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Genetic Diversity of Foodborne Pathogen Detected in Commercial Shellfish in Metropolitan Area
Biomed Sci Letters 2022;28:83-91
Published online June 30, 2022;  https://doi.org/10.15616/BSL.2022.28.2.83
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Jung Hun Park* and Kyu Bong Cho,* *
Correspondence to: Kyu Bong Cho. Department of Biomedical Laboratory Science, Shinhan University, Uijeongbu 11644, Korea.
Tel: +82-31-870-3712, Fax: +82-31-870-3719, e-mail: kbcho@shinhan.ac.kr
*Graduate student, **Professor.
Received May 11, 2022; Revised June 14, 2022; Accepted June 16, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
This study was performed to investigate the contamination status of microorganisms that cause food poisoning in shellfish distributed in the metropolitan area of South Korea. The analyses were conducted according to the sample type, season, and region. In particular, the genotype of food poisoning viruses that explosively cause collective food poisoning was analyzed. Total 483 each of four types of shellfish (Crassostrea gigas, Mytilus coruscus, Pectea albicans albicans, and Scapharca subcrenata) were collected from four distribution sites located in the metropolitan area. We investigated foodborne pathogen by multiplex PCR and RT/Nested PCR from shellfish. As a result, Vibrio parahaemolyticus and Bacillus cereus were detected in 13 and 21 samples and Norovirus (NoV) GI and GII were detected in 7 and 12 samples, respectively. V. parahaemolyticus and NoV GI and GII showed differences in types of shellfish and seasons, and no correlation was confirmed with regional differences. Also, as a result of genotyping for the detected NoV GI and GII, they were confirmed as NoV GI.7, GI.5 and GII.3, which are causes of collective food poisoning. Therefore, cross-infection with shellfish can cause food poisoning. In particular, attention must be paid to the handling and cooking of shellfish in organizations that implement group feeding, and it is necessary to establish a management system for microorganisms that cause food poisoning in the process of shellfish farming and distribution.
Keywords : Shellfish, Foodborne Pathogen, PCR, Phylogenetic analysis
꽌 濡

떇以묐룆 떇뭹뿉 쓽빐 泥대궡뿉 蹂묒썝泥닿 쑀엯릺뼱 利앹떇븯嫄곕굹 깮꽦맂 룆냼瑜 꽠痍⑦븯쓣 븣 諛쒖깮븯湲 븣臾몄뿉 媛먯뿼꽦(infection) 삉뒗 룆냼삎(intoxication) 吏덊솚쑝濡 遺꾨쪟맂떎(Kwun and Lee, 2007). 떇뭹쓽빟뭹븞쟾泥섏뿉꽌 二쇱슂 떇以묐룆 썝씤 誘몄깮臾쇰줈 吏젙븳 Vibrio parahaemolyticus, Bacillus cereus 벑 18醫 꽭洹좉낵 Norovirus (NoV), Sapovirus (SV) 벑 7醫 諛붿씠윭뒪뒗 二쇰줈 遺꾨-援ш컯 寃쎈줈(fecal-oral route)濡 媛먯뿼릺뒗뜲 듅엳 吏묐떒떇以묐룆 諛쒖깮뿉 븳 臾몄젣媛 몢릺怨 엳떎(Kang et al., 2018). 슦由щ굹씪뒗 떎뼇븳 빐궛臾쇱씠 以묒슂븳 떇옱猷뚮줈 궗슜릺뼱졇 솕쑝硫 쉶 媛숈씠 깮떇쑝濡쒕룄 留롮씠 꽠痍⑤릺뼱 뼱瑜섏뿉 븳 떇以묐룆 썝씤 誘몄깮臾쇱뿉 븳 뿰援ш 留롮씠 吏꾪뻾릺뿀떎(Koo et al., 2012; Kim et al., 2016; Ahn et al., 2016; Kang et al., 2018). 쇅援쓽 寃쎌슦뿉룄 떎뼇븳 뼱뙣瑜섏뿉 븯뿬 梨꾩랬遺꽣 꽠痍④퉴吏 떇以묐룆 썝씤 誘몄깮臾쇱뿉 븳 留롮 뿰援ъ 泥좎븳 愿由ш 씠猷⑥뼱吏怨 엳떎(Bellou et al., 2013; Elmahdi et al., 2016; Dumen et al., 2020). 諛섎㈃ 援궡뿉꽌 湲곗큹 뿰援ш 誘몃퉬븳 뙣瑜섎뒗 二쇰줈 뿰븞빐뿭뿉꽌 꽌떇븯硫 씤洹 二쇨굅吏, 媛異뺤궗쑁吏, 빞깮룞臾쇱꽌떇吏, 꽑諛뺢퀎瑜섏옣 벑뿉꽌 諛곗꽕맂 遺꾨 벑씠 媛뺤슦 벑쓽 쁺뼢쓣 諛쏆븘 泥대궡뿉 떇以묐룆 썝씤 誘몄깮臾쇨낵 룆꽦 臾쇱쭏뱾씠 異뺤쟻릺뼱 蹂대떎 뾼寃⑺븳 愿由ш 븘슂븯떎(Oh et al., 2012; Lee et al., 2010; Choi et al., 2012). 듅엳 떇以묐룆 썝씤 誘몄깮臾 以 V. parahaemolyticus, B. cereus뒗 援궡 吏묐떒떇以묐룆 諛쒖깮쓽 二쇱슂 썝씤쑝濡 떎닔쓽 솚옄媛 諛쒖깮븯쑝硫, NoV뒗 援궡肉먮쭔 븘땲씪 빐쇅뿉꽌 援댁쓣 留ㅺ컻泥대줈 븳 吏묐떒떇以묐룆쓽 썝씤쑝濡 蹂닿퀬릺뿀떎(Park et al., 2014; Chiu et al., 2020). 삉븳 SV뒗 援궡뿉꽌 뿰援ш 쟾臾 븷 肉먮쭔 븘땲씪 援궡쇅 븰援, 슂뼇떆꽕 諛 蹂묒썝 벑 떎닔쓽 湲곌뿉꽌 떇以묐룆 썝씤泥대줈 솗씤릺뿀떎(Becker-Dreps et al., 2019; Lee et al., 2021). 씠윭븳 臾몄젣뱾씠 몢맖뿉 뵲씪 援궡뿉꽌 깮떇븯뒗 뙣瑜섏뿉 븳 떇以묐룆 썝씤 꽭洹좉낵 諛붿씠윭뒪쓽 삤뿼떎깭뿉 븳 뿰援ъ쓽 以묒슂꽦씠 넂븘吏怨 엳떎. 슦由щ굹씪뒗 李멸뎬肉먮쭔 븘땲씪 솉빀, 媛由щ퉬, 뵾議곌컻 벑 떎뼇븳 뙣瑜섎 깮떇븯뒗 떇臾명솕媛 엳뼱꽌 洹몃줈 씤븳 떇以묐룆씠 諛쒖깮릺怨 엳쓣 寃껋쑝濡 삁痢〓릺吏留 뿭븰議곗궗쓽 遺議깆쑝濡 씠뿉 븳 蹂닿퀬媛 誘명씉븯떎. 삉븳 援궡 뙣瑜 깮궛吏뿭怨 궓遺吏뿭 쑀넻쁽옣쓽 李멸뎬뿉꽌 NoV 삤뿼뿉 븳 뿰援щ낫怨좊뒗 씪遺 엳쑝굹(Seo et al., 2012; Shin et al., 2014), 떎웾 쑀넻릺뒗 닔룄沅 吏뿭 쑀넻쁽옣뿉꽌쓽 떇以묐룆 썝씤 꽭洹 諛 諛붿씠윭뒪뿉 븳 뿰援щ뒗 蹂닿퀬맂 諛붽 뾾떎. 뵲씪꽌 遺꾩옄쟻 吏꾨떒쓣 넻븳 뙣瑜 깮궛吏뿭怨 쑀넻쁽옣쓽 떇以묐룆 썝씤 誘몄깮臾 삤뿼 쁽솴뿉 븳 젙솗븳 뙆븙씠 븘슂븳 떎젙씠떎. 蹂 뿰援ъ뿉꽌뒗 닔룄沅 吏뿭뿉 쑀넻릺怨 엳뒗 뙣瑜섏뿉 븳 떇以묐룆 썝씤 誘몄깮臾쇱쓽 삤뿼 쁽솴쓣 뙆븙븯怨 룺諛쒖쟻 吏묐떒 媛먯뿼쓣 쑀諛쒗븯뒗 떇以묐룆 썝씤 諛붿씠윭뒪쓽 삤뿼뿬遺 諛 쑀쟾삎쓣 遺꾩꽍븯怨좎옄 뿰援щ 떎떆븯떎.

