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Development of Molecular Diagnostic System with High Sensitivity for the Detection of Human Sapovirus from Water Environments
Biomed Sci Letters 2021;27:35-43
Published online March 31, 2021;  https://doi.org/10.15616/BSL.2021.27.1.35
© 2021 The Korean Society For Biomedical Laboratory Sciences.

Siwon Lee1,2,* , Kyung Seon Bae3,* * , Jin-Young Lee2,* * , Youn-Lee Joo3,* * , Ji-Hae Kim3,** and Kyung-A You3,,* * *

1Department of Biomedical Laboratory Science, Shinhan University, Uijeongbu 11644, Korea
2R&D Team, LSLK Co., Gimpo, Gyeonggi 10111, Korea
3Water Supply and Sewerage Research Division, National Institute of Environmental Research, Incheon 22689, Korea
Correspondence to: 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
*Professor, **Researcher, ***Senior Researcher.
Received January 15, 2021; Revised February 24, 2021; Accepted March 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 Sapovirus (HuSaV) is one of the major causes of acute gastroenteritis in humans, and it is used as a molecular diagnostic technique based on polymerase chain reaction (PCR) from humans, food, shellfish, and aquatic environments. In this study, the HuSaV diagnosis technique was used in an aquatic environment where a number of PCR inhibitors are included and pathogens, such as viruses, are estimated to exist at low concentration levels. HuSaV-specific primers are improved to detect 38 strains registered in the National Center for Biotechnology Information (NCBI). The established optimal condition and the composition, including the RT-nested PCR primers and SL® Non-specific reaction inhibitor, were found to have 100 times higher sensitivity based on HuSaV plasmid than the previously reported methods (100 ag based on HuSaV plasmid 1 ng/關L). Through an artificial infection test, the developed method was able to detect at least 1 fg/關L of HuSaV plasmid contaminated with total nucleic acid extracted from groundwater. In addition, RT-nested PCR primer sets for HuSaV detection can react, and a positive control is developed to verify false positives. This study is expected to be used as a HuSaV monitoring method in the future and applied to the safety response to HuSaV from water environments.
Keywords : Human Sapovirus, HuSaV, Positive control, SL® Non-specific reaction inhibitor, Water environments
Body

