title: Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.
abstract: The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.
authors.affiliation: Graduate School of Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University, Yongin 17104, Republic of Korea.
authors.forename: Mi-Ju
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Graduate School of Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University, Yongin 17104, Republic of Korea.
authors.forename: Shin-Young
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Graduate School of Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University, Yongin 17104, Republic of Korea.
authors.forename: Hyun-Joong
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Cheongju 28159, Republic of Korea.
authors.forename: Jeong Su
authors.initials: JS
authors.lastname: Lee
authors.affiliation: Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Cheongju 28159, Republic of Korea.
authors.forename: In Sun
authors.initials: IS
authors.lastname: Joo
authors.affiliation: Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Cheongju 28159, Republic of Korea.
authors.forename: Hyo Sun
authors.initials: HS
authors.lastname: Kwak
authors.affiliation: Graduate School of Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University, Yongin 17104, Republic of Korea.
authors.forename: Hae-Yeong
authors.initials: HY
authors.lastname: Kim
mesh_terms: D036022:Capsid Proteins / Q000235:genetics; D005516:Food Microbiology; D016679:Genome, Viral; D005838:Genotype; D030041:Hepatitis A virus / Q000235:genetics* / Q000302:isolation & purification*; D057230:Limit of Detection; D010446:Peptide Fragments / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D017422:Sequence Analysis, DNA; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D010446:Peptide Fragments; D012367:RNA, Viral; C096539:VP1 protein, hepatitis A virus; D015678:Viral Structural Proteins
keywords: Hepatitis A virus (HAV); epidemiological study; one-step duplex reverse transcription-polymerase chain reaction (RT-PCR); reverse transcription-quantitative polymerase chain reaction (RT-qPCR); simultaneous detection