Open AccessNaked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification— SYBR green I assays
Author(s) -
Singpanomchai Nuntita,
Akeda Yukihiro,
Tomono Kazunori,
Tamaru Aki,
Santanirand Pitak,
Ratthawongjirakul Panan
Publication year - 2019
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.22655
Subject(s) - recombinase polymerase amplification , sybr green i , agarose gel electrophoresis , amplicon , microbiology and biotechnology , mycobacterium tuberculosis , polymerase chain reaction , naked eye , biology , chemistry , virology , dna , tuberculosis , gene , genetics , medicine , detection limit , chromatography , pathology
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 , IS 6110 and IS 1081. 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 IS 6110 and IS 1081 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 IS 6110 and IS 1081 were 100%. The specificity of RPA ‐S was 100% for both targets; however, its sensitivities for IS 6110 and IS 1081 were 97.95% and 99.32%, respectively. The limits of detection of IS 6110 RPA ‐ AGE and RPA ‐S were 0.05 and 0.5 ng, respectively, while the LOD s of IS 1081 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.