옱猷 諛 諛⑸쾿

떆猷 닔吏

2020뀈 12썡遺꽣 2021뀈 9썡源뚯 닔룄沅뚯뿉 냼옱븳 4怨녹쓽 닔궛臾쇰룄留ㅼ떆옣뿉꽌 李멸뎬(Crassostrea gigas) 152媛, 솉빀(Mytilus coruscus) 124媛, 媛由щ퉬(Pectea albicans albicans) 106媛, 뵾議곌컻(Scapharca subcrenata) 101媛 벑 珥 483媛쒕 援щℓ븯쑝硫 떇以묐룆 썝씤 誘몄깮臾 寃궗瑜 쐞빐 깋옣긽깭瑜 쑀吏븯뿬 떎뿕떎濡 슫諛섑븯떎. 媛 떆猷뚮뒗 쓲瑜대뒗 臾쇱뿉 닔꽭븯뿬 몴硫댁쓽 遺덉닚臾쇱쓣 젣嫄고븯쑝硫 깋옣긽깭濡 蹂닿븯뿬 24떆媛 궡뿉 떎뿕쓣 吏꾪뻾븯떎.

떆猷 쟾 泥섎━

떆猷 쟾 泥섎━ 怨쇱젙 떇뭹쓽빟뭹븞쟾泥 2021뀈 떇以묐룆 썝씤議곗궗 떆뿕踰 媛씠뱶씪씤뿉 뵲씪 吏꾪뻾븯떎. 二쇱슂 떇以묐룆 썝씤 꽭洹좎씤 V. parahaemolyticus, B. cereus 벑 8醫낆쓣 遺꾨━븯湲 쐞빐 떆猷 珥 25 g怨 Triptic soy Broth Soybean-Casein Digest Medium (Becton, Dickinson and Company, Sparks, USA), Listeria Enrichment Broth (Becton, Dickinson and Company, Sparks, USA) 225 mL瑜 Bag-mixer濡 mix 썑 24, 48떆媛 룞븞 36℃뿉꽌 諛곗뼇븯뿬 DNA 異붿텧쓣 쐞븳 떆猷뚮줈 궗슜븯떎.

