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Development and Evaluation of a SYBR Green-Based, Real-time Polymerase Chain Reaction for Rapid and Specific Detection of Human Coxsackievirus B5
Biomed Sci Letters 2020;26:302-309
Published online December 31, 2020;  https://doi.org/10.15616/BSL.2020.26.4.302
© 2020 The Korean Society For Biomedical Laboratory Sciences.

Kyu Bong Cho,*

Department of Biomedical Laboratory Science, Shinhan University, Uijeongbu 11644, Korea
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
*Professor.
Received October 15, 2020; Revised November 27, 2020; Accepted November 30, 2020.
 Abstract
Human Coxsackievirus B5 (HuCoxV-B5) infection has been associated with various diseases such as myocarditis, aseptic meningitis, hand-foot-and mouth-disease, and insulin-dependent diabetes. HuCoxV-B5 is a virus transmitted through the fecal-oral route and is detected in clinics, aquatic environments, food, shellfish, etc. and is one of the more important viruses in public health because of its incidence rate reported worldwide. In this study, a combination of SYBR Green-based real-time PCR primers for molecular diagnosis including monitoring of HuCoxV-B5 was selected and the optimal reaction conditions were established. Compared with the previously reported TaqMan probe-based real-time PCR method, assessments including a sample applicability test were performed. Results showed that the real-time PCR method developed in this study was suitable for a molecular diagnostic technique for detecting HuCoxV-B5. This study is expected to contribute to efforts in responding to safety accidents in public health because the proposed method facilitates rapid diagnosis of clinical patients. It can also be used as a specific monitoring tool of HuCoxV-B5 in non-clinical areas such as aquatic environments among others.
Keywords : Human Coxsackievirus B5, HuCoxV-B5, Real-time PCR, SYBR green
꽌 濡

