Search for


TEXT SIZE

search for



CrossRef (0)
Mornitoring and Identification of Human Astrovirus from Groundwater in Korea Based on Highly Sensitive RT-nested PCR Primer Sets
Biomed Sci Letters 2021;27:255-263
Published online December 31, 2021;  https://doi.org/10.15616/BSL.2021.27.4.255
© 2021 The Korean Society For Biomedical Laboratory Sciences.

Siwon Lee1,*, Kyung Seon Bae2,**, Jihyun Park2,***, Jin-Ho Kim3,4,**, Jin-Young Lee1,***, Jiwon Choi1,5,****, Eung-Roh Park2,*** and Kyung-A You2,†,***

1R&D Team, LSLK Co. Ltd., Gimpo, Gyeonggi 10111, Korea
2Water Supply and Sewerage Research Division, National Institute of Environmental Research, Incheon 22689, Korea
3Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Chunam 31116, Korea
4Department of Chemistry, College of Science and Engineering, Dankook University, Chungnam 31116, Korea
5Department of Chemical and Biochemical Engineering, Dongguk University, Seoul 04620, Korea
Correspondence to: *Research director, **Researcher, ***Senior Researcher, ****Undergraduate student.
Corresponding author: Kyung-A You. Water Supply and Sewerage Research Division, National Institute of Environmental Research, Incheon 22689, Korea.
Tel: +82-32-560-8353, Fax : +82-32-563-7085, e-mail : angelka@korea.kr
Received September 16, 2021; Revised November 15, 2021; Accepted November 15, 2021.
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
Human Astrovirus (HuAstV) is an important gastrointestinal pathogen that is frequently reported worldwide. Monitoring of contaminated groundwater has been suggested since HuAstV is transmitted through the fecal-oral route. This study developed a test method based on conventional reverse transcription (RT)-nested polymerase chain reaction (PCR) that involves SL® non-specific reaction inhibitor for unknown non-specific amplification taking place in the groundwater environment. An optimal method for detecting HuAstV in groundwater sample through analysis and comparison against conventionally reported method was also suggested. The developed method enabled the production of nested PCR amplicon of 630 nt, which is a sufficient length for similarity analysis based on sequencing and genotyping. Amplicons suspected to be HuAstV were amplified in two out of the twenty groundwater samples collected in Korea, presenting 99.77% and 99.73% similarity against HuAstV 1 strain lhar/2011/kor (JN887820.1) in sequencing, respectively. These amplicons were identified as HuAstV 1.
Keywords : Human Astrovirus, HuAstV, Groundwater, SL® Non-specific reaction inhibitor
꽌 濡