二쇱슂 떇以묐룆 썝씤 諛붿씠윭뒪씤 NoV, SV瑜 遺꾨━븯湲 쐞빐 뙣瑜 以묒옣꽑 3~6 g뿉 3 mL쓽 Proteinase K (Sigma-Aldrich, Missouri, USA)瑜 遺꾩<븯怨 Tissue Homogenizer瑜 궗슜븯뿬 洹좎쭏솕 븯쑝硫 Shaking Incubation뿉꽌 37℃, 25 g濡 60遺꾧컙 諛섏쓳 썑 Water Bath뿉꽌 60℃濡 15遺꾧컙 異붽濡 諛섏쓳떆耳곕떎. 씠썑 4,000 g濡 썝떖遺꾨━븯뿬 긽痢듭븸 RNA 異붿텧쓣 쐞븳 떆猷뚮줈 궗슜븯떎.

Multiplex PCR뿉 쓽븳 二쇱슂 떇以묐룆 썝씤 꽭洹 寃궗

利앷퇏맂 떆猷뚮뒗 DNA Extraction Kit (Kogenebiotech, Seoul, Korea)뿉꽌 젣怨듯븯뒗 諛⑸쾿뿉 뵲씪 異붿텧븯쑝硫 떆猷 1 mL뿉 Lysis buffer A B瑜 媛곴컖 400 μL, 40 μL, Proteinase K RNase A瑜 10 μL뵫 꽔 썑 65℃濡 60遺꾧컙 빆삩닔議곗뿉꽌 諛섏쓳떆궓 썑 binding buffer 400 μL瑜 샎빀븯뿬 썝떖遺꾨━븯떎. 긽痢듭븸쓣 DNA binding column뿉 삷寃 14,328 g濡 썝떖遺꾨━ 썑 washing buffer 600 μL濡 꽭泥숉븯떎. 씠썑 elution buffer 100 μL瑜 꽔怨 3遺 룞븞 6,368 g濡 썝떖遺꾨━븯뿬 DNA瑜 쉶닔븯쑝硫 Microvolume Spectrophotometer (PhileKorea, Seoul, Korea)濡 닚룄 닔쑉쓣 痢≪젙 썑 Multiplex PCR쓽 二쇳삎 빑궛쑝濡 궗슜븯떎. 二쇱슂 떇以묐룆 썝씤 꽭洹좎쓽 듅씠쟻 봽씪씠癒몄 뼇꽦議곌뎔 Multiplex (8-plex) Pathogen Detection KIT (Kogene biotech, Seoul, Korea)뿉꽌 젣怨듬릺뒗 샎빀臾쇱쓣 궗슜븯떎.

Multiplex PCR쓣 닔뻾븯湲 쐞븯뿬 Primer Mix 5 μL, 2X Premix 10 μL, 二쇳삎 빑궛(total DNA 삉뒗 뼇꽦議곌뎔) 諛 硫멸퇏利앸쪟닔瑜 珥 volume 20 μL濡 諛섏쓳븯떎. 95℃뿉꽌 10遺꾧컙 珥덇린 蹂꽦떒怨 씠썑 95℃뿉꽌 30珥덇컙 蹂꽦, 60℃뿉꽌 30珥덇컙 寃고빀, 72℃뿉꽌 30珥덇컙 떊옣쓽 떒怨꾨 35쉶 諛섎났븯怨 72℃뿉꽌 10遺꾧컙 理쒖쥌 떊옣븯뿬 꽭洹 醫낅쪟蹂꾨줈 떎瑜 Size쓽 듅씠쟻 쑀쟾옄 떒렪쓣 利앺룺븯떎. PCR 利앺룺궛臾쇱 1.2% agarose gel뿉꽌 쟾湲곗쁺룞븯뿬 寃곌낵瑜 遺꾩꽍븯떎.

RT/Nested PCR뿉 쓽븳 二쇱슂 떇以묐룆 썝씤 諛붿씠윭뒪 寃궗

媛 떆猷뚮뒗 Universal RNA Extraction Kit (Bioneer Coporation, Daejeon, Korea)뿉꽌 젣怨듯븯뒗 諛⑸쾿뿉 뵲씪 異붿텧븯쑝硫 떆猷 500 μL뿉 RB buffer 500 μL瑜 꽔뼱 샎빀븳 썑 19,502 g濡 3遺꾧컙 썝떖遺꾨━븯떎. 긽痢듭븸쓣 99% Ethanol 200 μL 샎빀븯뿬 RNA瑜 移⑥쟾븯쑝硫 binding column쑝濡 삷寃 썝떖遺꾨━ 썑 700 μL쓽 RWA1 buffer 500 μL쓽 RWA2 buffer濡 꽭泥숉븯떎. 씠썑 ER buffer 100 μL瑜 꽔뼱 RNA瑜 쉶닔븯쑝硫 닚룄 닔쑉쓣 痢≪젙 썑 RT/Nested PCR쓽 二쇳삎 빑궛쑝濡 궗슜븯떎. NoV SV쓽 듅씠쟻 봽씪씠癒몃뒗 Table 1怨 媛숈씠 젣옉븯쑝硫, 뼇꽦議곌뎔 NoV GI (NCBI accession number JQ388274.1 湲곗 5,283~5,673), NoV GII (KX356908.1 湲곗 5,011~5,403) 諛 SV (KP298674.1 湲곗 4,440~6,436)쓽 뿼湲곗꽌뿴쓣 닔吏 썑 (二)Macrogen (Seoul, Korea)뿉 쓽猶고븯뿬 빑궛쓣 빀꽦븯떎.

