Optimization of ultra-fast convection polymerase chain reaction conditions for pathogen detection with nucleic acid lateral flow immunoassay
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Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to 3 µM or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, 2 µM of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.
Abstract
Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to 3 µM or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, 2 µM of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.
참고문헌 (16)
- Keith Pardee, "Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components", Cell, 165(5), pp.1255~1266 (2016), Elsevier BV[DOI]
- V. A. Brugman, "Detection of malaria sporozoites expelled during mosquito sugar feeding", Scientific Reports, 8(1), pp.7545~ (2018), Springer Science and Business Media LLC[DOI]
- Fumihiko Kawamori, "Evaluation of Diagnostic Assay for Rickettsioses Using Duplex Real-Time PCR in Multiple Laboratories in Japan", Japanese Journal of Infectious Diseases, 71(4), pp.267~273 (2018), Editorial Committee of Japanese Journal of Infectious Diseases, National Institute of Infectious Dis[DOI]
- Kyung-Young Song, "Ultra-fast DNA-based multiplex convection PCR method for meat species identification with possible on-site applications", Food Chemistry, 229, pp.341~346 (2017), Elsevier BV[DOI]
- Tae-Hoon Kim, "Development of a novel, rapid multiplex polymerase chain reaction assay for the detection and differentiation of Salmonella enterica serovars Enteritidis and Typhimurium using ultra-fast convection polymerase chain reaction", Foodborne Pathogens and Disease, 14(10), pp.580~586 (2017), Mary Ann Liebert Inc[DOI]
- Bin Liu, "Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis", Food Control, 27(1), pp.87~93 (2012), Elsevier BV[DOI]
- Biao Suo, "A multiplex real-time polymerase chain reaction for simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products", Foodborne Pathogens and Disease, 7(6), pp.619~628 (2010), Mary Ann Liebert Inc[DOI]
- Hatairat Kamphee, "Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay", PLOS ONE, 10(9), pp.e0137791~ (2015), Public Library of Science (PLoS)[DOI]
- Xiaohua He, "Identification of Salmonella enterica Typhimurium and variants using a novel multiplex PCR assay", Food Control, 65, pp.152~159 (2016), Elsevier BV[DOI]
- Keith Pardee, "Portable, On-Demand Biomolecular Manufacturing", Cell, 167(1), pp.248~259 (2016), Elsevier BV[DOI]
- Robert J. Meagher, "Engineering Paper-Based Sensors for Zika Virus", Trends in Molecular Medicine, 22(7), pp.529~530 (2016), Elsevier BV[DOI]
- Melissa K. Takahashi, "A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers", Nature Communications, 9(1), pp.3347~ (2018), Springer Science and Business Media LLC[DOI]
- Shan Shan, "Novel Strategies To Enhance Lateral Flow Immunoassay Sensitivity for Detecting Foodborne Pathogens", Journal of Agricultural and Food Chemistry, 63(3), pp.745~753 (2015), American Chemical Society (ACS)[DOI]
- Jyoti Singh, "Evaluation of gold nanoparticle based lateral flow assays for diagnosis of enterobacteriaceae members in food and water", Food Chemistry, 170, pp.470~483 (2015), Elsevier BV[DOI]
- Cong Wang, "Lateral flow strip for visual detection of K-ras mutations based on allele-specific PCR", Biotechnology Letters, 38(10), pp.1709~1714 (2016), Springer Science and Business Media LLC[DOI]
- Hongwei Zhang, "Rapid detection of methicillin-resistant Staphylococcus aureus in pork using a nucleic acid-based lateral flow immunoassay", International Journal of Food Microbiology, 243, pp.64~69 (2017), Elsevier BV[DOI]