Human Astrovirus (HuAstV)뒗 Astroviridae, Mamoastrovirus濡 遺꾨쪟릺硫, 8媛쒖쓽 serotypes씠 蹂닿퀬릺뿀떎(Krishna, 2005). 궗엺뿉寃 媛먯뿼맆 寃쎌슦 넻긽쟻 옞蹂듦린뒗 빟 3~4씪쓣 嫄곗퀜 吏덈퀝 湲곌컙 1~4씪濡, 떇슃遺吏, 怨좎뿴, 援ы넗, 蹂듯넻 벑쓣 룞諛섑븳 寃쎌쬆쓽 꽕궗媛 吏냽맂떎(Yi et al., 2004). 쟾 꽭怨꾩쟻쑝濡 5꽭 誘몃쭔 쁺븘쓽 寃쎌슦 빟 17뼲 嫄댁쓽 궗濡 52.5泥 紐낆쓽 궗留앹옄媛 諛쒖깮릺뿀怨, 9꽭 씠긽쓽 빟 90%媛 HuAstV뿉 븳 빆泥대 媛吏怨 엳뼱 궗엺쓽 옣愿怨 吏덈퀝 以 以묒슂븳 蹂묒썝泥대줈 蹂닿퀬릺怨 엳떎(Johnson et al., 2017; Hargest et al., 2020). 援쇅뿉꽌뒗 2017뀈 3썡 뒪럹씤쓽 뼱由곗씠吏묒뿉꽌뿉꽌 利앹긽씠 굹궃 엫긽 솚옄 以 6嫄닿낵 臾댁쬆긽 媛먯뿼옄 4嫄댁씠 蹂닿퀬(Parrón et al., 2021), 2017뀈 11썡 以묎뎅뿉꽌 12~17꽭 125紐(Tan et al., 2019)쓽 諛쒖깮씠 蹂닿퀬릺뿀쑝硫, 援궡뿉꽌뒗 옣뿼솚옄 以묒뿉꽌룄 빟 1.0~1.9%濡 궙 닔以씠뿀쑝굹, 理쒓렐 5뀈(2016~2020) 룞븞 2,521嫄댁쓽 엫긽 솚옄 蹂닿퀬濡 뿰 룊洹 빟 504紐낆쓽 HuAstV 媛먯뿼씠 蹂닿퀬릺硫댁꽌 二쇱슂 옣愿怨 諛붿씠윭뒪 以 빟 5.4% 젙룄瑜 李⑥븯怨 엳떎[Korea Centers for Disease Control and Prevention (KCDC), 2021]. HuAstV뒗 遺꾨-援ш컯 寃쎈줈濡 媛먯뿼릺誘濡 援, 솉빀 벑 뙣瑜섎줈遺꽣룄 媛먯뿼맆 닔 엳쑝硫, 2001~2012뀈 씪蹂 삤궗移댁뿉꽌 286嫄댁쓽 쐞옣뿼 諛쒕퀝 궗濡 以 88嫄(빟 30.7%)쓽 寃泥댁뿉꽌 寃異 諛 2014~2015뀈 씠깉由ъ븘쓽 솉빀 以 16.0~32.0% 닔以쑝濡 寃異쒕맂 諛 엳떎(Iritani et al., 2014; Fusco et al., 2017). 삉븳 HuAstV뒗 닔씤꽦 옣愿怨 諛붿씠윭뒪濡 吏븯닔 吏몴닔뿉꽌 빟 15씪 룞븞 媛먯뿼꽦씠 媛먯냼릺吏 븡븯怨, 紐 떖 룞븞 媛먯뿼꽦씠 쑀吏맂 寃껋씠 蹂닿퀬릺硫댁꽌(Espinosa et al., 2008), 냼洹쒕え 湲됱닔떆꽕, 留덉쓣긽닔 벑쓽 鍮 냼룆 삉뒗 냼룆 愿由ъ뿉 痍⑥빟븳 湲됱닔 떆꽕뿉꽌쓽 紐⑤땲꽣留 븘슂꽦씠 젣湲곕릺뿀쑝굹, 쁽옱 援궡 吏븯닔뿉꽌뒗 끂濡쒕컮씠윭뒪, 珥앸옣洹좉뎔 諛 옣洹 빆紐⑸쭔씠 紐⑤땲꽣留 릺怨 엳떎. 吏븯닔 벑 닔怨 솚寃쎌뿉꽌 닔씤꽦 諛붿씠윭뒪瑜 紐⑤땲꽣留 븯뒗 諛⑸쾿 떆猷 梨꾩랬, 깉由 諛 냽異 썑 珥 빑궛뿉꽌 以묓빀슚냼뿰뇙諛섏쓳(polymerase chain reaction; PCR) 湲곕컲쑝濡 듅씠쟻 蹂묒썝泥댁쓽 빑궛쓣 利앺룺븯뒗 諛⑸쾿씠 솢슜릺怨 엳怨, 씠 以묒뿉꽌룄 넂 븞젙꽦, 꼻 踰붿쐞쓽 궗슜옄 蹂댁쑀 諛 뿼湲곗꽌뿴 遺꾩꽍쓣 넻빐 genotyping씠 媛뒫븳 씪諛 PCR 諛⑸쾿쓣 솢슜릺怨 엳떎(Lee and Cho, 2019). 援궡뿉꽌룄 吏덈퀝愿由ъ껌, 떇뭹쓽빟뭹븞쟾泥 벑 湲곌怨 紐뉖챺 臾명뿄뿉꽌 PCR 湲곕컲쓽 寃궗踰뺤씠 솢슜릺怨 엳쑝굹[KCDC, 2015; Korea Ministry of Food and Drug Safety (KMFDS), 2015; Lee and Rho, 2020], 吏븯닔 벑 닔怨 솚寃 떆猷뚮줈쓽 쟻슜 뿰援щ뒗 誘명씉븯硫, 吏븯닔 以 諛붿씠윭뒪 빑궛쓽 誘몃웾 삤뿼 諛 떎닔쓽 PCR 빐臾쇱씠 룷븿맆 媛뒫꽦쑝濡 씤븯뿬 슦닔븳 寃異 誘쇨컧룄瑜 媛吏 nested PCR 닔以 씠긽쓽 寃異 誘쇨컧룄媛 븘슂븯떎(Lee and Cho, 2019; Lee et al., 2021). 븳렪, 援궡 엫긽 솚옄뿉 븳 뿰援ъ뿉 鍮꾪빐 吏븯닔뿉꽌쓽 HuAstV 紐⑤땲꽣留곸뿉 븳 뿰援щ뒗 誘명씉븯뿬 援궡 吏븯닔 以 HuAstV쓽 쁽솴, 寃異쒕릺뒗 쑀쟾삎, 諛쒖깮릺뒗 솚옄뱾怨쇱쓽 愿젴 뿰援 벑쓣 쐞빐꽌뒗 吏븯닔 以 HuAstV 紐⑤땲꽣留곸슜 寃궗 떆뒪뀥怨 紐⑤땲꽣留곸씠 븘슂븯떎. 뵲씪꽌 씠踰 뿰援ъ뿉꽌뒗 씪諛 뿭쟾궗(reverse transcription)-nested PCR 湲곕컲쓽 寃궗踰뺤쓣 媛쒕컻 諛 湲곗〈 蹂닿퀬맂 RT- 삉뒗 RT-nested PCR 諛⑸쾿怨 븿猿 鍮꾧탳‧遺꾩꽍븯쑝硫, 紐뉖챺 吏븯닔 떆猷뚯뿉꽌쓽 솢슜꽦쓣 룊媛 諛 寃異쒕맂 HuAstV쓽 쑀쟾삎쓣 遺꾩꽍븯떎.

옱猷 諛 諛⑸쾿

HuAstV 듅씠쟻 봽씪씠癒 議고빀 諛 諛붿씠윭뒪 빑궛 닔吏

HuAstV 듅씠쟻 寃고빀씠 媛뒫븳 씪諛 RT-PCR, 떎떆媛 RT-PCR, 벑삩利앺룺踰 벑쓽 젙諛⑺뼢怨 뿭諛⑺뼢 봽씪씠癒몃 annealing 삩룄 51~59℃(理쒖쟻 55℃)뿉꽌 諛섏쓳븷 닔 엳룄濡 蹂삎 諛 諛고빀븯뿬 20媛 썑蹂 RT-PCR 봽씪씠癒 議고빀쓣 援ъ꽦븯쑝硫, 씠 以 400~900 nucleotide (nt)쓽 利앺룺 궛臾 삎꽦씠 湲곕릺뒗 11媛쒕 썑蹂 RT-PCR 봽씪씠癒 議고빀쑝濡 븯떎(Table 1) [Lee and Rho, 2020; KCDC, 2015; KMFDS, 2015; National Institute of Environmental Research (NIER), 2016, 2017]. 븳렪, 誘멸뎅 援由쎌깮臾쇨났븰젙蹂댁꽱꽣(National Center for Biotechnology Information; NCBI) accession number JN887820瑜 湲곗쑝濡 HuAstV ORF 1b-2 gene (3,800~ 6,685 nt; 2,886 nt)쓽 빑궛 떒렪쓣 (二)Marcrogen (Seoul, Korea)뿉꽌 빀꽦븯뿬 썑蹂 RT-PCR 봽씪씠癒몃뱾씠 紐⑤몢 寃고빀 諛 諛섏쓳븷 닔 엳룄濡 븯떎. 삉븳 鍮 듅씠쟻 諛섏쓳 떆뿕뿉 궗슜븷 李멸퀬 諛붿씠윭뒪 6醫[Aichivirus A (AiV-A), Hepatitisvirus A (HAV), Hepatitisvirus E (HEV), Norovirus GII (NoV-GII), Rotavirus-A (RV-A) 諛 Sapovirus-GI (SaV-GI)]쓣 빑궛 떒렪쓣 븿猿 빀꽦 삉뒗 닔吏묓븯떎(Lee and Cho, 2019; Lee et al., 2021).

