Zhang Ruiqing, Ou Xichao, Sun Xiuli, Fan Guohao, Zhao Bing, Tian Fengyu, Li Fengyu, Shen Xinxin, Zhao Yanlin, Ma Xuejun
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
Front Microbiol. 2023 Apr 27;14:1141424. doi: 10.3389/fmicb.2023.1141424. eCollection 2023.
The World Health Organization (WHO) Global tuberculosis Report 2021 stated that rifampicin-resistant tuberculosis (RR-TB) remains a major public health threat. However, the in-practice diagnostic techniques for RR-TB have a variety of limitations including longer time, lack of sensitivity, and undetectable low proportion of heterogeneous drug resistance.
Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison.
The sensitivity of the MLP-RAP assay could reach 5 copies/μl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/μl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%.
MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.
世界卫生组织（WHO）《2021年全球结核病报告》指出，耐利福平结核病（RR-TB）仍然是一项重大的公共卫生威胁。然而，RR-TB的实际诊断技术存在多种局限性，包括时间较长、缺乏敏感性以及无法检测到低比例的异质性耐药。
在此，我们开发了一种基于多重锁核酸（LNA）探针的RAP方法（MLP-RAP），用于更灵敏地检测RR-TB的多个点突变及其异质性耐药。对从中国疾病预防控制中心国家结核病参比实验室收集的126株临床分离株和78份痰液样本进行了MLP-RAP检测。同时，还进行了qPCR以及巢式PCR产物的Sanger测序检测以作比较。
使用重组质粒时，MLP-RAP检测的灵敏度可达5拷贝/μl，比qPCR（100拷贝/μl）灵敏20倍。此外，对利福平异质性耐药的检测能力为5%。MLP-RAP检测对核酸提取的要求较低（煮沸法），置于荧光定量PCR仪中1小时内即可完成反应。临床评估结果表明，MLP-RAP方法对密码子516、526、531和533的覆盖特异性良好。78份煮沸痰液样本中有41份经MLP-RAP检测呈阳性，经巢式PCR产物的Sanger测序进一步证实，相反，qPCR仅能检测到32份样本。与巢式PCR产物的Sanger测序相比，MLP-RAP检测的特异性和灵敏度均为100%。
MLP-RAP检测能够高灵敏度和特异性地检测RR-TB感染，表明该检测方法在配备荧光定量PCR仪的普通实验室中具有用于快速、灵敏检测RR-TB的应用前景。