Human Sapovirus (HuSaV)뒗 Norovirus (NoV), Rotavirus A (RV-A), Astrovirus (AstV), enteric Adenovirus (eAdV) 븿猿 궗엺 湲됱꽦 쐞옣뿼쓽 二쇱슂 썝씤 諛붿씠윭뒪 以 븯굹씠떎(Oka et al., 2015; Hwang et al., 2015). 援궡뿉꽌뒗 쁺쑀븘뿉꽌 留 12꽭 뼱由곗씠뱾뿉寃 二쇰줈 諛쒖깮씠 蹂닿퀬릺뿀쑝硫, 8~12썡뿉 諛쒕퀝瑜좎씠 넂븯떎(Hwang et al., 2015; KCDC, 2019; Cho, 2018b). HuSaV뒗 遺꾨먭뎄媛 寃쎈줈濡 媛먯뿼 諛 삤뿼릺誘濡 궗엺, 떇뭹, 뙣瑜, 닔怨 솚寃 벑쑝濡쒕꽣 옞옱쟻 議댁옱 媛뒫꽦씠 엳쑝誘濡 媛 留ㅼ쭏뿉꽌쓽 紐⑤땲꽣留 벑 媛먯떆媛 以묒슂븯떎(KCDC, 2015; KMFDS, 2015; NIER, 2016). HuSaV 吏꾨떒 諛 紐⑤땲꽣留 諛⑸쾿 以묓빀슚냼뿰뇙諛섏쓳(polymerase chain reaction; PCR) 湲곕컲쓽 遺꾩옄吏꾨떒 湲곕쾿씠 二쇰줈 솢슜릺怨 엳떎(Cho, 2018a; Fukuda et al., 2006; Oka et al., 2006; Shigemoto et al., 2011). 洹몃윭굹 떆猷 以 吏븯닔 벑 닔怨 솚寃쎌 궗엺, 떇뭹, 뙣瑜 벑 떎瑜 留ㅼ쭏 鍮 蹂묒썝泥닿 긽쟻쑝濡 궙 닔以 냽룄 諛 떎뼇븳 쑀쟾옄삎쓽 議댁옱媛 異붿젙릺怨, 솚寃 떆猷뚮뒗 PCR 寃궗 떆 遺떇궛 벑 遺떇吏 臾쇱쭏, 誘몄궛, 湲덉냽씠삩, 뤃由ы럹, 뿼냼, 泥좊텇 벑 떎뼇븳 빐 臾쇱쭏쓣 룷븿븯怨 엳뼱 留ㅼ쭏쓽 듅꽦뿉 뵲瑜 怨좉컧룄 寃궗 湲곕쾿씠 븘슂븯떎(Cho, 2018b; Dalecka and Mezule, 2018; Schrader et al., 2012). 삉븳, 援궡 寃쎄린룄뿉꽌 理쒓렐 HuSaV-GI.3 쑀삎쓽 理쒖큹 蹂닿퀬(Cho et al., 2020)릺뒗 벑, 諛붿씠윭뒪쓽 蹂씠뿉 뵲瑜 genotyping쓽 以묒슂꽦씠 遺媛곷릺硫댁꽌 썑냽 쑀쟾삎 遺꾩꽍씠 媛뒫븳 conventional PCR쓽 솢슜씠 沅뚯옣릺怨 엳떎(NIER, 2016). 씠濡 씤빐 理쒓렐 떎뼇븳 醫낅쪟쓽 HuSaV 뿼湲곗꽌뿴씠 誘멸뎅援由쎌깮臾쇱젙蹂댁꽱꽣뿉 벑濡앸릺怨 엳쑝硫 湲곗〈 蹂닿퀬맂 紐뉖챺쓽 HuSaV 寃異쒖슜 RT-PCR 봽씪씠癒 議고빀 理쒓렐 벑濡앸맂 HuSaV쓽 뿼湲곗꽌뿴뿉 寃고빀븯吏 紐삵븷 媛뒫꽦씠 諛쒓껄릺뿀쑝굹, 吏븯닔 벑 닔怨 솚寃 以 옞옱쟻 議댁옱 媛뒫꽦씠 엳뒗 떎뼇븳 HuSaV 寃異쒖쓣 쐞븳 遺꾩옄吏꾨떒 湲곕컲 寃궗 湲곕쾿뿉 븳 뿰援щ뒗 誘명씉븳 긽솴씠떎. 븳렪, 遺꾩옄吏꾨떒 떆 寃궗쓽 떊猶곗꽦 뼢긽쓣 쐞빐 뼇꽦議 臾쇱쭏(i.e. viral plasmid, RNA transcript 벑)씠 궗슜릺怨 엳吏留, 寃궗 쟾 泥섎━ 벑 議곗옉 떆 뼇꽦議 臾쇱쭏濡쒕꽣쓽 삤뿼쑝濡 씤빐 쐞 뼇꽦(false positive)쓣 寃젙 븷 닔 엳뒗 떆뒪뀥씠 븘슂븯떎(Lee, 2013). 뵲씪꽌 씠踰 뿰援ъ뿉꽌뒗 HuSaV쓽 떎뼇븳 醫낅쪟쓽 뿼湲곗꽌뿴 遺李⑹씠 媛뒫븳 RT-PCR 諛 nested PCR 봽씪씠癒 議고빀쓣 媛쒕컻 諛 理쒖쟻쓽 利앺룺諛섏쓳쓣 쐞븳 議곌굔쓣 솗由쏀븯쑝硫, 쐞 뼇꽦 寃젙씠 媛뒫븳 옣移섍 룷븿맂 뼇긽議 臾쇱쭏쓣 媛쒕컻븯뒗 벑 닔怨 솚寃쎌뿉꽌 HuSaV 紐⑤땲꽣留곸쓣 쐞븳 遺꾩옄吏꾨떒 떆뒪뀥쓣 媛쒕컻븯떎.