Human Coxsackievirus (HuCoxV)뒗 Poliovirus, Parechovirus 벑怨 븿猿 二쇱슂 Enterovirus 洹몃9뿉 룷븿릺硫, 븳援, 誘멸뎅, 以묎뎅, 씠깉由ъ븘 벑 꽑吏꾧뎅쓣 룷븿븯뿬 쟾 꽭怨꾩쟻쑝濡 鍮덈쾲븯寃 蹂닿퀬릺怨 엳뼱 怨듭쨷 蹂닿굔븰쟻쑝濡 以묒슂븳 諛붿씠윭뒪 以 븯굹濡 瑗쏀엺떎(Wagenknecht et al., 1991; Chen et al., 2012; Hyeon et al., 2013; Fontana et al., 2019). HuCoxV뿉 룷븿맂 A B 以 B 洹몃9 媛먯뿼 떆 씪諛섏쟻쑝濡 臾댁쬆긽씠吏留, 닔留됱뿼, 뇤뿼, 떖洹쇱뿼, 寃쎈젴꽦 留덈퉬, 諛쒖쭊, 샇씉湲 吏덊솚 벑쓽 궗濡媛 蹂닿퀬릺뿀떎(Zhang et al., 1993; Triantafyllopoulou et al., 2004; Papa et al., 2006; Gullberg et al., 2010; Hu et al., 2012). 씠 以묒뿉꽌룄 B5삎(HuCoxV-B5) 궗엺뿉寃 媛옣 슦꽭븯寃 굹굹뒗 삁泥삎쑝濡 諛붿씠윭뒪꽦 떖洹쇱뿼쓽 씪諛섏쟻 썝씤, 궛諛쒖쟻쑝濡 諛쒕퀝븯뒗 솗옣꽦 떖洹 蹂묒쬆쓽 빟 25% 씠긽쓽 썝씤, 臾닿퇏꽦 닔留됱뿼, 떊寃쎌쟻 닔議깃뎄蹂, 씤뒓由 쓽議댁꽦 떦눊蹂 벑 떎뼇븳 吏덈퀝怨쇱쓽 뿰愿꽦씠 蹂닿퀬릺怨 엳떎(Papa et al., 2006; Gullberg et al., 2010; Hu et al., 2012). HuCoxV-B5 벑 Enterovirus 洹몃9뿉 룷븿릺뒗 諛붿씠윭뒪뱾 遺꾨-援ш컯 寃쎈줈濡 쟾뿼 삉뒗 삤뿼릺뒗 몴쟻씤 옣 愿怨 諛붿씠윭뒪씠硫, 엫긽 쇅 닔怨 솚寃, 떇뭹, 뙣瑜 벑뿉꽌룄 寃異쒕릺誘濡 떎뼇븳 留ㅼ쭏뿉꽌쓽 紐⑤땲꽣留곸 怨듭쨷 蹂닿굔븰쟻쑝濡 以묒슂븯떎(Cho, 2018; Lee and Cho, 2019). 洹몃윭굹 Enterovirus 洹몃9뿉 룷븿릺뒗 諛붿씠윭뒪뱾 빟 70醫낅쪟 씠긽쓽 삁泥삎쑝濡 援ъ꽦맂떎(Oberste et al., 1999). 씠뱾 遺遺 궗엺怨 愿젴맂 蹂묒썝꽦 諛붿씠윭뒪씠硫, 蹂닿퀬맂 Enterovirus 삁泥삎뱾쓽 吏㏃ 쑀쟾옄 떒렪뱾쓽 蹂듭옟븳 쑀뿰愿怨 벑쑝濡 듅젙 븳 醫낅쪟쓽 寃異쒕낫떎뒗 pan-enterovirus 벑 Enterovirus 洹몃9쓣 쟾泥댁쟻쑝濡 紐⑤땲꽣留 븷 닔 엳뒗 빆썝-빆泥 諛 遺꾩옄쟻 湲곗닠씠 뿰援щ릺뼱 솕떎(Lee and Jeong, 2004; Choi et al., 2013). 洹몃윭굹 媛 삁泥삎 蹂 떎瑜닿쾶 굹굹뒗 吏덈퀝, 쑀뻾, 嫄곕룞 듅꽦 벑쑝濡 媛쒕퀎쟻 紐⑤땲꽣留곸쓽 븘슂꽦씠 젣湲곕릺뿀쑝硫, 71삎 諛 68삎 Enterovirus 벑쓣 以묒떖쑝濡 媛쒕퀎쟻 吏꾨떒 湲곕쾿뱾씠 蹂닿퀬릺뿀떎(Tan et al., 2008). HuCoxV-B5 寃異쒖쓣 쐞빐 빆썝-빆泥 湲곕컲쓽 enzyme-linked immunosorbent assay (ELISA)踰뺤씠 궗슜릺뿀쑝굹(Kopecka et al., 1995), ELISA 湲곕쾿 쐞 뼇꽦, 寃異 誘쇨컧룄, 媛앷쟻 뙋룆 벑쓽 臾몄젣젏쑝濡 씤븯뿬 理쒓렐뿉뒗 遺꾩옄吏꾨떒 湲곕쾿뱾씠 蹂닿퀬릺怨 엳떎(Lee et al., 2013). Jaianand et al. (2011), Zhao et al. (2013)뿉 쓽빐 HuCoxV-B5 寃異쒖슜 벑삩利앺룺踰[loop-mediated isothermal amplification (LAMP)]씠 蹂닿퀬릺뿀쑝굹, LAMP뒗 븣 닔 뾾뒗 鍮 듅씠쟻 利앺룺쑝濡 씤븳 쐞 뼇꽦 諛섏쓳, 긽쟻쑝濡 怨좉쓽 鍮꾩슜, 醫곸 踰붿쐞쓽 궗슜옄 벑쓽 臾몄젣젏씠 蹂닿퀬맂 諛 엳떎(Hardinge and Murray, 2019; Schneider et al., 2019). Rezig et al. (2004) 벑뿉 쓽빐 씪諛 PCR 諛⑸쾿씠 蹂닿퀬릺뿀쑝굹, 誘몃웾 삤뿼 諛 떎닔쓽 PCR 빐 臾쇱쭏씠 룷븿맂 솚寃 떆猷뚯뿉꽌 슂援щ릺뒗 넂 듅씠꽦怨 슦닔븳 寃異 誘쇨컧룄 臾몄젣瑜 빐寃고븯湲 쐞빐꽌뒗 PCR 利앺룺 썑 利앺룺 궛臾쇱쓣 二쇳삎쑝濡 떎떆 븳 踰 利앺룺븯뒗 nested PCR쓽 솢슜, PCR 빐 臾쇱쭏쓣 媛먯냼떆궎뒗 reagent쓽 궗슜 벑쑝濡 빐寃고빐빞 븯硫, 긽쟻쑝濡 蹂듭옟븳 寃궗 怨쇱젙, 쟾湲곗쁺룞 벑쑝濡 빟 10떆媛 씠긽씠 냼슂릺뒗 븳怨꾩젏씠 엳뿀떎(Lee et al., 2015; Lee et al., 2017). 븳렪 real-time PCR 떒씪 怨쇱젙쑝濡 긽쟻쑝濡 媛꾪렪븯怨, 2떆媛 씠궡쓽 鍮좊Ⅸ 寃異쒖씠 媛뒫븯硫, nested PCR怨 룞벑 씠긽 닔以쓽 寃異 誘쇨컧룄瑜 媛吏 湲곕쾿쑝濡 理쒓렐 엫긽怨 鍮 엫긽뿉꽌 二쇱슂 遺꾩옄吏꾨떒 湲곕쾿쑝濡 솢슜릺怨 엳떎(Chen et al., 2017; Yoo et al., 2017; Corman et al., 2020). Mohamed et al. (2004) Kang et al. (2013)뿉 쓽빐 VP1 씪遺 쑀쟾옄 떒렪쓣 利앺룺븯뒗 real-time PCR 諛⑸쾿씠 蹂닿퀬릺뿀쑝굹, 듅씠꽦, 寃異 誘쇨컧룄, 떆猷 쟻슜꽦 룊媛 벑쓽 뿰援щ뒗 誘명씉븯떎.

