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Solid-phase recombinase polymerase amplification using an extremely low concentration of a solution primer for sensitive electrochemical detection of hepatitis B viral DNA

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Abstract
Recombinase polymerase amplification (RPA) is considered one of the best amplification methods for realizing a miniaturized diagnostic instrument; however, it is notably challenging to obtain low detection limits in solid-phase RPA. To overcome these difficulties, we combined solid-phase RPA with electrochemical detection and used a new concentration combination of three primers (surface-bound forward primer, solution reverse primer, and an extremely low concentration of solution forward primer). When solid-phase RPA was performed on an indium tin oxide (ITO) electrode modified with a surface-bound forward primer in a solution containing a biotin-terminated solution reverse primer, an extremely low concentration of a solution forward primer, and a template DNA or genomic DNA for a target gene of hepatitis B virus (HBV), amplification occurred mainly in solution until all the solution forward primers were consumed. Subsequently, DNA amplicons produced in solution participated in solid-phase amplification involving surface-bound forward primer and solution reverse primer. Afterward, neutravidin-conjugated DT-diaphorase (DT-D) was attached to a biotin-terminated DNA amplicon on the ITO electrode. Finally, chronocoulometric charges were measured using electrochemical-enzymatic redox cycling involving the ITO electrode, 1,4-naphthoquinone, DT-D, and reduced β-nicotinamide adenine dinucleotide. The detection limit for HBV was measured using microfabricated electrodes and was found to be approximately 0.1 fM. This proposed method demonstrated better amplification efficiency for HBV genomic DNA than solid-phase RPA without using additional solution primer and asymmetric solid-phase RPA.
Author(s)
김경호김광선김지현박선화양해식유병준전상용조혜진황상현Andi Muhammad IchzanChiew San FangPonnusamy Nandhakumar
Issued Date
2021
Type
Article
Keyword
Biosensing TechniquesBiotinDNADNAViral - geneticsElectric propertiesElectrochemical detectionGenetic researchHepatitis BHepatitis B - diagnosisHepatitis B virusHumansIndiumMedical collegesNiacinamideNucleic Acid Amplification TechniquesRecombinase polymerase amplificationRecombinasesRedox cyclingSensitivity and SpecificitySensorsSolid-phase amplification
DOI
10.1016/j.bios.2021.113065
URI
https://oak.ulsan.ac.kr/handle/2021.oak/8575
https://ulsan-primo.hosted.exlibrisgroup.com/primo-explore/fulldisplay?docid=TN_cdi_proquest_miscellaneous_2489263591&context=PC&vid=ULSAN&lang=ko_KR&search_scope=default_scope&adaptor=primo_central_multiple_fe&tab=default_tab&query=any,contains,Solid-phase%20recombinase%20polymerase%20amplification%20using%20an%20extremely%20low%20concentration%20of%20a%20solution%20primer%20for%20sensitive%20electrochemical%20detection%20of%20hepatitis%20B%20viral%20DNA&offset=0&pcAvailability=true
Publisher
BIOSENSORS & BIOELECTRONICS
Location
영국
Language
한국어
ISSN
0956-5663
Citation Volume
179
Citation Start Page
113065
Citation End Page
0
Appears in Collections:
Medicine > Medicine
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