RT/Nested PCR primer sets of Sapovirus and Norovirus GI · GII

Virus Target gene Type Name Sequence (5'→3') Polarity Location Size (bp) Remark
Sapovirus NS7-VP1 RT-PCR SV-F13 GAY YWG GCY CTC GCY ACC TAC + 5074~5094 803 KP298674.1
RT-PCR SV-R13 GGT GAN AYN CCA TTK TCC AT - 5857~5876
Nested PCR SV-F21 ANT AGT GTT TGA RAT GGA GGG + 5157~5177 435
Nested PCR SV-R2 GWG GGR TCA ACM CCW GGT GG - 5572~5591
Norovirus GI VP1 RT-PCR GI-F1M CTG CCC GAA TTY GTA AAT GAT GAT + 5339~5362 330 JQ388274.1
RT-PCR GI-R1M CCA ACC CAR CCA TTR TAC ATY TG - 5646~5668
Nested PCR GI-F2-U 1)AAT ACG ACT CAC TAT AGA TGA
TGA TGG CGT CTA AGG ACG C
+ 5355~5377 314
Nested PCR GI-R1M CCA ACC CAR CCA TTR TAC ATY TG - 5646~5668
Norovirus GII NS7-VP1 RT-PCR GII-F1M GGG AGG GCG ATC GCA ATC T + 5058~5076 341 KX356908.1
RT-PCR GII-R1M CCR CCI GCA TRI CCR TTR TAC AT - 5376~5398
Nested PCR GII-F3M TTG TGA ATG AAG ATG GCG TCG ART + 5088~5111 311
Nested PCR GII-R1M-U 2)GCG GAT AAC AAT TTC ACA CAG
GCC RCC IGC ART ICC RTT RTA CAT
- 5376~5398

1) Universal Primer (T7), AATACGACTCACTATAG (17 bp); 2) Universal Primer (M13R), GCGGATAACAATTTCACACAGG (22 bp)



RT PCR쓣 닔뻾븯湲 쐞븯뿬 AccuPower® RT-PCR PreMix (Bioneer Coporation, Daejeon, Korea), 25 pmol 냽룄쓽 젙諛⑺뼢 · 뿭諛⑺뼢 봽씪씠癒 2 μL, 二쇳삎 빑궛(total RNA 삉뒗 뼇꽦議곌뎔) 1 μL 諛 硫멸퇏利앸쪟닔 17 μL濡 珥 volume 20 μL濡 諛섏쓳븯떎. 45℃뿉꽌 60遺꾧컙 뿭쟾궗, 94℃뿉꽌 5遺꾧컙 珥덇린 蹂꽦떒怨 씠썑, 94℃뿉꽌 30珥덇컙 蹂꽦, 55℃뿉꽌 30珥덇컙 寃고빀, 72℃뿉꽌 1遺 30珥 룞븞 떊옣떒怨꾨 35쉶 諛섎났븯怨 72℃뿉꽌 7遺꾧컙 理쒖쥌 떊옣븯떎. Nested PCR쓣 닔뻾븯湲 쐞븯뿬 AccuPower® HotStart PreMix with UDG (Bioneer Coporation), 25 pmol 냽룄쓽 젙諛⑺뼢 · 뿭諛⑺뼢 봽씪씠癒 2 μL, 二쇳삎 빑궛(RT PCR 궛臾) 1 μL 諛 硫멸퇏利앸쪟닔 17 μL濡 珥 volume 20 μL濡 諛섏쓳븯떎. 94℃뿉꽌 5遺꾧컙 珥덇린 蹂꽦떒怨 씠썑, 94℃뿉꽌 30珥덇컙 蹂꽦, 55℃뿉꽌 30珥덇컙 寃고빀, 72℃뿉꽌 1遺 30珥덇컙 떊옣떒怨꾨 35쉶 諛섎났븯怨, 72℃뿉꽌 7遺꾧컙 理쒖쥌 떊옣븯떎. PCR 利앺룺궛臾쇱 1.2% agarose gel뿉꽌 쟾湲곗쁺룞븯뿬 寃곌낵瑜 遺꾩꽍븯떎.

二쇱슂 떇以묐룆 썝씤 諛붿씠윭뒪쓽 뿼湲곗꽌뿴 遺꾩꽍

쟾湲곗쁺룞 寃곌낵뿉꽌 떒씪 諛대뱶媛 솗씤맂 떆猷뚯쓽 寃쎌슦 Total Fragment DNA Purification Kit (iNtRON Biotechnology, Seongnam, Korea)쓽 PCR purification Protocol뿉 뵲씪 젙젣븯떎. Nested PCR 궛臾쇱쓣 5X BNL Buffer 샎빀 썑 column쑝濡 삷寃 11,000 g濡 30珥덇컙 썝떖遺꾨━븯떎. 750 μL쓽 washing Buffer (added EtOH)瑜 궗슜븯뿬 꽭泥 썑 elution buffer瑜 泥④븯뿬 DNA瑜 젙젣븯떎. 硫떚 諛대뱶媛 솗씤맂 떆猷뚯쓽 寃쎌슦 gel extraction 諛⑸쾿뿉 뵲씪 젙젣븯떎. 뿼湲곗꽌뿴 遺꾩꽍씠 븘슂븳 諛대뱶 쐞移섏쓽 agarose gel쓣 UV 긽뿉꽌 옒씪궡뼱 500 μL쓽 BNL buffer 샎빀븳 썑 빆삩닔議곗뿉꽌 55℃뿉꽌 10遺꾧컙 諛섏쓳떆耳곕떎. 씠썑 column뿉 삷寃 11,000 g濡 30珥덇컙 썝떖遺꾨━ 썑 washing buffer elution buffer瑜 궗슜븯뿬 DNA瑜 젙젣븯떎.