HuSaV뒗 NCBI accession number KP298674 4,440-6,439 돱겢젅삤씠뱶(nucleotide; nt)瑜 湲곗쑝濡 2,000 nt 諛 李멸퀬諛붿씠윭뒪 6醫(Aichivirus A, Astrovirus, Hepatitisvirus A, Hepatitisvirus E, NorovirusRotavirus-A)쓽 쑀쟾옄 떒렪쓣 빀꽦븯떎[Marcrogen (Seoul, Korea)]. HuSaV 寃異쒖슜 썑蹂 RT-PCR 諛 nested PCR 봽씪씠癒 議고빀 湲곗〈 蹂닿퀬맂 諛⑸쾿뱾[Khamrin et al., 2011; Kitajima et al., 2010; Korea Centers for Disease Control and Prevention (KCDC), 2015; Korea Ministry of Food and Drug Safety (KMFDS), 2015; Kumthip et al., 2020; Liu et al., 2015; Oka et al., 2015; Shigemoto et al., 2011; Thwiny et al., 2015]쓣 湲곗쑝濡 벑濡앸맂 38媛 醫낅쪟쓽 HuSaV 쑀삎뿉 寃고빀씠 媛뒫븯룄濡 돱겢젅삤씠뱶 젅꽣肄붾뱶濡 蹂삎븯떎. 蹂삎븳 PCR 봽씪씠癒몃뒗 Oligo Calculator version 3.27濡 self annealing, potential hairpin formation 벑쓣 젏寃븯뿬 理쒖젙 젙諛⑺뼢 3媛 諛 뿭諛⑺뼢 2媛쒖쓽 PCR 봽씪씠癒몃뱾쓣 꽕怨 諛 젣옉 諛 꽕怨꾪븳 봽씪씠癒몃뱾쓣 議고빀븳 6媛쒖쓽 썑蹂 RT-PCR 봽씪씠癒 議고빀쓣 援ъ꽦븯떎(Table 1). HuSaV 寃異쒖슜 6媛쒖쓽 썑蹂 RT-PCR 봽씪씠癒 議고빀뿉 븳 寃異 誘쇨컧룄 諛 李멸퀬 諛붿씠윭뒪 6醫 빑궛뿉 븳 鍮 듅씠쟻 諛섏쓳쓣 寃젙븯떎. HuSaV plasmid 1 ng/關L 湲곗 10-3뿉꽌 10-8源뚯 씗꽍븯뿬 寃異 誘쇨컧룄瑜 遺꾩꽍븯쑝硫, PCR 議곗꽦怨 議곌굔 援由쏀솚寃쎄낵븰썝 닔씤꽦諛붿씠윭뒪 寃궗踰뺢낵 룞씪븯寃 닔뻾븯떎(NIER, 2016). HuSaV 寃異쒖슜 썑蹂 RT-PCR 봽씪씠癒 議고빀뿉꽌뒗 10-3뿉꽌 10-5 닔以쓽 寃異 誘쇨컧룄媛 굹궗쑝硫, 씠 以 媛옣 슦닔븳 寃異 誘쇨컧룄瑜 蹂댁씤 썑蹂 RT-PCR 봽씪씠癒 議고빀 #1怨 #5떎. #1怨 #5뿉 븯뿬 10-5뿉꽌 10-6 源뚯쓽 寃異 誘쇨컧룄瑜 異붽濡 遺꾩꽍븳 寃곌낵 781 nt쓽 궛臾쇱씠 삎꽦릺뒗 #1瑜 꽑諛쒗븯떎(Fig. 1A). RT-PCR 봽씪씠癒 議고빀 #1 李멸퀬諛붿씠윭뒪 6醫낆뿉 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡븘 HuSaV 留뚯쓣 듅씠쟻쑝濡 寃異쒗븷 닔 엳쓣 寃껋쑝濡 異붿젙릺뿀떎(옄猷 誘 젣怨). 삉븳, 吏븯닔 벑 떆猷뚯뿉꽌 궙 닔以쓽 HuSaV 삤뿼쓽 寃쎌슦 怨좉컧룄 寃異쒖쓣 쐞븯뿬 RT-PCR 봽씪씠癒 議고빀 #1쓽 궛臾쇰줈遺꽣 利앺룺씠 媛뒫븳 nested PCR 봽씪씠癒 議고빀쓣 꽑諛쒗븯떎. RT-PCR 봽씪씠癒 議고빀 #1濡쒕꽣 3媛쒖쓽 썑蹂 nested PCR 봽씪씠癒 議고빀(#1-1, #1-2 諛 #1-3)쓣 꽕怨꾪븯쑝硫, 寃異 誘쇨컧룄瑜 遺꾩꽍븳 寃곌낵 491 nt瑜 理쒖쥌 궛臾쇰줈 븯뒗 #1-1씠 10-7 닔以쑝濡 떎瑜 2媛 議고빀뿉 鍮꾪빐 빟 10諛 닔以 슦닔븯寃 굹궗떎(Fig. 1B). HuSaV plasmid瑜 二쇳삎쑝濡 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 RT-nested 봽씪씠癒 議고빀쓣 궗슜븯뿬 理쒖쥌 利앺룺맂 궛臾쇱쓣 뿼湲곗꽌뿴 遺꾩꽍븳 寃곌낵, HuSaV (NCBI accession number KP298674) 100.0% 寃곌낵媛 굹궗떎(옄猷 誘 젣怨). 븳렪 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 HuSaV 寃異쒖슜 RT-PCR 諛 nested PCR 봽씪씠癒 議고빀 湲곗〈 蹂닿퀬맂 7媛쒖쓽 RT-PCR 諛 RT-nested PCR 諛⑸쾿뿉 鍮꾪빐 RT-PCR 닔以뿉꽌 빟 룞벑-1000諛 씠긽, nested PCR 닔以뿉꽌뒗 빟 100~1,000諛 슦닔븳 寃異 誘쇨컧룄媛 굹궓뿉 뵲씪(Fig. 2) 떆猷 궡 궙 닔以쓽 HuSaV 議댁옱 떆뿉룄 寃異쒖씠 湲곕맂떎.

