Evaluation of digital PCR assay in detection of M.tuberculosis IS6110 and IS1081 in tuberculosis patients plasma
Evaluation of digital PCR assay in detection of M.tuberculosis IS6110 and IS1081 in tuberculosis patients plasma
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数字PCR检测结核病患者血浆中结核分枝杆菌IS6110和IS1081的评价
DOI：
10.1186/s12879-020-05375-y
复制
发表时间：
2020-09-07
影响因子：
3.7
通讯作者：
Zhang, Zongde
中科院分区：
文献类型：
--
作者：
Lyu, Lingna;Li, Zihui;Zhang, Zongde
背景
结核病仍然是一个重大的诊断和治疗挑战，全世界涂片和培养阴性发病率很高。传统的诊断试验费时费力，灵敏度低。数字聚合酶链式反应是一种可以检测相对较低丰度的靶序列并获得其绝对拷贝数的新技术。
方法
我们利用250多个样本评估了dPCR在结核病诊断中的准确性，并首次选择了结核分枝杆菌特异的IS1081和广泛使用的IS6110作为dPCR的扩增目标。使用QuantaSoft版本1.7.4.0917(BioRad)计算目标DNA的定量，并使用SPSS版本13.0软件(SPSS Inc.，芝加哥，伊利诺伊州，美国)进行统计分析。
结果
IS6110-dPCR的敏感性高于IS1081，其敏感性和特异性分别为40.6%和93.4%。将结核分枝杆菌DNA拷贝数高的个体因素分为双侧结核、肺外结核和播散型结核时，IS6110和IS1081-dPCR对播散型结核的敏感性最高(IS6110,100%；IS1081，68.8%)，而肺外结核(IS6110，50.0%；IS1081，39.3%)略高于双侧(IS6110，43.3%；IS1081，33.3%)。与传统的结核病诊断试验相比，IS6110IS1081-dPCR联合检测的敏感性不如涂片显微镜或分支杆菌培养，但高于IS6110qPCR(p0.05)，能检出47.4%的涂片阴性肺结核患者。
结论
本研究提示，血浆IS6110-dPCR是一种快速、中等准确度、创伤较小的检测结核分枝杆菌DNA的方法，IS6110-IS1081-dPCR有可能辅助诊断涂阳肺结核。
Background
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.
Methods
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.
Results
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.
Conclusion
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.