Information of development combination and reference RT-nested PCR primer sets for the detection of human Astrovirus

Division PCR type Primer information Location* Length (nt) References
Name Sequence (5'→3') Mer (nt) Start End
Candidate RT-nested PCR primer sets in this study RT-PCR primer set #1 RT-PCR HAstV_F30 CTGACCTTGAATCACTCCAT 20 4045 4064 848 NIER, 2017
Mon270R TCAGATGCATTGTCATTGGT 20 4873 4892 KMFDS, 2015
RT-PCR primer set #2 RT-PCR HAstV_F40 GGAGCACTGCCTYTCRCG 18 4133 4150 868 NIER, 2017
AstV_3_B3 CACTCTGAAGCAAGTTCAA 19 4982 5000 Lee and Rho, 2020
RT-PCR primer set #3 RT-PCR HAstV_F40 GGAGCACTGCCTYTCRCG 18 4133 4150 760 NIER, 2017
Mon270R TCAGATGCATTGTCATTGGT 20 4873 4892 KMFDS, 2015
RT-PCR primer set #4 RT-PCR AstV_1_F3 AGGACCAAAGAAGTGTGAT 19 4226 4244 775 Lee and Rho, 2020
AstV_3_B3 CACTCTGAAGCAAGTTCAA 19 4982 5000
RT-PCR primer set #5 RT-PCR AstV_1_F3 AGGACCAAAGAAGTGTGAT 19 4226 4244 841 Lee and Rho, 2020
HAstV_R40 ACTTGMYTRTCTGTTGAYTT 20 5047 5066 NIER, 2017
RT-PCR primer set #6 RT-PCR HAstV_F50 AAGTGTGATGGCTAGCAAGT 20 4236 4255 765 NIER, 2017
AstV_3_B3 CACTCTGAAGCAAGTTCAA 19 4982 5000 Lee and Rho, 2020
RT-PCR primer set #7 RT-PCR HAstV_F50 AAGTGTGATGGCTAGCAAGT 20 4236 4255 831 NIER, 2017
HAstV_R40 ACTTGMYTRTCTGTTGAYTT 20 5047 5066
RT-PCR primer set #8 RT-PCR F3m GCARGTHACTGTTGAGGTC 19 4263 4281 738 This study
AstV_3_B3 CACTCTGAAGCAAGTTCAA 19 4982 5000 Lee and Rho, 2020
RT-PCR primer set #9 RT-PCR F3m GCARGTHACTGTTGAGGTC 19 4263 4281 804 This study
HAstV_R40 ACTTGMYTRTCTGTTGAYTT 20 5047 5066 NIER, 2017
RT-PCR primer set #10 RT-PCR F3m GCARGTHACTGTTGAGGTC 19 4263 4281 886 This study
HAstV_R30 AAGTGCTGTCCGAGCAAART 20 5129 5148 NIER, 2017
RT-PCR primer set #11 RT-PCR F3m GCARGTHACTGTTGAGGTC 19 4263 4281 630 This study
Mon270R TCAGATGCATTGTCATTGGT 20 4873 4892 KMFDS, 2015
Nested PCR primer set #4-1 Nested PCR F3m GCARGTHACTGTTGAGGTC 19 4263 4281 738 This study
AstV_3_B3 CACTCTGAAGCAAGTTCAA 19 4982 5000 Lee and Rho, 2020
Nested PCR primer set #4-2 and #8-1 Nested PCR F3m GCARGTHACTGTTGAGGTC 19 4263 4281 630 This study
Mon270R TCAGATGCATTGTCATTGGT 20 4873 4892 KMFDS, 2015
Reference RT- or RT-nested PCR primer sets Ref. set A RT-PCR HuAstV-2-B3 ACAACTCAGGAAACAAGGT 19 4443 4461 558 Lee and Rho, 2020
HuAstV-3-B3 CACTCTGAAGCAAGTTCAA 19 4982 5000
Nested PCR HuAstV-3-F3 GTAAGCACCTTGATGTTACA 20 4778 4797 223
HuAstV-3-B3 CACTCTGAAGCAAGTTCAA 19 4982 5000
Ref. set B RT-PCR HuAstV-3-F3 GTAAGCACCTTGATGTTACA 20 4778 4797 223 Lee and Rho, 2020
HuAstV-3-B3 CACTCTGAAGCAAGTTCAA 19 4982 5000
Ref. set C RT-PCR Mon 269 CAACTCAGGAAACAGGGTGT 20 4444 4463 449 KMFDS, 2015
Mon 270 TCAGATGCATTGTCATTGGT 20 4873 4892

