传统的诊断检测耗时且灵敏度低。数字 PCR 是一种新型技术，可检测相对低丰度的靶序列，并获得靶序列的绝对拷贝数。
我们使用超过 250 个标本评估了 dPCR 在结核病诊断中的准确性，并且首次选择了 M.tuberculosis 特异性 IS1081 作为扩增靶点，而不是广泛使用的 IS6110。使用 QuantaSoft Version 1.7.4.0917（BioRad）计算目标 DNA 的定量，使用 SPSS 版本 13.0 软件（SPSS Inc.，芝加哥，IL，USA）进行统计分析。
IS6110-dPCR 比 IS1081 更敏感，其灵敏度和特异性分别为 40.6%和 93.4%。当我们根据个人因素对血浆中高拷贝数 M.tuberculosis 衍生 DNA 进行分类时：双侧结核病、肺外结核病和播散性结核病，IS6110 和 IS1081-dPCR 的灵敏度在播散性结核病患者中最高（IS6110，100%；IS1081，68.8%），而在肺外结核病患者中略高于双侧结核病患者（IS6110，50.0%；IS1081，39.3%）。与传统的结核病诊断检测相比，联合检测 IS6110 和 IS1081-dPCR 的灵敏度不如涂片显微镜或分枝杆菌培养，但高于 IS6110 qPCR（p<0.05），并能够检测到 47.4%的涂片阴性结核病患者。
本研究表明，血浆 IS6110-dPCR 是一种快速、中度准确且微创的方法，可检测结核病患者血浆中的 M.tuberculosis DNA，IS6110 和 IS1081-dPCR 具有辅助诊断涂片阴性结核病的潜力。
