Nuntita Singpanomchai1, Yukihiro Akeda2, Kazunori Tomono2, Aki Tamaru3, Pitak Santanirand4, Panan Ratthawongjirakul5. 1. 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. 2. Division of Infection Control and Prevention, Osaka University Hospital, Osaka University, Osaka, Japan. 3. Department of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan. 4. Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. 5. Research Group of Innovative Diagnosis of Antimicrobial Resistance, Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
Abstract
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.
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.
Authors: Christian A Ganoza; Jessica N Ricaldi; José Chauca; Gabriel Rojas; César Munayco; Juan Agapito; Juan Carlos Palomino; Humberto Guerra Journal: J Med Microbiol Date: 2008-09 Impact factor: 2.472
Authors: David S Boyle; Ruth McNerney; Hwee Teng Low; Brandon Troy Leader; Ailyn C Pérez-Osorio; Jessica C Meyer; Denise M O'Sullivan; David G Brooks; Olaf Piepenburg; Matthew S Forrest Journal: PLoS One Date: 2014-08-13 Impact factor: 3.240