Browse > Article
http://dx.doi.org/10.11620/IJOB.2019.44.1.8

Optimization of ultra-fast convection polymerase chain reaction conditions for pathogen detection with nucleic acid lateral flow immunoassay  

Kim, Tae-Hoon (Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University)
Hwang, Hyun Jin (R&D Center, Ahram Biosystems Inc.)
Kim, Jeong Hee (Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University)
Publication Information
International Journal of Oral Biology / v.44, no.1, 2019 , pp. 8-13 More about this Journal
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 $3{\mu}M$ or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, $2{\mu}M$ 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.
Keywords
Molecular diagnostics; Ultra-fast convection polymerase chain reaction; Nucleic acid lateral flow immunoassay;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Wang C, Chen X, Wu Y, Li H, Wang Y, Pan X, Tang T, Liu Z, Li X. Lateral flow strip for visual detection of K-ras mutations based on allele-specific PCR. Biotechnol Lett 2016;38:1709-14. doi: 10.1007/s10529-016-2161-9.   DOI
2 Zhang H, Ma L, Ma L, Hua MZ, Wang S, Lu X. Rapid detection of methicillin-resistant Staphylococcus aureus in pork using a nucleic acid-based lateral flow immunoassay. Int J Food Microbiol 2017;243:64-9. doi: 10.1016/j.ijfoodmicro.2016.12.003.   DOI
3 Pardee K, Green AA, Takahashi MK, Braff D, Lambert G, Lee JW, Ferrante T, Ma D, Donghia N, Fan M, Daringer NM, Bosch I, Dudley DM, O'Connor DH, Gehrke L, Collins JJ. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell 2016;165:1255-66. doi: 10.1016/j.cell.2016.04.059.   DOI
4 Brugman VA, Kristan M, Gibbins MP, Angrisano F, Sala KA, Dessens JT, Blagborough AM, Walker T. Detection of malaria sporozoites expelled during mosquito sugar feeding. Sci Rep 2018;8:7545. doi: 10.1038/s41598-018-26010-6.   DOI
5 Liu B, Zhou X, Zhang L, Liu W, Dan X, Shi C, Shi X. Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis. Food Control 2012;27:87-93. doi: 10.1016/j.foodcont.2012.01.062.   DOI
6 Kawamori F, Shimazu Y, Sato H, Monma N, Ikegaya A, Yamamoto S, Fujita H, Morita H, Tamaki Y, Takamoto N, Su H, Shimada M, Shimamura Y, Masuda S, Ando S, Ohashi N. Evaluation of diagnostic assay for Rickettsioses using duplex real-time PCR in multiple laboratories in Japan. Jpn J Infect Dis 2018;71:267-73. doi: 10.7883/yoken.JJID.2017.447.   DOI
7 Song KY, Hwang HJ, Kim JH. Ultra-fast DNA-based multiplex convection PCR method for meat species identification with possible on-site applications. Food Chem 2017;229:341-6. doi: 10.1016/j.foodchem.2017.02.085.   DOI
8 Kim TH, Hwang HJ, Kim JH. 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 Pathog Dis 2017;14:580-6. doi: 10.1089/fpd.2017.2290.   DOI
9 Kamphee H, Chaiprasert A, Prammananan T, Wiriyachaiporn N, Kanchanatavee A, Dharakul T. Rapid molecular detection of multidrug-resistant tuberculosis by PCR-nucleic acid lateral flow immunoassay. PLoS One 2015;10:e0137791. doi: 10.1371/journal.pone.0137791.   DOI
10 Suo B, He Y, Tu SI, Shi X. A multiplex real-time polymerase chain reaction for simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products. Foodborne Pathog Dis 2010;7:619-28. doi: 10.1089/fpd.2009.0430.   DOI
11 He X, Xu X, Li K, Liu B, Yue T. Identification of Salmonella enterica Typhimurium and variants using a novel multiplex PCR assay. Food Control 2016;65:152-9. doi: 10.1016/j.foodcont.2016.01.015.   DOI
12 Shan S, Lai W, Xiong Y, Wei H, Xu H. Novel strategies to enhance lateral flow immunoassay sensitivity for detecting foodborne pathogens. J Agric Food Chem 2015;63:745-53. doi: 10.1021/jf5046415.   DOI
13 Pardee K, Slomovic S, Nguyen PQ, Lee JW, Donghia N, Burrill D, Ferrante T, McSorley FR, Furuta Y, Vernet A, Lewandowski M, Boddy CN, Joshi NS, Collins JJ. Portable, on-demand biomolecular manufacturing. Cell 2016;167:248-59. doi: 10.1016/j.cell.2016.09.013.   DOI
14 Meagher RJ, Negrete OA, Van Rompay KK. Engineering paper-based sensors for Zika virus. Trends Mol Med 2016;22;529-30. doi: 10.1016/j.molmed.2016.05.009.   DOI
15 Takahashi MK, Tan X, Dy AJ, Braff D, Akana RT, Furuta Y, Donghia N, Ananthakrishnan A, Collins JJ. A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nat Commun 2018;9:3347. doi: 10.1038/s41467-018-05864-4.   DOI
16 Singh J, Sharma S, Nara S. Evaluation of gold nanoparticle based lateral flow assays for diagnosis of enterobacteriaceae members in food and water. Food Chem 2015;170:470-83. doi: 10.1016/j.foodchem.2014.08.092.   DOI