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KMID : 0545120160260081398
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Journal of Microbiology and Biotechnology
2016 Volume.26 No. 8 p.1398 ~ p.1403
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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
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Kim Mi-Ju
Lee Shin-Young
Kim Hyun-Joong
Lee Jeong-Su
Joo In-Sun
Kwak Hyo-Sun
Kim Hae-Yeong
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Abstract
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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 × 101 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 × 102 copies/20 g fresh lettuce, 9.7 × 103 copies/20 g frozen strawberries, and 4.1 × 103 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.
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KEYWORD
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Hepatitis A virus (HAV), simultaneous detection, epidemiological study, one-step duplex reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
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