RT- and nested PCR information for the detection of human Sapovirus (HuSaV)

Division PCR type Primer information Length (nt) References Remark

Name Sequence (5'→5') Mer (nt) Location*

Start End
Candidate RT-nested PCR primer ses in this study RT-PCR primer set #1 RT-PCR SaV-F5098m GCYTGGTTYATAGGTGGTAC 20 5,098 5,117 781 This study SV-F11 base modified
SaV-R5878m CWGGTGAIMHICCATTKTCCAT 22 5,857 5,878 SV-R1 base modified

RT-PCR primer set #2 RT-PCR SaV-F5157m AITAGTGTTTGARATGGAGGG 21 5,157 5,177 435 This study SV-F21 base modified
SV-R2 GWGGGRTCAACMCCWGGTGG 20 5,572 5,591 [2,3] Same as reference

RT-PCR primer set #3 RT-PCR SaV-F5101m TGGTTYATAGGTGGTRCAG 19 5,101 5,119 778 This study SV-F11 base modified
SaV-R5878m CWGGTGAIMHICCATTKTCCAT 22 5,857 5,878 SV-R1 base modified

RT-PCR primer set #4 RT-PCR SaV-F5101m TGGTTYATAGGTGGTRCAG 19 5,101 5,119 491 This study SV-F11 base modified
SV-R2 GWGGGRTCAACMCCWGGTGG 20 5,572 5,591 [2,3] Same as reference

RT-PCR primer set #5 RT-PCR SaV-F5098m GCYTGGTTYATAGGTGGTAC 20 5,098 5,117 494 This study SV-F11 base modified
SV-R2 GWGGGRTCAACMCCWGGTGG 20 5,572 5,591 [2,3] Same as reference

Nested PCR primer set #1-1 Nested PCR SaV-F5101m TGGTTYATAGGTGGTRCAG 19 5,101 5,119 491 This study SV-F11 base modified
SV-R2 GWGGGRTCAACMCCWGGTGG 20 5,572 5,591 [2,3] Same as reference


Nested PCR primer set #1-2 SaV-F5098m GCYTGGTTYATAGGTGGTAC 20 5,098 5,117 494 This study SV-F11 base modified
SV-R2 GWGGGRTCAACMCCWGGTGG 20 5,572 5,591 [2,3] Same as reference


Nested PCR primer set #1-3 SaV-F5157m AITAGTGTTTGARATGGAGGG 21 5,157 5,177 435 This study SV-F21 base modified
SV-R2 GWGGGRTCAACMCCWGGTGG 20 5,572 5,591 [2,3] Same as reference

Reference RT or RT-nested PCR primer sets Ref.#1 RT-PCR SV-F21 (5157-5177) ANTAGTGTTTGARATGGAGGG 21 5,157 5,177 722 Shigemoto et al., 2011
SV-R1 (5857-5878) CWGGTGAMACMCCATTKTCCAT 22 5,857 5,878

Ref.#2 RT-PCR SV-F11 (5098-5117) GCYTTGTTYATAGGTGGTAC 20 5,098 5,117 781 Oka et al., 2015
SV-R1 (5857-5878) CWGCTGAMACMCCATTKTCCAT 22 5,857 5,878

Nested PCR SV-F21 (5157-5177) ANTAGTGTTTGARATGGAGGG 21 5,157 5,177 435
SV-R2 (5572-5591) GWGGGRTCAACMCCWGGTGG 20 5,572 5,591

Ref.#3 RT-PCR SV-F11 (5098-5117) GCYTTGTTYATAGGTGGTAC 20 5,098 5,117 781 Kitajima et al., 2010
SV-R1 (5857-5878) CWGCTGAMACMCCATTKTCCAT 22 5,857 5,878

Reference RT or RT-nested PCR primer sets Ref.#3 Nested PCR 1245Rfwd (5161-5177) TAGTGTTTGARATGGAGGG 19 5,159 5,177 433 Kitajima et al., 2010
SV-R2 (5572-5591) GWGGGRTCAACMCCWGGTGG 20 5,572 5,591

Ref.#4 RT-PCR SLV5317 CTCGCCACCTACRAWGCBTGGTT 23 5,083 5,105 434 Kumthip et al., 2020
SLV5749 CGGRCYTCAAAVSTACCBCCCCA 23 5,494 5,516

Ref.#5 RT-PCR SV-F14 (5074-) GAACAAGCTGTGGCATGCTAC 21 5,074 5,094 803 Liu et al., 2015
SV-R14 (-5876) GGTGAGMMYCCATTCTCCAT 20 5,857 5,876

