노로바이러스의 검출과 HRM을 이용한 유전적 다양성 분석

Abstract

In this study, we conducted a phylogenetic analysis of noroviruses in water and oyster samples. Feline calicivirus (FCV) strain F9, a known norovirus surrogate, was spiked into the digestive gland of oysters, and different methods of concentrating noroviruses in the oyster digestive gland were compared. The different procedures, viral elution with glycine buffer, disruption of tissue by sonication, and precipitation ultracentrifugation, based on polyethylene glycol (PEG) precipitation did not differ in the band density of gel electrophoresis of the PCR amplicon of the spiked FCV strain F9. Due to time considerations and the accessibility of reagents and instruments, we used the viral elution with glycine buffer and PEG treatment procedures. Water samples from Dong Brook were ultracentrifuged to concentrate the virus. Oyster samples were from Tongyeong seashore. PCR was conducted on the noroviruses in the samples, using primer sets designed in this laboratory to be more sensitive and specific in amplification. Proportions of genogroup I and genogroup II in noroviruses detected in Dong Brook water were 74.47% and 67.6%, respectively, and in the Tongyeong oyster samples the proportions were 57.14%, respectively. Phylogenetic analysis of the Dong Brook noroviruses showed that they appeared to be members of GI/1, GI/2, GI/4, GI/5, GI/9, and GI/10 in genogroup I and GII/4, GII/5, GII/11 and GII/13 in genogroup II. The major genotypes were GI/4 (5/12), GI/5 (4/12), and GII/4 (4/10). To confirm the presence of genetic variation, possibly derived from degenerate primers targeting different regions for cDNA synthesis and PCR, we compared nucleotide sequences of the cloned fragments. Then, to examine whether norovirus variations were more numerous than those derived from nucleic acid sequencing using the cloned PCR amplicon, we applied high-resolution melting (HRM) analysis to analyze greater numbers of cloned colonies. As a result, we were able to detect various norovirus genotypes, using primers for different cDNA syntheses and PCR. Compared with sequencing as a genotyping method, HRM analysis is a simple, more rapid method to differentiate norovirus genotypes and allowed us to group noroviruses from even a single sample.