젙젣맂 DNA뒗 (二)Macrogen (Seoul, Korea)뿉 뿼湲곗꽌뿴 遺꾩꽍쓣 쓽猶고븯쑝硫, 쑀쟾삎 遺꾩꽍쓣 쐞빐 NoV GI.1~9 뿼湲곗꽌뿴 28媛, NoV GII.1~NA2 뿼湲곗꽌뿴 36媛쒖 議곌뎔쑝濡 Sapovirus (SV), Human Rotavirus (HuRV), Hepatitis A virus (HAV) 諛 Human Astrovirus (HuAstV)쓽 뿼湲곗꽌뿴쓣 닔吏묓븯떎. 뼇諛⑺뼢쑝濡 遺꾩꽍맂 뿼湲곗꽌뿴怨 닔吏묓븳 뿼湲곗꽌뿴쓣 빀爾 Bioedit ver.7.2.6쑝濡 multiple sequence alignment瑜 吏꾪뻾븯떎. 씠썑 NCBI BLAST瑜 궗슜븯뿬 쑀궗꽦쓣 遺꾩꽍븯쑝硫, RIVM Norovirus Genotyping tool ver.2.0쓣 씠슜븯뿬 꽭遺 쑀쟾삎쓣 솗씤븯떎. 理쒖쥌쟻쑝濡 MEGA sofrware ver.3쑝濡 Neighbor-joining湲곕컲 phylogenetic tree瑜 젣옉븯뿬 遺꾩꽍븯떎.

寃 怨

二쇱슂 떇以묐룆 썝씤 꽭洹 議곗궗

떇以묐룆 썝씤 꽭洹좎쓣 議곗궗븯湲 쐞빐 multiplex PCR쓣 닔뻾븯뿬 뙣瑜 醫낅쪟蹂꾨줈 遺꾩꽍븳 寃곌낵 Fig. 1怨 媛숈씠 李멸뎬, 솉빀 諛 媛由щ퉬뿉꽌 媛곴컖 50媛 以 7媛(14%), 100媛 以 12媛(12%) 諛 80媛 以 2媛(2.5%)뿉꽌 B. cereus媛 寃異쒕릺뿀쑝硫, 삉븳 솉빀 100媛 以 5媛(5%), 媛由щ퉬 80媛 以 8媛(10%)뿉꽌 V. parahaemolyticus媛 寃異쒕릺뿀떎. 씠쇅쓽 뙣瑜섏뿉꽌뒗 떇以묐룆 썝씤 꽭洹좎씠 寃異쒕릺吏 븡븯떎. 썝궛吏蹂꾨줈 遺꾩꽍븳 寃곌낵 Fig. 1怨 媛숈씠 넻쁺씠 썝궛吏씤 뙣瑜 80媛 以 17媛(21.2%)쓽 떆猷뚯뿉꽌 B. cereus媛 寃異쒕릺뿀쑝硫 怨좏씎씠 썝궛吏씤 뙣瑜 90媛 以 4媛(4.4%)뿉꽌 B. cereus媛, 13媛(14.4%)뿉꽌 V. parahaemolyticus媛 寃異쒕릺뿀떎. 씠쇅쓽 썝궛吏뿉꽌뒗 떇以묐룆 썝씤 꽭洹좎씠 寃異쒕릺吏 븡븯떎. 怨꾩젅蹂꾨줈 遺꾩꽍븳 寃곌낵 Table 2, Fig. 1怨 媛숈씠 12썡~2썡(寃⑥슱~遊)뿉 닔吏묓븳 李멸뎬 50媛 以 7媛(14%)뿉꽌 B. cereus媛 寃異쒕릺뿀떎. 7썡~9썡(뿬由~媛쓣)뿉 닔吏묓븳 솉빀, 媛由щ퉬뿉꽌 媛곴컖 100媛 以 17媛(17%)쓽 B. cereus 80媛 以 10媛(12.5%)쓽 V. parahaemolyticus媛 寃異쒕릺뿀떎. 씠쇅쓽 떆猷뚯뿉꽌뒗 떇以묐룆 썝씤 꽭洹좎씠 寃異쒕릺吏 븡븯떎.

Seasonal difference of Foodborne Bacteria in Shellfish

Season December ~ Februray July ~ September Total
Sample Crassostrea gigas Mytilus coruscus Pectea albicans albicans Scapharca subcrenata Crassostrea gigas Mytilus coruscus Pectea albicans albicans Scapharca subcrenata
Bacteria Salmonella spp. - - - - - - - - -
Listeria monocytogenes - - - - - - - - -
Bacillus cereus 7 (14) - - - - 12 (12) 2 (2.5) - 21 (6.7)
Escherichia coli O157 - - - - - - - - -
Yersinia enterocolitica - - - - - - - - -
Vibrio parahaemolyticus - - - - - 5 (5) 8 (10) - 13 (4.1)
Staphylococcus aureus - - - - - - - - -
Shigella spp. - - - - - - - - -
Total 7 (14) - - - - 17 (17) 10 (12.5) - 34 (10.9)


Fig. 1. Multiplex PCR of Foodborne Bacteria from Shellfish. M, 100 bp Ladder maker; P1, Positive Control DNA 1(B. cereus, amplicon size 303 nt); P2, Positive Control DNA 2 (V. parahaemolyticus, amplicon size 375 nt); Pannel A, Lane 1~10, Crassostrea gigas (Tongyeong) Sample 1~10; Pannel B, Lane 1~10, Mytilus coruscus (Tongyeong) Sample 1~10; Pannel C, Lane 1~10, Mytilus coruscus (Goheung) Sample 1~10; Pannel D, Lane 1~10, Pectea albicans albicans (Goheung) Sample 1~10; N, Negative Control.