뵲씪꽌 씠踰 뿰援ъ뿉꽌뒗 HuCoxV-B5瑜 떊냽 諛 듅씠쟻쑝濡 寃異쒗븷 닔 엳뒗 real-time PCR 諛⑸쾿쓣 媛쒕컻븯쑝硫, 솚寃 떆猷 벑뿉꽌쓽 솢슜꽦쓣 룊媛븯떎.

옱猷 諛 諛⑸쾿

봽씪씠癒 꽕怨 諛 諛붿씠윭뒪 빑궛 닔吏

HuCoxV-B5 듅씠쟻 寃異쒖쓣 쐞븳 봽씪씠癒 議고빀 Mohamed et al. (2004)瑜 援ъ꽦 삁젙씤 PCR 議곗꽦臾쇨낵 議곌굔뿉 留욎텛뼱 蹂삎 諛 옱꽕怨 븯쑝硫 (Table 1 and Fig. 1), Oligo Calculator version 3.27濡 옞옱쟻 hairpin 삎꽦, self annealing 벑쓣 寃젙븯떎. 븳렪, HuCoxV-B5 5'-UTR 遺遺꾩쓣 긽쑝濡 빑궛 떒렪쓣 利앺룺븯뒗 寃궗踰뺤쓣 怨좎븞븯湲 쐞븯뿬 National Center for Biotechnology Information (NCBI) accession number JX843811 湲곗 449-609 nucleotide (nt) (161 nt)瑜 留덊겕濡쒖젨(Seoul, Korea)뿉 쓽猶고븯뿬 떒씪 媛떏쑝濡 빀꽦 諛 쑀愿 湲곌뿉꽌 HuCoxV-B5 cDNA瑜 닔吏묓븯떎. 삉븳 鍮 듅씠쟻 諛섏쓳뿉 궗슜븷 李멸퀬 諛붿씠윭뒪 15醫[Adenovirus 41 (AdV-41), Aichivirus A (AiV-A), Aichivirus B (AiV-B), Aichivirus C (AiV-C), Astrovirus (AstV), Enterovirus 71 (EV-71), Enterovirus 68 (EV-68), Hepatitisvirus A (HAV), Hepatitisvirus E (HEV), Norovirus GI (NoV-GI), Norovirus GII (NoV-GII), Orthoreovirus segment S (OrV-S), Parachovirus A (PeV-A), Poliovirus type 3 (PV-type3) 諛 Sapovirus (SaV)]쓽 빑궛 뿼湲곗꽌뿴 씪遺瑜 떒씪 媛떏쓽 plasmid濡 빀꽦븯떎.

