Sun Chunrong, Wang Chaohong, Xiao Fei, Jia Nan, Huang Xiaolan, Fu Jin, Zhang Yu, Zhou Juan, Wang Guirong, Wang Yi
Experiment Research Center, Capital Institute of Pediatrics, Beijing, 100020, PR China.
Department of Clinical Laboratory, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, 101125, PR China.
Heliyon. 2024 May 24;10(11):e31901. doi: 10.1016/j.heliyon.2024.e31901. eCollection 2024 Jun 15.
Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the sis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 min) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.
世界卫生组织（WHO）推荐的用于结核分枝杆菌（MTB）检测的核酸检测分子技术被认为有潜力实现准确的结核病（TB）报告。在本研究中，开发了一种将实时（rt）荧光技术与多重交叉置换扩增（MCDA）相结合的新方法，用于快速、灵敏且特异的MTB检测（称为MTB-rt-MCDA）。根据rt-MCDA检测原理，设计了一组十条引物用于MCDA反应，其中一条引物带有限制性内切酶识别位点、一个荧光团和一个猝灭剂，以实现实时荧光检测。MTB-rt-MCDA检测在实时荧光平台上于优化条件（67°C，40分钟）下进行。MTB-rt-MCDA检测能够准确鉴定MTB菌株，与其他细菌无交叉反应。MTB-rt-MCDA检测的最低可检测基因组DNA浓度为25 fg/μl。我们采用从临床病例痰液中提取的基因组DNA模板来验证该检测方法的实际适用性，MTB-rt-MCDA检测的检测能力与Xpert方法以及基于MCDA的生物传感器检测相当，且优于涂片显微镜检查法。MTB-rt-MCDA检测的完整过程，包括DNA的快速提取和rt-MCDA检测，耗时不到1小时。总之，所提出的MTB-rt-MCDA检测为MTB感染的快速筛查提供了一种有效且简便的选择。