二쇱슂 떇以묐룆 썝씤 諛붿씠윭뒪 議곗궗

떇以묐룆 썝씤 諛붿씠윭뒪 議곗궗瑜 쐞빐 RT/Nested PCR쓣 닔뻾븯뿬 뙣瑜 醫낅쪟蹂꾨줈 遺꾩꽍븳 寃곌낵 紐⑤뱺 뙣瑜섏뿉꽌 SV뒗 寃異쒕릺吏 븡븯쑝硫, 李멸뎬 102媛 以 7媛(6.8%)뿉꽌 NoV GI, 12媛(11.7%)뿉꽌 NoV GII媛 寃異쒕릺뿀떎. 썝궛吏蹂꾨줈 遺꾩꽍븳 寃곌낵 李멸뎬 102媛 以 넻쁺씠 썝궛吏씤 1媛(0.9%) 떆猷뚯뿉꽌 NoV GI씠 寃異쒕릺뿀쑝硫, 怨좏씎씠 썝궛吏씤 1媛(0.9%) 떆猷뚯뿉꽌 NoV GII媛 寃異쒕릺뿀떎. 삉븳 李멸뎬 102媛 以 뿬닔媛 썝궛吏씤 3媛(2.9%)쓽 떆猷뚯뿉꽌 NoV GI씠 寃異쒕릺뿀쑝硫, 9媛(8.8%) 떆猷뚯뿉꽌 NoV GII媛 寃異쒕릺뿀떎. 李멸뎬 102媛 以 씤泥쒖씠 썝궛吏씤 寃쎌슦 3媛(2.9%) 떆猷뚯뿉꽌 NoV GI씠 寃異쒕릺뿀쑝硫, 2媛(1.9%) 떆猷뚯뿉꽌 NoV GII媛 寃異쒕릺뿀떎. 怨꾩젅蹂꾨줈 遺꾩꽍븳 寃곌낵 Table 3, Fig. 2 媛숈씠 12썡~2썡(寃⑥슱~遊)뿉뒗 102媛 以 7媛(6.8%)쓽 李멸뎬뿉꽌 NoV GI씠 寃異쒕릺뿀쑝硫, 12媛(11.7%)쓽 李멸뎬뿉꽌 NoV GII媛 寃異쒕릺뿀떎. 7썡~9썡(뿬由~媛쓣)뿉뒗 SV 諛 NoV GI, GII媛 寃異쒕릺吏 븡븯떎.

Seasonal difference of Sapovirus and Norovirus GI · GII in Shellfish

Season December ~ February July ~ September Total
Sample Crassostrea gigas Mytilus coruscus Pectea albicans albicans Scapharca subcrenata Crassostrea gigas Mytilus coruscus Pectea albicans albicans Scapharca subcrenata
Virus Sapovirus - - - - - - - - -
Norovirus GI 7 (6.8) - - - - - - - 7 (6.8)
Norovirus GII 12 (11.7) - - - - - - - 12 (11.7)
Total 19 (18.6) - - - - - - - -


Fig. 2. Nested PCR of Norovirus GI · GII from Shellfish. M, 100 bp Ladder maker; Pannel A, P, Positive Control (Norovirus GI Plasmid); Lane 1, Crassostrea gigas (Tongyeong) sample; Lane 2~3, Crassostrea gigas (Incheon) sample; Pannel B, P, Positive Control (Norovirus GII Plasmid); Lane 1, Crassostrea gigas (Incheon) sample; PN, RT PCR Negative Control; N, Negative Control.

二쇱슂 떇以묐룆 썝씤 諛붿씠윭뒪 怨꾪넻닔 遺꾩꽍

遺꾩꽍맂 NoV 뿼湲곗꽌뿴뿉 븳 NCBI BLAST 寃곌낵 Sample #1~2뒗 NoV GI.3 99.19%, 98.58%, Sample #3 NoV GI.5 100%, Sample #4뒗 NoV GII 98.99%쓽 쑀궗꽦씠 솗씤릺뿀떎. RIVM Norovirus Genotyping tool 遺꾩꽍 寃곌낵 Sample #1~2뒗 NoV GI.7, Sample #3 NoV GI.5, Sample #4뒗 NoV GII.3濡 굹궗쑝硫, Fig. 3怨 媛숈씠 Sample #1~2뒗 99% 쑀궗꽦씤 NoV GI.3蹂대떎 100% 쑀궗꽦씤 NoV GI.7 (AY675555, AJ844469)怨 쑀뿰愿怨꾧 넂븯쑝硫, Sample #3 NoV GI.5 (AB039774, AJ277614, AF414406, AM263418), Sample #4뒗 NoV GII.3 (EU187437, U22498) 쑀뿰愿怨꾧 넂쓬쓣 솗씤븯떎. 씠쇅쓽 Sample 뿼湲곗꽌뿴 遺꾩꽍 寃곌낵 noise signal 벑 鍮꾪듅씠쟻 利앺룺씠 솗씤릺뼱 怨꾪넻닔 遺꾩꽍뿉꽌 젣쇅븯떎.