Specific PCR primer sets for the detection of HuCoxV-B5

Set # Primer name Sequence (5'-3') Mer (nt) Location Product size (nt) Reference

Start Stop
1 CoxV-B5_F452 CCGGCCCCTGAATGCGGCTA 20 452 471 149 -
CoxV-B5_R600 TGTCACCATAAGCAGCCAGTGTAGG 25 576 600
2 CoxV-B5_F452 CCGGCCCCTGAATGCGGCTA 20 452 471 153 -
CoxV-B5_R604 CAATTGTCACCATAAGCAGCCAG 23 582 604
3 CoxV-B5_F454 GGCCCCTGAATGCGGCTA 18 454 471 147 -
CoxV-B5_R600 TGTCACCATAAGCAGCCAGTGTAGG 25 576 600
4 CoxV-B5_F454 GGCCCCTGAATGCGGCTA 18 454 471 151 -
CoxV-B5_R604 CAATTGTCACCATAAGCAGCCAG 23 582 604

Ref. #1 NMF1 GCCCCTGAATGCGGC 15 455 469 149 Mohamed et al., 2004
NMR1 AATTGTCACCATAAGCAGC 19 585 603
TaqMan® probe HEX-CGGAACCGACTACTTTGGGTGTCCGT-BHQ1 26 537 562
Ref. #2 CVB F AAACCCAAACATGTGAAGGCGTGG 24 3,166 3,189 118 Kang et al., 2013
CVB R TGGTAATGTTTGAGCGCGTTGTGG 24 3,260 3,283
CVB P HEX-ACCGCCGAGGCTATGTCAATATGAGA-BHQ1 26 3,198 3,223

Fig. 1. SYBR Green-based, real-time PCR primer map for the detection of Human Coxsackievirus B5 (HuCoxV-B5).