*Based on NCBI accession number JN887820



RT-nested PCR 봽씪씠癒 議고빀 꽑諛 諛 李멸퀬 봽씪씠癒 鍮꾧탳

HuAstV瑜 듅씠쟻쑝濡 寃異쒗븷 닔 엳쓣 寃껋쑝濡 異붿젙릺뒗 11媛 썑蹂 RT-PCR 봽씪씠癒 議고빀쓣 긽쑝濡 븯뿬 ⅰ) HuAstV 빑궛 떒렪뿉 듅씠쟻 諛섏쓳 깮꽦 뿬遺, ⅱ) HuAstV 빑궛뿉 븳 寃異 誘쇨컧룄 諛 ⅲ) 李멸퀬 諛붿씠윭뒪 빑궛 떒렪쓽 鍮 듅씠쟻 諛섏쓳 깮꽦 뿬遺瑜 솗씤븯떎. ⅰ~ⅲ) 怨쇱젙쓣 넻빐 HuAstV 寃異쒖슜 씪諛 RT-PCR 봽씪씠癒 議고빀쓣 꽑諛쒗븯쑝떎. ⅳ) 쐞쓽 ⅱ) 怨쇱젙쓽 RT-PCR 궛臾쇱쓣 二쇳삎쑝濡 nested PCR 봽씪씠癒몃 꽑諛 諛 寃異 誘쇨컧룄瑜 遺꾩꽍븯쑝硫, ⅴ) 理쒖쥌 꽑諛쒗븳 nested PCR 봽씪씠癒 議고빀쓽 李멸퀬 諛붿씠윭뒪 鍮 듅씠쟻 諛섏쓳 깮꽦 뿬遺瑜 솗씤븯떎. ⅵ) 湲곗〈 蹂닿퀬맂 RT-PCR 봽씪씠癒 議고빀뱾쓽 寃異 誘쇨컧룄 諛 李멸퀬 諛붿씠윭뒪뿉 鍮 듅씠쟻 諛섏쓳 깮꽦 뿬遺瑜 솗씤븯떎. 븳렪, RT-PCR 議곗꽦 AccuPower® RT/PCR PreMix (Bioneer, Daejeon, Korea) dry type, 젙諛⑺뼢 諛 뿭諛⑺뼢 PCR 봽씪씠癒 媛 25 p濡 1 μL(珥 2 μL), nucleic acid free water 17 μL, template 1 μL濡 珥 20 μL濡, 議곌굔 뿭쟾궗(42℃, 60遺), 珥덇린 蹂꽦(95℃, 10遺), 35쉶 諛섎났(95℃뿉꽌 45珥, 55℃뿉꽌 60珥, 72℃뿉꽌 60珥), 理쒖쥌 떊옣(72℃, 5遺)쑝濡 븯떎(Lee and Cho, 2019; Lee et al., 2021). Nested PCR AccuPower® HotStart PCR PreMix (Bioneer) dry type쓣 궗슜븯뿬 RT-PCR怨 룞씪븳 議곗꽦 諛 뿭쟾궗 怨쇱젙쓣 젣쇅븳 룞씪 議곌굔쑝濡 븯떎(Lee and Cho, 2019; Lee et al., 2021). 븳렪, 鍮꾧탳 봽씪씠癒 議고빀 젙蹂대뒗 Table 1怨 媛숈쑝硫, 湲곗〈 蹂닿퀬맂 議곌굔 諛 議곗꽦怨 룞씪븯寃 븯떎(Lee and Rho, 2020; KCDC, 2015; KMFDS, 2015; NIER, 2016, 2017).

吏븯닔 떆猷뚯뿉꽌쓽 쟻슜꽦 寃넗 諛 紐⑤땲꽣留

援由쏀솚寃쎄낵븰썝 怨좎떆 젣 2017-50뿉 뵲씪 쟾援 냼洹쒕え湲됱닔떆꽕怨 留덉쓣긽닔룄瑜 긽쑝濡 떆猷 20媛쒕 梨꾩랬븯떎(옄猷 誘 젣怨). 떎뿕떎濡 씠룞븯뿬 깉由 諛 냽異 썑 RNeasy Mini Kit (Qiagen, Germany)瑜 궗슜븯뿬 냽異뺤븸뿉꽌 total RNA瑜 異붿텧븯떎. 씠踰 뿰援ъ뿉꽌 理쒖쥌 꽑諛쒗븳 RT-PCR 諛 nested PCR 봽씪씠癒 議고빀, 鍮꾧탳 봽씪씠癒 議고빀뱾 以 媛옣 슦닔븳 寃異 誘쇨컧룄媛 굹궃 1媛(議고빀A) 諛 湲곌뿉꽌 젣怨듯븯뒗 봽씪씠癒 1媛(議고빀C)쑝濡 珥 3媛 議고빀뱾濡 吏븯닔 以 HuAstV 紐⑤땲꽣留 諛 쟻슜꽦쓣 寃넗븯떎. 꽑諛 봽씪씠癒몃 궗슜븳 RT-PCR AccuPower® RT/PCR PreMix (Bioneer), nested PCR AccuPower® HotStart PCR PreMix (Bioneer)瑜 궗슜븯쑝굹, 吏븯닔 벑 솚寃 떆猷뚯뿉꽌 굹굹뒗 鍮 듅씠쟻 諛섏쓳뼲젣瑜 쐞빐 RT- 諛 nested PCR 紐⑤몢 議곗꽦臾쇰줈 SL® Non-specific reaction inhibitor (LSLK, Gyeonggi, Korea) 3 μL瑜 룷븿븯뿬 珥 20 μL濡 諛섏쓳븯떎(Lee and Cho, 2019). 븳렪, 鍮꾧탳 봽씪씠癒 議고빀 湲곗〈 蹂닿퀬맂 議곌굔 諛 議곗꽦怨 룞씪븯寃 븯怨(Lee and Rho, 2020; KCDC, 2015; KMFDS, 2015; NIER, 2016, 2017), 궛臾쇰뱾 紐⑤몢 1.2% agarose gel뿉꽌 쟾湲곗쁺룞븯쑝硫, 몢 媛쒖쓽 諛⑸쾿쑝濡 吏븯닔뿉꽌 紐⑤땲꽣留곷맂 寃곌낵瑜 鍮꾧탳븯떎. 븳렪, 떆猷 以 異붿젙 뼇꽦 뿼湲곗꽌뿴씠 굹궃 諛대뱶瑜 MEGAquick-spinTM Plus Total Fragment DNA Purification Kit (iNtRON, Gyeonggi, Korea)濡 젙젣 썑 (二)Marcrogen뿉꽌 sequencing븯떎. 뿼湲곗꽌뿴쓣 NCBI BLAST濡 쑀궗꽦쓣 遺꾩꽍븯떎. 븳렪, 쑀쟾삎 遺꾩꽍쓣 쐞븯뿬 HuAstV genotype 1-8뿉 룷븿릺뒗 32媛 taxa 諛 븘썐 洹몃9쑝濡 닔씤꽦 옣愿怨 諛붿씠윭뒪 4醫[HAV (NC_001489.1), HEV (NC_001434.1), human Enterovirus A (NC_001612.1) 諛 AiV-A (NC_001918.1)]쓣 닔吏 썑, BioEdit ver. 7.1.3濡 떎以 뿼湲곗꽌뿴 젙젹븯쑝硫, MEGA software ver. 6.03쓣 궗슜븯뿬 Neighbor-joining phylogenetic tree瑜 援ъ텞븯떎(Hall, 1999; Tamura et al., 2013).