Fig. 3. Phylogenetic tree of Norovirus GI · GII in Shellfish. Pannel A, Sample #1, Crassostrea gigas (Tongyeong); Sample #2~3, Crassostrea gigas (Incheon); Pannel B, Sample #4, Crassostrea gigas (Incheon).
怨 李

蹂 뿰援ъ뿉꽌뒗 援궡뿉꽌 떎뼇븳 떇옄옱濡 솢슜릺硫 二쇰줈 깮떇쑝濡 留롮 꽠痍④ 씠猷⑥뼱吏뒗 뿬윭 醫낅쪟쓽 뙣瑜섏뿉 븳 誘몄깮臾쇳븰쟻 븞쟾꽦쓣 솗씤븯怨좎옄 떇以묐룆 썝씤 誘몄깮臾쇱쓽 遺꾪룷瑜 鍮꾧탳遺꾩꽍쓣 븯쑝硫 듅엳 룺諛쒖쟻쑝濡 떇以묐룆쓣 쑀諛쒗븯뒗 諛붿씠윭뒪쓽 遺꾪룷 諛 쑀쟾삎쓣 遺꾩꽍븯怨좎옄 븯떎. 뿰援 寃곌낵, V. parahaemolyticus쓽 寃쎌슦 솉빀뿉꽌 5%, 媛由щ퉬뿉꽌 10%媛 寃異쒕릺뼱 湲곗〈 援궡 李멸뎬쓣 긽쑝濡 븳 뿰援щ낫떎뒗 궙븯떎(Kang et al., 2016). 湲고 뤃뱶 17.5%, 以묎뎅 33.3% 벑쓽 援쇅 蹂닿퀬蹂대떎룄 긽쟻쑝濡 궙寃 寃異쒕릺뿀떎(Lopatek et al., 2015; Xie et al., 2016). 理쒓렐 援궡뿉꽌 蹂닿퀬맂 뼱瑜섏뿉꽌쓽 V. parahaemolyticus 寃異쒕쪧 16.8% 蹂대떎룄 궙寃 遺꾪룷븿씠 솗씤릺뿀떎(Lee et al., 2019). 씠윭븳 寃쏀뼢 V. parahaemolyticus쓽 궙 寃異쒕쪧뿉룄 援먯감 媛먯뿼쑝濡 씤븳 吏묐떒떇以묐룆쓽 媛뒫꽦씠 議댁옱븯湲곗뿉 吏냽쟻씤 紐⑤땲꽣留곸씠 븘닔쟻씠硫, 씠踰 뿰援ъ뿉꽌 寃異쒗븳 toxR 쑀쟾옄 씠쇅뿉룄 鍮덈쾲븯寃 蹂닿퀬릺뒗 trh, tdh 쑀쟾옄뿉 븳 썑냽뿰援ш 吏꾪뻾맂떎硫 二쇱슂 蹂묒썝꽦 씤옄瑜 媛吏뒗 V. parahaemolyticus뿉 븳 泥닿퀎쟻씤 愿由ш 맆 寃껋쑝濡 궗猷뚮맂떎. 삉븳 B. cereus쓽 삤뿼뿬遺瑜 遺꾩꽍븳 寃곌낵, 李멸뎬뿉꽌 14%, 솉빀뿉꽌 12%, 媛由щ퉬뿉꽌 2.5%媛 寃異쒕릺뼱 湲곗〈 援궡 李멸뎬쓣 긽쑝濡 븳 B. cereus쓽 寃異쒕쪧蹂대떎 蹂대떎 궙寃 寃異쒕릺뿀쑝硫(Kim et al., 2017), 理쒓렐 留뚯뿉꽌 蹂닿퀬븳 2.5%쓽 寃異쒕쪧 蹂대떎뒗 援궡 뙣瑜섍 넂寃 삤뿼릺뼱 엳쓬씠 솗씤릺뿀떎(Hsu et al., 2021). 씠쇅뿉룄 援쇅쓽 뼱瑜, 媛묎컖瑜, 몢議깅쪟瑜 긽쑝濡 遺꾩꽍맂 38%濡 蹂대떎뒗 썾뵮 궙 寃異쒕쪧쓣 굹궡뿀떎(Rahmati and Labbe, 2008). 씠윭븳 寃쏀뼢 삤뿼맂 넗뼇쓽 B. cereus媛 떎뼇븳 닔궛臾 몴硫댁씠굹 泥대궡濡 異뺤쟻릺뼱 쑀넻怨쇱젙뿉꽌 援먯감 삤뿼씠 吏꾪뻾맆 寃껋쑝濡 궗猷뚮릺硫 옉뾽솚寃쎌뿉 븳 泥좎븳 쐞깮愿由ш 븘슂븷 寃껋쑝濡 뙋떒맂떎. SV쓽 寃쎌슦 理쒓렐 援궡뿉꽌 25꽭 誘몃쭔怨 50꽭 씠긽쓽 援誘쇱뿉꽌 媛곴컖 16%, 12%쓽 떇以묐룆 諛쒖깮瑜좎씠 蹂닿퀬릺怨 엳쑝硫, 援궡 怨좊벑븰援먯뿉꽌룄 17嫄댁쓽 SV뿉 쓽븳 떇以묐룆 諛쒖깮씠 蹂닿퀬릺뿀쑝굹 씠踰 뙣瑜섎 긽쑝濡 븳 뿰援ъ뿉꽌뒗 寃異쒕릺吏 븡븘 뙣瑜섏쓽 뿰愿꽦 솗씤릺吏 븡븯떎(Li et al., 2020; Cho et al., 2020). 