봽씪씠癒 꽑諛

HuCoxV-B5뿉 듅씠쟻 諛섏쓳씠 異붿젙릺뒗 4媛쒖쓽 썑蹂 봽씪씠癒 議고빀쓽 듅씠쟻 諛섏쓳 諛 寃異 誘쇨컧룄瑜 遺꾩꽍븯떎. HuCoxV-B5 plasmid 1 ng/μL 湲곗쑝濡 10-6 (1 fg/μL)源뚯 10諛 떒怨 씗꽍븯쑝硫, cDNA뒗 1,000, 500, 250, 100, 50, 10 copies 諛 1 copy濡 씗꽍븯떎. HuCoxV-B5 plasmid cDNA瑜 씗꽍븸쓣 二쇳삎쑝濡 SYBR Green 湲곕컲쓽 real-time PCR쓣 닔뻾븯쑝硫, 議곗꽦 iTaqTM Universal SYBR® Green Supermix (Biorad, USA) 10 μL, 젙 諛⑺뼢 諛 뿭 諛⑺뼢 봽씪씠癒 2 μL (25 pmol 1 μL, each), 二쇳삎 빑궛 1 μL 諛 nucleic acid free water 7 μL濡 珥 20 μL濡 븯떎. Real-time PCR 議곌굔 95꼦뿉꽌 5遺꾧컙 1쉶 珥덇린 蹂꽦 썑, 95꼦뿉꽌 15珥, 61꼦뿉꽌 60珥덈 35쉶 諛섎났븯떎. 삉븳 듅씠쟻 諛섏쓳 諛 寃異 誘쇨컧룄뿉꽌 슦닔븳 寃곌낵瑜 蹂댁씤 봽씪씠癒 議고빀쓣 긽쑝濡 鍮 듅씠쟻 諛섏쓳쓣 닔뻾븯떎. 二쇳삎 빑궛쑝濡 positive control [HuCoxV plasmid (100 pg/μL)], negative control [nucleic acid free water], 李멸퀬 諛붿씠윭뒪 15醫 빑궛(AdV-41, AiV-A, AiV-B, AiV-C, AstV, EV-71, EV-68, HAV, HEV, NoV-GI, NoV-GII, OrV-S, PeV-A, PV-type 3 諛 SaV)쓣 二쇳삎쑝濡 諛섏쓳븯떎. 諛섏쓳 議곗꽦 諛 議곌굔 듅씠쟻 諛섏쓳怨 룞씪븯寃 븯떎.

꽑諛 봽씪씠癒몄쓽 룊媛

꽑諛쒗븳 real-time PCR 봽씪씠癒몄쓽 룊媛瑜 쐞븯뿬 湲곗〈 蹂닿퀬맂 HuCoxV-B5 寃異쒖슜 real-time PCR 諛⑸쾿怨 寃異 誘쇨컧룄 諛 듅씠꽦쓣 鍮꾧탳븯떎(Table 1). Real-time PCR 議곗꽦 cDNA瑜 二쇳삎쑝濡 궗슜 諛 媛쒕컻 봽씪씠癒 議곌굔怨 룞씪븯寃 븯쑝硫, 議곌굔 媛곴컖쓽 蹂닿퀬맂 諛⑸쾿怨 룞씪븯寃 븯떎(Mohamed et al., 2004; Kang et al., 2013). 삉븳 떆猷 쟻슜꽦 떆뿕쓣 쐞븯뿬 솚寃쎈 援由쏀솚寃쎄낵븰썝 2007-2016 愿젴 紐⑤땲꽣留 옄猷(Lee et al., 2018)瑜 諛뷀깢쑝濡 삤뿼씠 삁긽릺뒗 吏뿭 以 10媛 吏젏쓽 吏븯닔 떆猷뚮 臾댁옉쐞 梨꾩랬븯쑝硫, 떎뿕떎濡 씠룞 썑 깉由 諛 냽異뺤쓣 닔뻾븯떎. 냽異 떆猷뚯뿉꽌 QIAamp® Viral RNA Mini (Qiagen, Germany)濡 total RNA瑜 異붿텧 썑, ReverTra Ace-α-TM (Toyobo, Japan)쓣 궗슜븯뿬 cDNA濡 빀꽦븯쑝硫, 紐⑤몢 젣뭹쓽 留ㅻ돱뼹뿉 뵲씪 닔뻾븯떎. 떆猷 梨꾩랬뿉꽌 빑궛 異붿텧源뚯쓽 怨쇱젙 솚寃쎈 援由쏀솚寃쎄낵븰썝 怨좎떆 젣 2017-50샇뿉 쓽嫄고븯뿬 닔뻾븯떎. 꽑諛쒕맂 real-time PCR 봽씪씠癒 議고빀쓣 궗슜븯뿬 吏븯닔 떆猷뚯쓽 cDNA濡쒕꽣 HuCoxV-B5瑜 寃異쒗븯떎. 삉븳 꽑諛쒗븳 봽씪씠癒 議고빀쓣 씪諛 PCR濡 蹂삎[95꼦뿉꽌 5遺꾧컙 1쉶 珥덇린 蹂꽦, 35쉶 諛섎났(95꼦뿉꽌 45珥, 61꼦뿉꽌 60珥 諛 72꼦뿉꽌 60珥), 72꼦뿉꽌 5遺꾧컙 理쒖쥌 떊옣]븯뿬 HuCoxV-B5瑜 寃異쒗븯떎. 삉븳 씪諛 PCR쓽 寃異 誘쇨컧룄媛 real-time PCR 蹂대떎 궙 寃껋 媛먯븞븯뿬 HuCoxV-B5_F452怨 HuCoxV-B5_R604 봽씪씠癒몃 씠슜 1李 PCR 利앺룺 썑, 利앺룺 궛臾쇱뿉꽌 꽑諛 봽씪씠癒몃 궗슜븯뿬 nested PCR쓣 닔뻾븯떎. 솚寃 떆猷뚯뿉꽌 PCR 諛 nested PCR 떆 議곗꽦臾쇰줈 紐⑤몢 鍮 듅씠쟻 諛대뱶 뼲젣 臾쇱쭏씤 SL® Nonspecific reaction inhibitor (NSRI; LSLK, Korea)瑜 3 μL瑜 룷븿븯뿬 諛섏쓳븯떎. 븳렪 nested PCR 利앺룺 궛臾쇰뱾쓣 MEGAquick-spinTM Plus Total Fragment DNA Purification Kit (INtRON, Korea)瑜 궗슜븯뿬 젙젣 썑 留덊겕濡쒖젨(https://dna.macrogen.com/main.do)뿉꽌 뿼湲곗꽌뿴 遺꾩꽍븯쑝硫, NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)濡 쑀궗꽦쓣 솗씤븯떎.

