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KMID : 0364820110470010117
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Korean Journal of Microbiology
2011 Volume.47 No. 1 p.117 ~ p.123
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Rapid Detection Method for Human Rotavirus from Vegetables by a Combination of Filtration and Integrated Cell Culture/Real-Time Reverse Transcription PCR
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Hyeon Ji-Yeon
Chon Jung-Whan
Song- Kwang-young
Hwang In-Gyun
Kwak Hyo-Sun
Lee Jung-soo
Kim Moo-Sang
Lee Chung-Bok
Seo Kun-Ho
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Abstract
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The purpose of this study was to evaluate and compare different elution and concentration methods for optimization of human rotavirus (HRV) detection method using real-time RT-PCR and cell culture techniques. The leafy vegetable samples (lettuce, Chinese cabbage) were artificially inoculated with HRV. Viruses were extracted from the vegetables by two different elution buffers, buffer A (100 mM Tris-HCl, 50 mM glycine, 3% beef extract, pH 9.5) and buffer B (250 mM Threonine, 300 mM NaCl, pH 9.5), and the extracted viruses were concentrated by filtration and PEG precipitation equentially. To determine infectivity of the viruses, the viruses recovered from the samples were infected to the MA-104 cells, and integrated cell culture real-time RT-PCR was performed at 1, 48, 72, 96, 120, 144, 168 h post-infection (p.i.). The elution buffer A was more efficient in extracting the virus from the produce samples tested than the buffer B, 29.54% and 18.32% of recoveries, respectively. The sensitivity of real-time RT-PCR method was markedly improved when the virus was concentrated by the filtration method. When the viruses were eluted and concentrated by buffer A and filtration, espectively, the average recovery rate was approximately 51.89%. When the viruses recovered from samples were infected to MA-104 cell, infectious HRV was detected within 48 h p.i. by ICC/real-time RT-PCR, whereas cytopathic effects were not observed until 72 h p.i. The optimized detection method evaluated in this study could be useful for rapid and reliable detection of HRV in fresh produce products and applied for detection of other food-borne viruses.
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KEYWORD
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cell culture, filtration, integrated cell culture/real-time reverse transcription PCR, rotavirus, vegetables
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