씠泥섎읆 湲곗〈 援궡뿉꽌 寃異쒕맂 SV뒗 諛붿씠윭뒪 蹂씠濡 씤븳 떎뼇븳 쑀쟾삎씠 솗씤릺뿀쑝硫 씠뿉 뵲瑜 湲됱꽦쐞옣뿼씠 諛쒖깮븷 솗瑜좎씠 넂븘吏먯뿉 뵲씪 吏냽쟻씤 紐⑤땲꽣留곸씠 븘닔쟻씠떎. 삉븳 NoV쓽 삤뿼뿬遺瑜 遺꾩꽍븳 寃곌낵 李멸뎬뿉꽌 18.6%媛 寃異쒕릺뼱 湲곗〈 援궡 李멸뎬쓣 긽쑝濡 븳 뿰援ъ뿉꽌쓽 寃異쒕쪧 38%뿉 鍮꾪븯뿬 궙寃 寃異쒕릺뿀쑝硫(Shin et al., 2014), 씠깉由ъ븘, 뒪럹씤뿉꽌쓽 14.2%, 12.2% 蹂대떎뒗 넂 寃異쒕쪧쓣 굹궡뿀怨 떛媛룷瑜댁쓽 30%쓽 寃異쒕쪧 蹂대떎뒗 궙寃 寃異쒕릺뼱 뼇떇옣 諛 쑀넻怨쇱젙뿉꽌쓽 NoV뿉 븳 愿由ш 援쇅蹂대떎뒗 옒 씠猷⑥뼱吏怨 엳쓬씠 솗씤릺뿀떎(La Bella et al., 2017; de Oliveira-Tozetto et al., 2021; Ho et al., 2015). NoV쓽 愿由ш 옒 씠猷⑥뼱吏怨 엳쓬뿉룄 吏냽쟻쑝濡 떇以묐룆 諛쒖깮씠 蹂닿퀬릺뒗뜲 씠뒗 듅엳 諛붿씠윭뒪濡 씤븳 吏덈퀝 솗궛냽룄媛 留ㅼ슦 鍮좊Ⅴ湲 븣臾몄뿉 援먯감 媛먯뿼 벑쓽 썝씤쓣 뙆븙븯뒗 寃 以묒슂븯硫 泥좎븳 뿭븰議곗궗媛 븘닔쟻씠떎. NoV뒗 GI~GX쓽 쑀쟾삎씠 議댁옱븯硫 씠 以 GI, GII, GIV뒗 씤泥댁뿉 媛먯뿼릺硫 吏묐떒떇以묐룆쓣 쑀諛쒗븯뒗뜲 씠쓽 議댁옱뿬遺瑜 遺꾩꽍븯뒗 寃껋 떇以묐룆 쑀諛 썝씤 諛붿씠윭뒪濡쒖꽌쓽 뿭븷 뿬遺瑜 뙋蹂꾪븯뒗뜲 留ㅼ슦 以묒슂븯떎(Chhabra et al., 2019; Han and Chung, 2017). 蹂 뿰援ъ뿉꽌 寃異쒕맂 NoV뿉 븯뿬 쑀쟾삎쓣 遺꾩꽍븳 寃곌낵 GI.7, GI.5 諛 GII.3濡 솗씤릺뿀떎. 援쇅뿉꽌 遺꾩꽍맂 쑀쟾삎 二쇰줈 GI.2, GI.3, GII.17쑝濡 李⑥씠媛 엳뿀쑝硫(Ho et al., 2015; Chiu et al., 2020; Cheng et al., 2017), 2013뀈 援궡 怨좊벑븰援 8紐낆뿉寃뚯꽌 諛쒖깮븳 떇以묐룆쓽 NoV 쑀쟾삎씤 GII.4, GII.11 諛 GII.14, 援쇅쓽 吏묐떒떇以묐룆 썝씤泥댁씤 NoV GII.4룄 李⑥씠瑜 굹깉떎(Cho et al., 2016; Widdowson et al., 2004; Bull et al., 2006; Park et al., 2010). 븯吏留 理쒓렐 NoV GI.7, GI.5 諛 GII.3뿉 븳 吏묐떒떇以묐룆 諛쒖깮씠 蹂닿퀬릺뼱 寃異쒕맂 쑀쟾삎씠 떇以묐룆쓣 쑀諛쒗븯뒗 二쇱슂 긽엫쓣 븣 닔 엳뿀떎(Marques Mendanha de Oliveira et al., 2014; Li et al., 2018; Fu et al., 2021). 씠濡 씤븯뿬 뙣瑜섎줈 씤븳 援먯감 媛먯뿼쓽 媛뒫꽦씠 긽議댄븯硫 듅엳 吏묐떒 湲됱떇쓣 떆뻾븯뒗 湲곌, 븰援 벑쓽 뙣瑜 痍④툒 諛 議곕━ 떆 二쇱쓽瑜 슂븯怨 뙣瑜 뼇떇怨 쑀넻怨쇱젙뿉꽌쓽 떇以묐룆 쑀諛 誘몄깮臾쇱뿉 븳 븞쟾꽦 솗蹂 諛 泥닿퀎쟻 愿由щ 쐞븳 吏냽쟻씤 뿰援ш 븘슂븷 寃껋쑝濡 궗猷뚮맂떎.

ACKNOWLEDGEMENT

None.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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