寃곌낵 諛 怨좎같

HuAstV RT-nested PCR 봽씪씠癒 議고빀 꽑諛 諛 李멸퀬 봽씪씠癒 議고빀怨쇱쓽 鍮꾧탳

HuAstV 듅씠쟻 利앺룺씠 異붿젙릺뒗 11媛 썑蹂 RT-PCR 봽씪씠癒 議고빀뱾 以 3媛 議고빀(#04, #08 諛 #11)뿉꽌 775, 738 諛 630 nt濡 湲곕릺뒗 겕湲곗쓽 amplicon씠 삎꽦릺뿀떎(Fig. 1). 諛섏쓳씠 슦닔븯怨 궛臾쇱쓽 겕湲곌 긽쟻쑝濡 겙 #04 #08쓣 꽑諛쒗븯뿬 寃異 誘쇨컧룄 諛 鍮 듅씠쟻 諛섏쓳 뿬遺瑜 遺꾩꽍븳 寃곌낵 議고빀 #04뒗 10 fg/μL, 議고빀 #08 1 fg/μL 굹궗쑝硫, 몢 媛 議고빀 紐⑤몢 李멸퀬 諛붿씠윭뒪 빑궛 6醫낆뿉 븳 鍮 듅씠쟻 諛섏쓳 뾾쓬뿉 뵲씪 吏븯닔뿉꽌 닔씤꽦 옣愿怨 諛붿씠윭뒪瑜 寃異쒗븷 닔 엳뒗 쟻빀븳 寃異 誘쇨컧룄 닔以쓣 媛吏 RT-PCR 봽씪씠癒 議고빀쑝濡 異붿젙릺뿀떎(Fig. 2) (Lee and Cho, 2019; Lee et al., 2021). RT-PCR 議고빀 #04 (775 nt)濡 遺꽣뒗 2媛[RT-PCR 봽씪씠癒 議고빀 #08(= nested PCR 봽씪씠癒 議고빀 #04-1) 諛 #11(= nested PCR 봽씪씠癒 議고빀 #04-2)], 議고빀 #08 (738 nt)濡 遺꽣뒗 1媛[RT-PCR 봽씪씠癒 議고빀 #11(= nested PCR 봽씪씠癒 議고빀 #08-1)]쓽 썑蹂 nested PCR 봽씪씠癒 議고빀쓽 寃異 誘쇨컧룄瑜 遺꾩꽍븯떎. 꽭 媛쒖쓽 썑蹂 nested PCR 議고빀 紐⑤몢 HuAstV瑜 1 fg/μL 닔以源뚯 寃異쒗븷 닔 엳뿀쑝굹 議고빀 #08-1뿉꽌뒗 諛섏쓳씠 빟뻽怨 鍮 듅씠쟻 諛대뱶媛 븿猿 굹궗쑝硫, 議고빀 #04-1怨 #04-2뿉꽌뒗 룞씪븳 寃곌낵媛 굹궓뿉 뵲씪 RT-PCR 봽씪씠癒 궛臾 겕湲곗씤 775 nt 援щ텇씠 릺뒗 #04-2 (630 nt)瑜 꽑諛쒗븯떎(Fig. 3A). 꽑諛쒗븳 nested PCR 봽씪씠癒 議고빀쓽 李멸퀬 諛붿씠윭뒪 6醫낆뿉 븳 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡쓬뿉 뵲씪 理쒖쥌 봽씪씠癒 議고빀쑝濡 꽑諛쒗븯떎(Fig. 3B). 븳렪, 3뙇쓽 鍮꾧탳 봽씪씠癒 議고빀뱾 以, 議고빀 A B뒗 RT-PCR뿉꽌뒗 100 fg/μL, nested PCR뿉꽌 10 fg/μL 닔以, 鍮꾧탳 봽씪씠癒 議고빀 C뿉꽌뒗 RT-PCR뿉꽌 10 fg/μL濡 굹궗쑝硫, 李멸퀬 諛붿씠윭뒪 6醫낆쓽 빑궛뿉 븳 鍮 듅씠쟻 諛섏쓳 紐⑤몢 굹굹吏 븡븯떎(Fig. 4). 씠踰 뿰援ъ뿉꽌 꽑諛쒗븳 RT-nested PCR 봽씪씠癒 議고빀쓣 룷븿븳 議곗꽦臾 諛 議곌굔쓣 솢슜븷 寃쎌슦, 鍮꾧탳 봽씪씠癒 議고빀 A B뿉 鍮꾪빐 룞벑 떆媛 鍮 RT- 諛 nested PCR뿉꽌 빟 10諛 닔以쓽 寃異 誘쇨컧룄 뼢긽맆 닔 엳怨, 鍮꾧탳 봽씪씠癒 議고빀 C뒗 RT-PCR 닔以뿉꽌뒗 룞벑븳 寃異 誘쇨컧룄씠吏留 利앺룺 궛臾쇱쓽 겕湲곌 긽쟻쑝濡 겕떎. 삉븳 꽑諛 nested PCR 솢슜 떆 빟 2떆媛꾩쓽 諛섏쓳 떆媛꾩씠 異붽릺吏留 10諛 넂 寃異 誘쇨컧룄 뼢긽씠 湲곕맂떎.

