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Journal of Clinical Microbiology, April 2003, p. 1548-1557, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1548-1557.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real-Time Quantitative Reverse Transcription-PCR

Tsutomu Kageyama,1* Shigeyuki Kojima,1 Michiyo Shinohara,2 Kazue Uchida,2 Shuetsu Fukushi,1 Fuminori B. Hoshino,1 Naokazu Takeda,3 and Kazuhiko Katayama3

Section of Infectious Disease, R&D Center, BML, Kawagoe, Saitama 350-1101,1 Saitama Institute of Public Health, Saitama, Saitama 338-0824,2 Department of Virus Diseases and Vaccine Control, National Institute of Infectious Diseases, Musashi-murayama, Tokyo 208-0011, Japan3

Received 15 April 2002/ Returned for modification 28 September 2002/ Accepted 6 January 2003

We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.

* Corresponding author. Mailing address: Section of Infectious Disease, R&D Center, BML, Matoba 1361-1, Kawagoe, Saitama 350-1101, Japan. Phone: 81-49-232-0440. Fax: 81-49-232-5480. E-mail: tkage{at}alk.co.jp.

Journal of Clinical Microbiology, April 2003, p. 1548-1557, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1548-1557.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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