Reverse transcription-quantitative PCR assays for genotype-specific detection of human noroviruses in clinical and environmental samples

Mohan Amarasiri; Masaaki Kitajima; Akiho Miyamura; Ricardo Santos; Silvia Monteiro; Takayuki Miura; Shinobu Kazama; Satoshi Okabe; Daisuke Sano

doi:10.1016/j.ijheh.2018.02.008

Reverse transcription-quantitative PCR assays for genotype-specific detection of human noroviruses in clinical and environmental samples

Int J Hyg Environ Health. 2018 Apr;221(3):578-585. doi: 10.1016/j.ijheh.2018.02.008. Epub 2018 Mar 2.

Authors

Mohan Amarasiri¹, Masaaki Kitajima², Akiho Miyamura², Ricardo Santos³, Silvia Monteiro³, Takayuki Miura⁴, Shinobu Kazama⁵, Satoshi Okabe², Daisuke Sano⁶

Affiliations

¹ Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan; Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13, West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan. Electronic address: mohan.amarasiri.b5@tohoku.ac.jp.
² Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13, West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan.
³ Laboratorio Analises, Instituto Superior Tecnico, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.
⁴ Department of Environmental Health, National Institute of Public Health, 2-3-6 Minami, Wako, Saitama 351-0197, Japan.
⁵ Center for Simulation Sciences and Informational Biology, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
⁶ Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.

PMID: 29496455
DOI: 10.1016/j.ijheh.2018.02.008

Abstract

Circulation of human noroviruses in water environments is suspected to be genotype-dependent, but the established primer and probe sets for noroviruses are usually genogroup-specific, which do not allow to compare the genotype-specific properties, such as persistence in water environments and resistance to disinfectants. In this study, quantitative PCR assays were designed for genotype-specific quantification of four epidemiologically important genotypes, GII.3, GII.4, GII.6, and GII.17. Developed assays were tested using norovirus positive stool samples which were previously confirmed to present target genotypes of this study. The results were 100% in accordance with the previous results. Effect of the co-existence of multiple genotypes in a sample on the target genotype quantification was evaluated using composite stool samples and wastewater samples containing multiple genotypes and the presence of non-target genotypes didn't affect the quantification of target genotype. Sensitivity and specificity was 100% for all four assays developed in this study with no cross-reactions between genotypes demonstrating the validity of our assays and their applicability to clinical and environmental samples.

Keywords: Genotype; Monitoring; Norovirus; Primers; Quantification; RT-qPCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Caliciviridae Infections / diagnosis*
Caliciviridae Infections / virology
Environmental Monitoring
Feces / virology*
Gastroenteritis / diagnosis
Gastroenteritis / virology
Genotype*
Humans
Norovirus* / genetics
Norovirus* / growth & development
Real-Time Polymerase Chain Reaction
Reverse Transcription*
Sensitivity and Specificity
Wastewater / virology*
Water

Substances

Waste Water
Water