Nested PCR SV-F22 (5154-) SMWAWTAGTGTTTGARATG 19 5,154 5,172 438
SV-R2 (-5591) GWGGGRTCAACMCCWGGTGG 20 5,572 5,591

Ref.#6 RT-PCR SR80 TGGGATTCTACACAAAACCC 20 4,366 4,385 320 Thwiny et al., 2015
JV33 GTGTANATGCARTCATCACC 20 4,666 4,685

Ref.#7 RT-PCR SLV5317 CTCGCCACCTACRAWGCBTGGTT 23 5,083 5,105 100 Khamrin et al., 2011
SMP-R CMWWCCCCTCCATYTCAAACAC 22 5,161 5,182

*Based on NCBI accession number KP298674.1



Fig. 1. Sensitivity test of candidate RT-PCR and nested primer sets for the detection of HuSaV. (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 test of seven previously reported RT-PCR or RT-nested PCR primer sets for the detection of HuSaV. M, 100 bp Ladder marker (Enzynomics); -3 ~ -8, template dilution value from 1 ng/μL HuSaV plasmid; N, negative control.

媛쒕컻븳 PCR 諛 nested PCR쓽 떆猷 寃젙쓣 쐞븯뿬, 援由쏀솚寃쎄낵븰썝 怨좎떆 젣 2017-50뿉 뵲씪 吏븯닔 떆猷 20젏쓣 梨꾩랬, 깉由 諛 냽異뺥븯쑝硫, 理쒖쥌 냽異뺤븸쑝濡쒕꽣 RNeasy Mini Kit (Qiagen, Germany)쓽 留ㅻ돱뼹뿉 뵲씪 total RNA瑜 異붿텧븯떎. RT-PCR AccuPower RT/PCR PreMix (Bioneer, Daejeon, Korea), nested PCR AccuPower HotStart PCR PreMix (Bioneer)瑜 궗슜븯怨, 솚寃 떆猷뚯뿉꽌 굹굹뒗 鍮 듅씠쟻 諛섏쓳뼲젣瑜 쐞빐 SL Non-specific reaction inhibitor (LSLK, Gyeonggi, Korea) 3 關L瑜 룷븿븯뿬 理쒖쥌 20 關L volume쑝濡 諛섏쓳븯떎. RT-nested PCR 寃곌낵, 20媛 吏븯닔 떆猷뚯뿉꽌 遺꾩꽍 寃곌낵 紐⑤몢 쓬꽦쑝濡 遺꾩꽍맖뿉 뵲씪(Fig. 3A), HuSaV plasmid쓽 吏븯닔뿉꽌 異붿텧븳 빑궛뿉 씤쐞媛먯뿼 븯뿬 떆猷 궡 뼇꽦씤 寃쎌슦쓽 옉룞 媛뒫꽦 諛 寃異 誘쇨컧룄瑜 遺꾩꽍븯떎. 吏븯닔 떆猷#1뿉꽌 12 preps.쓽 total RNA瑜 異붿텧븯뿬 720 關L瑜 솗蹂댄븯怨, 45 關L RNA瑜 8媛 깉濡쒖슫 tube뿉 遺꾩< 썑 1 ng/關L plasmid 5 關L瑜 떆옉쑝濡 10諛 떒怨 씗꽍븯떎. 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 RT-nested PCR 봽씪씠癒 議고빀쓣 룷븿븳 議곗꽦臾 諛 議곌굔쑝濡 씤쐞쟻 媛먯뿼 떆猷뚯뿉 븳 寃異 誘쇨컧룄瑜 遺꾩꽍븳 寃곌낵 RT-PCR뿉꽌 빟 10-4 닔以, nested PCR뿉꽌 빟 10-6 닔以쑝濡 굹궗쑝硫, 닚닔븳 plasmid 議곌굔 鍮 떆猷 궡뿉꽌뒗 寃異 誘쇨컧룄媛 빟 10諛 닔以 媛먯냼븯떎(Fig. 3B). 븳렪, 媛쒕컻븳 떆뿕踰뺤쓽 諛몃━뜲씠뀡쓣 쐞빐 떎瑜 궇 떎瑜 寃궗옄 諛 떎瑜 湲곌쓽 뿰援ъ옄뿉 쓽빐 듅씠꽦 諛 寃異 誘쇨컧룄 벑뿉 빐 2쉶 異붽 諛섎났 떆뿕븯뿬 珥 3쉶 떆뿕 寃곌낵 룞씪븳 寃곌낵媛 굹궗떎(옄猷 誘 젣怨).