Fig. 1. Specific reaction of candidate RT-PCR primer sets for the detection of human Astrovirus (HuAstV). #01-#11, combination of candidate RT-PCR primer sets. A, candidate RT-PCR primer sets. B, candidate nested PCR primer sets. M, 100 bp Ladder marker (Enzynomics, Korea); -3 ~ -8, template dilution value from 1 ng/μL HuSaV plasmid; N, negative control, PN, PCR negative control.

Fig. 2. Sensitivity and non-specific reaction tests of two candidate RT-PCR primer sets. (A) RT-PCR primer set #04 (Amplicon size 775 nt). (B) RT-PCR primer set #08 (738 nt).

Fig. 3. Sensitivity and non-specific reaction tests of candidate nested PCR primer sets. (A) Two candidate nested PCR primer sets from RT-PCR #04 and #08. (B) non-specific reaction of selectived nested PCR primer set #04-2 (630 nt).

Fig. 4. Sensitivity and non-specific reaction tests of reference RT- or RT-nested PCR primer sets.

吏븯닔 떆猷뚯뿉꽌쓽 쟻슜꽦 寃넗 諛 紐⑤땲꽣留

쟾援 냼洹쒕え湲됱닔떆꽕怨 留덉쓣긽닔룄쓽 吏븯닔 20媛쒖뿉꽌 異붿텧븳 total RNA濡쒕꽣 꽑諛쒗븳 RT-nested PCR 議고빀쑝濡 諛섏쓳븯쓣 븣 떆猷 #1怨 #2뿉꽌 630 nt쓽 異붿젙 뼇꽦 諛대뱶媛 굹궗쑝굹, 諛섎㈃ 鍮꾧탳 봽씪씠癒 議고빀 A뿉꽌뒗 떆猷 2뿉꽌, 鍮꾧탳 봽씪씠癒 議고빀 C뿉꽌뒗 遺遺꾩쓽 떆猷뚯뿉꽌 鍮 듅씠쟻 諛대뱶 걣由쇱씠 굹궗떎(Fig. 5). 씠寃껋 吏븯닔 벑 솚寃 떆猷 궡 븣 닔 뾾뒗 PCR inhobotors濡 씤빐 닚닔븳 빑궛留 議댁옱븯뒗 긽깭뿉 鍮꾪빐 빟 100諛 닔以源뚯 寃異 誘쇨컧룄媛 빐맆 닔룄 엳쑝硫, 븣 닔 뾾뒗 鍮 듅씠쟻 諛섏쓳씠 떎닔 굹굹뒗 寃껋쑝濡 꽕紐낅맆 닔 엳떎(Lee and Cho, 2019; Lee et al., 2021). 꽑諛 봽씪씠癒 議고빀 鍮꾧탳 봽씪씠癒 議고빀뱾뿉꽌 寃異쒗븯吏 紐삵븳 2媛 떆猷 궡 HuAstV 듅씠쟻 빑궛쓣 寃異쒗븷 닔 엳뿀怨, 利앺룺맂 궛臾쇱쓽 뿼湲곗꽌뿴쓽 NCBI BLAST븳 寃곌낵 떆猷 #1怨 #2뒗 HuAstV 1 strain lhar/2011/kor (JN887820.1) 媛곴컖 99.77% 99.73%쓽 쑀궗꽦씠 굹궗쑝硫, 몢 떆猷 媛 뿼湲곗꽌뿴쓽 쑀궗꽦 99.64%떎. Genotyping 寃곌낵 紐⑤몢 AstV genotype 1濡 굹궗쑝硫, HuAstV strain lhar/2011/kor (JN887820.1), HuAstV type strain Dresden (AY720892.1) 諛 HuAstV isolate JZ (KF211475.1)怨 쑀뿰愿怨꾧 넂븯떎(Fig. 6). 븳렪, 鍮꾧탳 봽씪씠癒 議고빀 A C뿉꽌 利앺룺맂 鍮 듅씠쟻 諛대뱶 以 利앺룺 겕湲곗 쑀궗븳 諛대뱶瑜 gel-purification 썑 뿼湲곗꽌뿴 遺꾩꽍쓣 닔뻾븯쑝굹 noise signal 벑쑝濡 紐⑤몢 鍮 듅씠쟻 利앺룺쑝濡 솗씤릺뿀떎. 씠뿉 뵲씪 씠踰 뿰援ъ뿉꽌 꽑諛쒗븳 RT-nested PCR 봽씪씠癒 議고빀 湲곗〈 蹂닿퀬맂 鍮꾧탳 봽씪씠癒몃뱾뿉 鍮꾪빐 吏븯닔뿉꽌쓽 솢슜꽦씠 긽쟻쑝濡 슜씠븯뿬 HuAstV 紐⑤땲꽣留곸슜쑝濡 쟻빀 븷 寃껋쑝濡 蹂댁씠硫, 寃異 誘쇨컧룄媛 슦닔븳 봽씪씠癒몃뱾쓽 議고빀怨 븿猿 鍮 듅씠쟻 뼲젣 臾쇱쭏쓣 룷븿븳 議곗꽦臾쇱쓽 씠쑀濡 異붿젙맂떎.

Fig. 5. Sample tests of developed RT-nested PCR primer set and two reference RT- or RT-nested PCR primer sets. 1~20, groundwater sample number.

