Singpanomchai Nuntita, Akeda Yukihiro, Tomono Kazunori, Tamaru Aki, Santanirand Pitak, Ratthawongjirakul Panan
Program of Molecular sciences in Medical Microbiology and Immunology, Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
Division of Infection Control and Prevention, Osaka University Hospital, Osaka University, Osaka, Japan.
J Clin Lab Anal. 2019 Feb;33(2):e22655. doi: 10.1002/jcla.22655. Epub 2018 Aug 20.
Rapid diagnosis of Mycobacterium tuberculosis (Mtb) is key to controlling the spread of tuberculosis, which is a global health concern. In this study, isothermal recombinase polymerase amplification (RPA) was developed to detect specific targets of Mtb, IS6110 and IS1081. Additionally, SYBR Green I was used for endpoint detection of the RPA products by the naked eye.
A total of 146 genomic Mtb DNA samples and 24 genomic nontuberculous mycobacteria (NTM) DNA samples were amplified at IS6110 and IS1081 by RPA. After a complete amplification, the RPA amplicons were examined by agarose gel electrophoresis (RPA-AGE) and SYBR Green I (RPA-S) assays. The performance of the RPA assays was evaluated by comparing them to a conventional PCR.
The RPA assay demonstrated to have a good capability to differentiate Mtb from NTM with a very short turnaround time at a constant temperature. Compared to conventional PCR, the sensitivities and specificities of RPA-AGE for IS6110 and IS1081 were 100%. The specificity of RPA-S was 100% for both targets; however, its sensitivities for IS6110 and IS1081 were 97.95% and 99.32%, respectively. The limits of detection of IS6110 RPA-AGE and RPA-S were 0.05 and 0.5 ng, respectively, while the LODs of IS1081 RPA-AGE and RPA-S were 0.00005 and 0.05 ng, respectively. Both RPA assays showed a satisfying diagnostic specificity, with no cross-reaction with other bacteria.
A rapid, sensitive, naked eye RPA assay can be integrated into point-of-care diagnosis for Mtb detection, especially in remote areas where laboratory instrument resources are limited.
结核分枝杆菌（Mtb）的快速诊断是控制结核病传播的关键，结核病是一个全球关注的健康问题。在本研究中，开发了等温重组酶聚合酶扩增（RPA）技术来检测Mtb的特异性靶标IS6110和IS1081。此外，使用SYBR Green I通过肉眼对RPA产物进行终点检测。
通过RPA在IS6110和IS1081处对总共146份基因组Mtb DNA样本和24份基因组非结核分枝杆菌（NTM）DNA样本进行扩增。完全扩增后，通过琼脂糖凝胶电泳（RPA-AGE）和SYBR Green I（RPA-S）检测对RPA扩增子进行检测。通过与传统PCR比较来评估RPA检测的性能。
RPA检测显示出在恒定温度下以非常短的周转时间区分Mtb和NTM的良好能力。与传统PCR相比，RPA-AGE对IS6110和IS1081的敏感性和特异性均为100%。RPA-S对两个靶标的特异性均为100%；然而，其对IS6110和IS1081的敏感性分别为97.95%和99.32%。IS6110 RPA-AGE和RPA-S的检测限分别为0.05和0.5 ng，而IS1081 RPA-AGE和RPA-S的检测限分别为0.00005和0.05 ng。两种RPA检测均显示出令人满意的诊断特异性，与其他细菌无交叉反应。
一种快速、灵敏、肉眼可见的RPA检测可整合到用于Mtb检测的即时诊断中，特别是在实验室仪器资源有限的偏远地区。