Fig. 3. Sample and artificial infection tests of developed RT-nested PCR primer set for the detection of HuSaV in this study. (A) HuSaV detection using the composition including RT-nested PCR primer sets and conditions developed in this study. (B) Artificial infection tests of 20 groundwater samples. M, 100 bp Ladder marker (Enzynomics); -1 ~ -7, template dilution value from 1 ng/μL HuSaV plasmid; N, negative control, PN, PCR negative control.

븳렪, 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 RT-PCR 諛 nested PCR 봽씪씠癒 議고빀씠 諛섏쓳븷 닔 엳뒗 뼇꽦議 臾쇱쭏쓣 젣옉븯떎. 떎뿕 떆 쐞 뼇꽦쓣 젏寃븷 닔 엳룄濡 利앺룺 궛臾쇱쓽 겕湲곕 떎瑜닿쾶 븯怨, nested PCR 利앺룺 썑 4醫낅쪟쓽 젣븳슚냼媛 諛섏쓳븷 닔 엳뒗 듅젙 뿼湲곗꽌뿴쓣 룷븿븯뒗 諛⑸쾿(Lee et al., 2011; Lee, 2013)쓣 쟻슜븯뿬 꽕怨 썑 (二) Macrogen뿉꽌 쑀쟾옄 빀꽦븯떎(Fig. 4A). 젣옉븳 뼇꽦議 臾쇱쭏쓣 1 pg/關L濡 씗꽍 썑 媛쒕컻븳 PCR 諛 nested PCR 봽씪씠癒 議고빀쓽 諛섏쓳꽦쓣 젏寃븯떎. Nested PCR 利앺룺 썑 궛臾쇱쓣 EcoRV (GAT/ATC) (New England Biolabs, Boston, USA)뿉 泥섎━ 썑, 2% agarose gel뿉 쟾湲곗쁺룞 븯쑝硫 UV 븯뿉꽌 遺꾩꽍븯떎. PCR 寃곌낵 500 nt, nested PCR 寃곌낵 450 nt쓽 듅젙 諛대뱶媛 삎꽦릺뿀떎. 1st PCR 利앺룺 떆 781 nt, 2nd PCR 利앺룺 떆 491 nt媛 굹굹뒗 湲곗〈 HuSaV plasmid 諛 떆猷뚯뿉꽌 利앺룺씠 삁긽릺뒗 HuSaV 쑀쟾옄 떒렪쓽 겕湲곗 李⑥씠媛 굹궗쑝硫, 媛쒕컻븳 뼇꽦議 臾쇱쭏 nested PCR 利앺룺 궛臾쇱뿉 젣븳슚냼 EcoRV 泥섎━ 寃곌낵 2醫낅쪟 겕湲(322 諛 172 nt)쓽 諛대뱶媛 굹궗떎(Fig. 4B). 씠뿉 뵲씪 媛쒕컻븳 뼇꽦議 臾쇱쭏뿉꽌 삤뿼씠 씪뼱굹 쐞 뼇꽦 諛섏쓳씠 굹굹硫 1st PCR 떆 겕湲곕줈 寃젙씠 媛뒫븯硫, 2nd PCR 떆 궛臾쇱쓽 젣븳슚냼 EcoRV 泥섎━濡 諛대뱶쓽 젅떒쓣 솗씤븯뿬 쐞 뼇꽦뿉 븳 뿬遺瑜 솗씤븷 닔 엳쓣 寃껋쑝濡 궗猷뚮맂떎.

Fig. 4. The positive control design information and restriction enzyme treatment result developed in this study. (A) Design information of positive control capable of false positive test via restriction enzyme cutting from nested PCR product and amplicon size thereof. (B) Restriction enzyme EcoRV digestion of plasmid and development of positive control using the nested PCR amplicons as a template. M, 100 bp DNA Ladder marker (Enzynomics); P, plasmid; dP, developed positive control; N, negative control; PN, PCR negative control; Pt, plasmid treatment; dPt, developed positive control treatment.