Fig. 6. Neighbor-joining tree showing the phylogenetic position of two HuAstV positive samples based on amplicon sequence using selectived nested PCR primer set (F3m and Mon270R). Bootstrap values (< 50%) based on 1,000 replications are shown. Waterborn enteric viruses [Hepatitisvirus A (NC_001489.1), Hepatitisvirus E (NC_001434.1), human Enterovirus A (NC_001612.1) and human Aichivirus A (NC_001918.1)] were used as out groups. Bar, 0.1 nucleotide substitutions.

吏븯닔뿉꽌 HuAstV 紐⑤땲꽣留곷쾿쑝濡 솢슜

씠踰 뿰援ъ뿉꽌뒗 湲곕낯 蹂닿퀬맂 conventional PCR 湲곕컲쓽 諛⑸쾿 鍮 吏븯닔 떆猷뚯뿉꽌 怨좉컧룄濡 鍮 듅씠쟻 諛섏쓳 뾾씠 HuAstV쓽 빑궛쓣 紐⑤땲꽣留 븷 닔 엳뒗 RT-PCR 諛 nested PCR 봽씪씠癒 議고빀쓣 꽑諛쒗븯쑝硫, 씠瑜 솢슜븯뿬 吏븯닔 以 HuAstV瑜 紐⑤땲꽣留 븯뒗 諛⑸쾿 떎쓬怨 媛숇떎; ⅰ) 援由쏀솚寃쎄낵븰썝 怨좎떆 젣 2017-50뿉 뵲씪 吏븯닔 떆猷뚮 梨꾩랬 썑 븘씠뒪 諛뺤뒪뿉 떞븘 떎뿕떎濡 씠룞븯뿬, ⅱ) 깉由 諛 냽異뺤쓣 떆뻾븳떎. ⅲ) 냽異뺤븸 140 μL濡쒕꽣 RNeasy Mini Kit (Qiagen) 삉뒗 룞벑 꽦뒫쓽 kit瑜 궗슜븯뿬 total RNA瑜 異붿텧 諛 nanodrop 벑쑝濡 쉶닔맂 빑궛쓽 냽룄瑜 痢≪젙븳떎. ⅳ) 봽씪씠癒 議고빀AstV_1_F3 (5'-AGG ACC AAA GAA GTG TGA T-3') 諛 AstV_3_B3 (5'-CAC TCT GAA GCA AGT TCA A-3') (775 nt)쓣 궗슜븯뿬 RT-PCR쓣 닔뻾 썑 1.2% agarose gel뿉 윭떇 諛 gel documentation system뿉꽌 솗씤븳떎. 씠븣 HuAstV 빑궛쓣 뼇꽦議곌뎔쓽 二쇳삎쑝濡, 쓬꽦議곌뎔쑝濡쒕뒗 nucleic acid free water瑜 궗슜븳떎. ⅴ) 봽씪씠癒 議고빀 F3m (5'-GCA RGT HAC TGT TGA GGT C-3') 諛 MON 270R (5'-TCA GAT GCA TTG TCA TTG GT-3') (630 nt)瑜 궗슜븯뿬 nested PCR쓣 닔뻾 썑 1.2% agarose gel뿉 윭떇 諛 gel documentation system뿉꽌 솗씤븳떎. 씠븣 뼇꽦議곌뎔쑝濡쒕뒗 RT-PCR 뼇꽦議곌뎔쓽 利앺룺궛臾, RT-PCR negative쓽 利앺룺뿬遺 寃젙쓣 쐞빐 RT-PCR 쓬꽦議곌뎔쓽 利앺룺궛臾, nested PCR 쓬꽦議곌뎔쑝濡 nucleic acid free water瑜 궗슜븳떎. ⅵ) Nested PCR 寃곌낵 빟 630 nt쓽 諛대뱶媛 삎꽦릺硫 '異붿젙 뼇꽦'쑝濡 뙋젙븯怨 뿼湲곗꽌뿴 遺꾩꽍쓣 닔뻾븳떎. 뿼湲곗꽌뿴 遺꾩꽍 떆 鍮 듅씠쟻 諛대뱶媛 굹굹硫 빟 630 nt쓽 諛대뱶留뚯쓣 gel-purification븯뿬 sequencing븯硫, 떊猶 媛뒫븳 뿼湲곗꽌뿴 遺遺꾩쓣 痍⑦븯뿬 NCBI BLAST瑜 넻빐 query sequence쓽 similarity瑜 솗씤븳떎. ⅶ) BioEdit, MEGA 벑 software packages瑜 넻빐 maximum likelihood 삉뒗 neighbor joining 湲곕컲쓽 tree making method濡 genotyping쓣 닔뻾븯뿬 理쒖쥌 HuAstV 뼇꽦쑝濡 솗젙븷 닔 엳떎.

