KMID : 0603820110170030191
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Journal of Experimental & Biomedical Science 2011 Volume.17 No. 3 p.191 ~ p.196
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Real-time Nucleic Acid Sequence Based Amplification (Real-time NASBA) for Detection of Norovirus
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Lee In-Soo
Choi Dong-Hyuk Lim Jae-Won Cho Yoon-Jung Bang Hye-Eun Jin Hyun-Woo Choi Yeon-Im Park Sang-Jung Kim Sung-Hyun Lee Hye-Young Kim Tae-Ue Jeong Hye-Sook Cheon Doo-Sung
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Abstract
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Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least 106 viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup ¥° (G¥°) and genogroup ¥± (G¥±) using standard viral RNA. For real-time NASBA assay which can detected noroV G¥° and G¥±, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV G¥° and G¥±. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV G¥° and G¥± were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.
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KEYWORD
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Norovirus, Real-time NASBA, RT-PCR
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