Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification-SYBR green I assays
Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification-SYBR green I assays
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DOI：
10.1002/jcla.22655
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发表时间：
2019-02-01
影响因子：
2.7
通讯作者：
Ratthawongjirakul, Panan
中科院分区：
文献类型：
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作者：
Singpanomchai, Nuntita;Akeda, Yukihiro;Ratthawongjirakul, Panan
背景结核分枝杆菌（Mycobacterium tuberculosis，Mtb）的快速诊断是控制结核病传播的关键，结核病是一个全球性的健康问题。本研究开发了等温重组酶聚合酶扩增（RPA）来检测Mtb、IS 6110和IS 1081的特异性靶点。此外，SYBR绿色I用于RPA产品的肉眼终点检测。方法采用RPA技术，在IS 6110和IS 1081上对146份结核分枝杆菌基因组DNA和24份非结核分枝杆菌基因组DNA进行扩增。在完全扩增后，通过琼脂糖凝胶电泳（RPA-AGE）和SYBR绿色I（RPA-S）测定来检查RPA扩增子。RPA测定的性能通过将其与常规PCR进行比较来评价。结果RPA检测方法具有良好的区分结核分枝杆菌和非结核分枝杆菌的能力，且在恒温条件下周转时间很短。与常规PCR相比，RPA-AGE对IS 6110和IS 1081的敏感性和特异性均为100%。RPA-S对两种靶点的特异性均为100%，而对IS 6110和IS 1081的敏感性分别为97.95%和99.32%。IS 6110 RPA-AGE和RPA-S的检出限分别为0.05和0.5 ng，IS 1081 RPA-AGE和RPA-S的检出限分别为0.00005和0.05 ng。两种RPA检测方法均显示出令人满意的诊断特异性，与其他细菌无交叉反应。结论快速、灵敏、裸眼RPA检测可用于结核分枝杆菌的床旁诊断，尤其是在实验室仪器资源有限的偏远地区。
Background 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. Method 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. Results 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. Conclusion 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.