븳렪, 솚寃쎈 援由쏀솚寃쎄낵븰썝뿉꽌 냼洹쒕え 닔룄떆꽕, 留덉쓣긽닔룄 벑쓽 쓬슜 諛 鍮꾩쓬슜 吏븯닔 떆猷뚯뿉꽌 끂濡쒕컮씠윭뒪 留뚯쓣 湲곗빆紐⑹쑝濡 紐⑤땲꽣留곸쓣 떆뻾븯怨 엳떎. 洹몃윭굹 닔씤꽦 옣愿怨 諛붿씠윭뒪뒗 빟 150뿬 醫낆씠 議댁옱븯硫, 궗엺 쇅뿉룄 닔怨, 떇뭹, 뙣瑜 벑 떎뼇븳 留ㅼ쭏濡쒕꽣 媛먯뿼씠 씠猷⑥뼱吏 닔 엳뼱 以묒슂븳 蹂묒썝泥대줈 遺꾨쪟 諛 蹂닿퀬릺硫댁꽌(KCDC, 2015; KMFDS, 2015; NIER, 2016, 2017), 吏덈퀝愿由ъ껌怨 떇빟泥섎 以묒떖쑝濡 떆룄 蹂닿굔솚寃쎌뿰援ъ썝쓽 쑀愿 떆猷뚯뿉 誘멸퇋젣 닔씤꽦 옣愿怨 諛붿씠윭뒪 以 二쇱슂븳 HAV, HEV, SaV, RV-A enteric Adenovirus 븿猿 HuAstV 벑쓽 紐⑤땲꽣留곸쓣 沅뚭퀬븯怨 엳쑝硫, 솚寃쎈 援由쏀솚寃쎄낵븰썝뿉꽌룄 吏븯닔뿉꽌 HuAstV 벑 誘 洹쒖젣 닔씤꽦 諛붿씠윭뒪뿉 븳 뿰援щ 닔뻾븯怨 엳떎. 洹몃윭굹 吏븯닔 떆猷뚮뒗 梨꾩랬, 깉由 諛 냽異 벑 쟾 泥섎━뿉 留롮 떆媛, 鍮꾩슜 諛 끂룞젰씠 븘슂븯硫(Lee and Cho, 2019; Lee et al., 2021), 씠踰 뿰援ъ뿉꽌룄 20媛 닔以쓽 吏븯닔 떆猷뚯뿉꽌 紐⑤땲꽣留곸쓣 닔뻾븯떎. 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 諛⑸쾿쓽 寃利, 援궡 吏븯닔 벑 닔怨꾩뿉 옞옱쟻쑝濡 議댁옱븯뒗 HuAstV쓽 諛쒖깮썝 愿由, 궗쟾쓳쓣 쐞븳 洹쇨굅옄猷뚯쓽 援ъ텞 벑쓣 쐞빐꽌뒗 뜑슧 떎뼇븳 吏븯닔 떆猷뚮줈遺꽣 紐⑤땲꽣留곸씠 븘슂븷 寃껋쑝濡 蹂댁씤떎.

ACKNOWLEDGEMENT

This work was supported by a grant from the National Institute of Environmental Research (NIER), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIER-2020-01-01-003).

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

References
  1. Espinosa AC, Mazari-Hiriart M, Espinosa R, Maruri-Avidal L, M챕ndez E, Arias CF. Infectivity and genome persistence of rotavirus and astrovirus in groundwater and surface water. Water Res. 2008. 42: 2618-2628.
    Pubmed CrossRef
  2. Fusco G, Di Bartolo I, Cioffi BCioffi B et al. Prevalence of foodborne viruses in mussels in Southern Italy. Food Environ Virol. 2017. 9: 187-194.
    Pubmed CrossRef
  3. Hall T. BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser. 1999. 41: 95-98.
  4. Hargest V, Sharp B, Livingston BLivingston B et al. Astrovirus replication is inhibited by nitazoxanide in vitro and in vivo. J Virol. 2020. 94: e01706-19.
    Pubmed KoreaMed CrossRef
  5. Iritani N, Kaida A, Abe NAbe N et al. Detection and genetic characterization of human enteric viruses in oyster-associated gastroenteritis outbreaks between 2001 and 2012 in Osaka City, Japan. J Med Virol. 2014. 86: 2019-2025.
    Pubmed CrossRef
  6. Johnson C, Hargest V, Cortez V, Meliopoulos VA, Schultz-Cherry S. Astrovirus pathogenesis. Viruses. 2017. 9: 22.
    Pubmed KoreaMed CrossRef
  7. Korea Centers for Disease Control and Prevention (KCDC). Practical guidelines for laboratory diagnosis of waterborne food-borne diseases. 2015. pp. 88-89. KCDC. Chungcheongbuk-do, Korea.
  8. Korea Centers for Disease Control and Prevention (KCDC). Infectious disease potal. http://www.kdca.go.kr/npt/biz/npp/iss/gastrointestinalStatisticsMain.do. accessed 12 August 2021. 2021.
  9. Korea Ministry of Food and Drug Safety (KMFDS). Test method of food poisoning cause investigation. 2015. pp. 223-227. Chungcheongbuk-do, Korea.
  10. Krishna NK. Identification of structural domains involved in astrovirus capsid biology. Viral Immunol. 2005. 18: 17-26.
    Pubmed KoreaMed CrossRef
  11. Lee JY, Rho JY. Development of diagnostic method for human astrovirus with rapid, specific and high sensitivity using loop-mediated isothermal amplification method. Korean J Agric Sci. 2020. 47: 173-182.
  12. Lee S, Cho KB. Development of reverse transcription semi-nested PCR primer pairs for the specific and highly sensitive detection of human aichivirus A1. Biomed Sci Letters. 2019. 25: 331-338.
    CrossRef
  13. Lee S, Bae KS, Lee JY, Joo YL, Kim JH, You KA. Development of molecular diagnostic system with high sensitivity for the detection of human sapovirus from water environments. Biomed Sci Letters. 2021. 27: 35-43.
    CrossRef
  14. National Institute of Environmental Research. Development and verification of genetically diagnostic method for the detection of non-regulated viruses from water environment (I). 2016. pp. 1-21. NIER. Incheon, Korea.
  15. National Institute of Environmental Research. Development and verification of genetically diagnost2004ic method for the detection of non-regulated viruses from water environment (뀫). 2017. pp. 1-23. NIER. Incheon, Korea.
  16. Parr처n I, Plasencia E, Cornejo-S찼nchez TCornejo-S찼nchez T et al. Human astrovirus outbreak in a daycare center and propagation among household contacts. Viruses. 2021. 13: 1100.
    Pubmed KoreaMed CrossRef
  17. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013. 30: 2725-2729.
    Pubmed KoreaMed CrossRef
  18. Tan Y, He WT, Chen MM, Mo JJ, Ju Y, Chen M. An outbreak of human astrovirus lineage 1b in a middle school in Guangxi, Southern China in 2017. Chin Med J. 2019. 132: 336.
    Pubmed KoreaMed CrossRef
  19. Yi J, Lee JK, Chung EH, Cho DH, Kim EC. An outbreak of astrovirus infection of newborns with hemorrhagic diarrhea in a neonatal unit. Korean J Clin Microbiol. 2004. 7: 55-58.