씠踰 뿰援ъ뿉꽌뒗 엫긽, 떇뭹, 吏븯닔 諛 鍮 냼룆닔 벑 떎뼇븳 솚寃쎌뿉꽌 떊냽븯怨 듅씠꽦씠 넂쑝硫, 떎뼇븳 HuSaV 룷븿 븯쐞遺꾨쪟援곗쓽 寃異쒖씠 媛뒫븳 蹂대떎 슚怨쇱쟻씤 吏꾨떒슜 RT-nested PCR 봽씪씠癒 議고빀쓣 媛쒕컻븯怨좎옄 븯떎. 쁽옱 HuSaV쓽 吏꾨떒 듅씠쟻 諛섏쓳, 寃異 誘쇨컧룄, 遺꾩꽍 궃씠룄, 鍮꾩슜 벑쓣 怨좊젮븳 real-time qPCR, 벑삩利앺룺踰(Loop-mediated isothermal amplification; LAMP), multiplex reverse-transcription PCR 벑씠 蹂닿퀬릺뼱 엳떎(Cho et al., 2018; Fukuda et al., 2006; Oka et al., 2006; Shigemoto et al., 2011). 씠뱾 以 real-time qPCR쓽 寃쎌슦, Taq-man probe 諛⑸쾿씠 씪諛섏쟻쑝濡 媛옣 留롮씠 궗슜릺怨 엳쑝硫(Ponchel et al., 2003) 듅씠꽦, 寃異 誘쇨컧룄媛 넂怨 떊냽븳 吏꾨떒씠 媛뒫븳 옣젏쓣 媛吏怨 엳뼱 떎뼇븳 遺꾩빞뿉꽌 솢슜씠 蹂닿퀬(Mano et al., 2014; Botes et al., 2013; Bhullar et al., 2014) 릺怨 엳떎. 洹몃윭굹 빐떦 뵆옯뤌 利앺룺 궛臾쇰줈 뿼湲곗꽌뿴 遺꾩꽍쓣 븷 닔 뾾뒗 떒젏씠 엳떎(Cho et al., 2018). 벑삩利앺룺踰뺤쓽 寃쎌슦, 鍮 듅씠쟻 쐞 뼇꽦, 넂 鍮꾩슜 벑쓽 떒젏씠 蹂닿퀬릺怨 엳뒗 벑(Smith and Osborn, 2009) PCR 寃궗 湲곕쾿뿉 떎뼇븳 옣떒젏씠 議댁옱븯뒗 寃껋씠 듅吏뺤씠떎. 씠踰 뿰援ъ뿉꽌 吏꾨떒 諛⑸쾿쑝濡 궗슜븳 conventional PCR쓽 寃쎌슦, 삤옖 湲곌컙 留롮 뿰援ъ옄뱾뿉 쓽빐 븞젙꽦씠 寃利앸릺뿀怨, 뿼湲곗꽌뿴 遺꾩꽍씠 媛뒫븯硫, 긽쟻쑝濡 鍮꾩슜, 寃궗옄쓽 닕젴룄, 遺꾩꽍 궃씠룄 痢〓㈃뿉꽌 떎瑜 PCR 寃궗 湲곕쾿 蹂대떎 옣젏쓣 媛吏怨 엳뼱, 몴以寃궗踰뺤쑝濡 媛옣 留롮씠 궗슜릺怨 엳뒗 諛⑸쾿씠떎(KCDC, 2015; KMFDS, 2015; NIER, 2016). 씠뿉 뵲씪 씠踰 뿰援ъ뿉꽌뒗, HuSaV쓽 genotyping쓣 넻븳 썑냽 쑀쟾삎 遺꾩꽍쓣 쐞빐 conventional RT-nested PCR 諛⑸쾿 湲곕컲쓽 湲곗닠쓣 媛쒕컻븯떎. 蹂 뿰援ъ뿉꽌 媛쒕컻븳 RT-nested PCR 봽씪씠癒몃뒗 HuSaV 38媛 븯쐞遺꾨쪟援곗쓽 variation쓣 寃異쒗븷 닔 엳룄濡 꽕怨꾪븯떎. 湲곗〈 蹂닿퀬맂 PCR 봽씪씠癒 鍮 RT-PCR 젙諛⑺뼢 봽씪씠癒몃뒗 2 nt variation, RT-PCR 뿭諛⑺뼢 봽씪씠癒몃뒗 6 nt variation, nested PCR 젙諛⑺뼢 봽씪씠癒몃뒗 2 nt variation쓣 而ㅻ쾭븿뿉 뵲씪 湲곗〈 諛⑸쾿 鍮 꼻 닔以쓽 HuSaV 寃고빀씠 異붿젙릺뿀떎(옄猷 誘 젣怨). 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 諛⑸쾿 紐⑤땲꽣留 寃궗湲곌컙쓣 嫄곗퀜 뼢썑 吏븯닔 벑 닔怨 솚寃쎌뿉꽌 HuSaV 寃異쒖쓣 쐞븳 諛⑸쾿 벑쑝濡 솢슜꽦씠 湲곕맂떎.

