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Journal of Clinical Microbiology, February 2009, p. 435-440, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01247-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development of a Rapid High-Throughput Method for High-Resolution Melting Analysis for Routine Detection and Genotyping of Noroviruses{triangledown}

Etsuko Tajiri-Utagawa,1* Masayuki Hara,1,2 Kuniaki Takahashi,3 Mayumi Watanabe,2 and Takaji Wakita1

Second Department of Virology, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0012,1 Department of Virology, Kitasato Research Center for Environmental Science, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa 228-8555,2 Roche Diagnostics K.K., 2-6-1 Shiba, Tokyo 105-0014, Japan3

Received 2 July 2008/ Returned for modification 8 August 2008/ Accepted 2 December 2008

We developed a simple, rapid, high-throughput detection and genotyping method for noroviruses using real-time reverse transcription-PCR (RT-PCR) and high-resolution melting (HRM) analysis to create a difference plot. The capsid gene was amplified by real-time RT-PCR in the presence of ResoLight HRM dye, a saturating DNA dye. Following optimization of the HRM assay conditions, the major norovirus genotypes were selected. Because we had only small quantities of the patient stool samples used in this study, we constructed plasmids for each genotype and used these to optimize the HRM assay. We selected six stool samples, each positive for one of the six dominant subtypes of noroviruses that have been circulating in Japan, namely, genotypes 4, 8, and 9 from genogroup 1 and genotypes 3, 4, and 10 from genogroup 2. The specific high-resolution derivate plot of the HRM assay for each plasmid was constructed by subtracting the melting-curve shape of the plasmid from the reference or base curve. The RNAs extracted from 14 clinical samples positive for small round structured viruses were then directly analyzed using the HRM assay. The HRM data from the clinical RNA samples corresponded with the genotype results obtained by RT-PCR and sequencing of the clinical samples. In addition, the HRM data from the clinical RNA samples corresponded with the HRM data from the six reference plasmid DNAs, indicating that this assay is useful for the direct detection and genotyping of noroviruses in clinical samples. This assay requires no multiplexing or hybridization probes and provides a new approach to the genetic screening of noroviruses in clinical virology laboratories.

* Corresponding author. Mailing address: Laboratory of Diarrhea, Second Department of Virology, National Institute of Infectious Diseases, 4-7-1 Gakuen, Mushashimurayama-shi, Tokyo 208-0012, Japan. Phone: 81-42-561-0771. Fax: 81-42-561-4729. E-mail: etu{at}nih.go.jp

{triangledown} Published ahead of print on 10 December 2008.

Journal of Clinical Microbiology, February 2009, p. 435-440, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01247-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Henke-Gendo, C., Harste, G., Juergens-Saathoff, B., Mattner, F., Deppe, H., Heim, A. (2009). New Real-Time PCR Detects Prolonged Norovirus Excretion in Highly Immunosuppressed Patients and Children. J. Clin. Microbiol. 47: 2855-2862 [Abstract] [Full Text]  


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