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. Botes M, de Kwaadsteniet M, Cloete TE. Application of quantitative PCR for the detection of microorganisms in water. Anal Bioanal Chem. 2013. 405: 91-108.
    Pubmed KoreaMed CrossRef
  2. Bhullar SS, Chandak NH, Purohit HPurohit H et al. Determination of viral load by quantitative real-time PCR in herpes simplex encephalitis patients. Intervirology. 2014. 57: 1-7.
    Pubmed CrossRef
  3. Cho KB. Development of nested PCR primer set for the specific and highly sensitive detection of human parvovirus B19. Biomed Sci Lett. 2018a. 24: 390-397.
    CrossRef
  4. Cho KB. Construction of Improved PCR primer set for the detection of human enteric adenovirus 41. Biomed Sci Lett. 2018b. 24: 230-238.
    CrossRef
  5. Cho SR, Lee DY, Jung SJung S et al. Pathogen surveillance of acute viral gastroenteritis in Korea in 2017. Public Health Weekly Report. 2018. 11: 1374-1380.
  6. Cho SR, Yun SJ, Chae SJChae SJ et al. An outbreak associated with sapovirus GI.3 in an elementary school in Gyeonggi-do, Korea. J Korean Med Sci. 2020. 35: e281.
    Pubmed KoreaMed CrossRef
  7. Dalecka B, Mezule L. Study of potential PCR inhibitors in drinking water for Escherichia coli identification. Agron Res. 2018. 16: 1351-1359.
  8. Fukuda S, Takao S, Kuwayama M, Shimazu Y, Miyazaki K. Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol. 2006. 44: 1376-1381.
    Pubmed KoreaMed CrossRef
  9. Hwnag BM, Lee DY, Chung GT, Yoo CK. Laboratory surveillance of viral acute gastroenteritis in Korea, 2014. Public Health Weekly Report. 2015. 8: 1172-1177.
  10. Khamrin P, Okame M, Thongprachum AThongprachum A et al. A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea. J Virol Methods. 2011. 173: 390-393.
    Pubmed CrossRef
  11. Kitajima M, Oka T, Haramoto EHaramoto E et al. Detection and genetic analysis of human sapoviruses in river water in Japan. Appl Environ Microbiol. 2010. 76: 2461-2467.
    Pubmed KoreaMed CrossRef
  12. 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.
  13. Korea Centers for Disease Control and Prevention (KCDC). Weekly sentinel surveillance report, PHWR, 2019. pp. 2283. KCDC, Chungcheongbuk-do, Korea.
  14. Korea Ministry of Food and Drug Safety (KMFDS). Test method of food poisoning cause investigation, 2015. pp. 223-227. KMFDS, Chungcheongbuk-do, Korea.
  15. Kumthip K, Khamrin P, Ushijima HUshijima H et al. Genetic recombination and diversity of sapovirus in pediatric patients with acute gastroenteritis in Thailand, 2010-2018. PeerJ. 2020. 8: e8520.
    Pubmed KoreaMed CrossRef
  16. Lee S. A study of molecular biological detection methods for seed-transmitted viruses in quarantine. Ph. D. thesis, 2013. Dankook University, Cheonan, Chungcheongnam-do, Korea.
  17. Lee SG, Lee SH, Park SWPark SW et al. Standardized positive controls for detection of norovirus by reverse transcription PCR. Virol J. 2011. 260: 1-8.
    Pubmed KoreaMed CrossRef
  18. Liu X, Yamamoto D, Saito MSaito M et al. Molecular detection and characterization of sapovirus in hospitalized children with acute gastroenteritis in the Philippines. J Clin Virol. 2015. 68: 83-88.
    Pubmed CrossRef
  19. Mano J, Hatano S, Futo SFuto S et al. Development of direct real-time PCR system applicable to a wide range of foods and agricultural products. Shokuhin Eiseigaku Zasshi. 2014. 55: 25-33.
    Pubmed CrossRef
  20. NIER. 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.
  21. Oka T, Katayama K, Hansman GSHansman GS et al. Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction. J Med Virol. 2006. 78: 1347-1353.
    Pubmed CrossRef
  22. Oka T, Wang Q, Katayama K, Saif LJ. Comprehensive review of human sapoviruses. Clin Microbiol Rev. 2015. 28: 32-53.
    Pubmed KoreaMed CrossRef
  23. Ponchel F, Toomes C, Bransfield KBransfield K et al. Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol. 2003. 3: 18.
    Pubmed KoreaMed CrossRef
  24. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012. 113: 1014-1026.
    Pubmed CrossRef
  25. Shigemoto N, Fukuda S, Tanizawa YTanizawa Y et al. Detection of norovirus, sapovirus, and human astrovirus in fecal specimens using a multiplex reverse transcription-PCR with fluorescent dye-labeled primers. Microbiol Immunol. 2011. 55: 369-372.
    Pubmed CrossRef
  26. Smith CJ, Osborn AM. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol. 2009. 67: 6-20.
    Pubmed CrossRef
  27. Thwiny H, Hasony H. Molecular detection of human sapovirus from healthy and hospitalized children with acute gastroenteritis in Basrah. JIARM. 2015. 3: 393-403.