寃곌낵 諛 怨좎같

珥 4媛쒖쓽 썑蹂 봽씪씠癒 議고빀 紐⑤몢 HuCoxV-B5 plasmid cDNA뿉꽌 듅씠쟻 諛섏쓳씠 굹궗쑝硫, plasmid 1~ 100 pg/μL, cDNA 250~1,000 copies쓽 寃異 誘쇨컧룄媛 굹궗떎. 씠 以 썑蹂 봽씪씠癒 議고빀 #2媛 plasmid 1 pg/μL, cDNA 250 copies濡 媛옣 슦닔븳 寃異 誘쇨컧룄瑜 蹂댁쑝硫, 議고빀 #3怨 議고빀 #4뒗 議고빀 #2뿉 鍮꾪빐 plasmid 湲곗 빟 10諛, cDNA 湲곗 빟 2諛 닔以 궙 寃異 誘쇨컧룄瑜 蹂댁떎. 븳렪 議고빀 #1 plasmid 湲곗 #3怨 #4 룞벑 닔以쓣 蹂댁쑝굹 cDNA뿉꽌 긽쟻쑝濡 궙 닔以쓽 寃異 誘쇨컧룄媛 굹궗떎. 븳렪, single threshold 옄룞怨꾩궛 諛⑹떇쓣 湲곗쑝濡 븳 cycle quantification value (Cq) 媛믪 plasmid 100 pg/μL 湲곗 빟 28.05~28.09, 10 pg/μL 湲곗 빟 30.89~31.20 닔以쑝濡 굹궗쑝硫 cDNA 1,000 copies 湲곗 빟 26.10~31.17, 500 copies 湲곗 27.72~32.93 닔以쑝濡 굹궗떎(Fig. 2). HuCoxV-B5 plasmid cDNA뿉꽌 Cq 媛믪쓣 룷븿븳 寃異 誘쇨컧룄 벑쓣 怨좊젮븯뿬 議고빀 #2, #3 諛 #4瑜 꽑諛쒗븯떎. 삉븳 꽑諛쒗븳 3媛쒖쓽 썑蹂 봽씪씠癒 議고빀뱾쓣 긽쑝濡 李멸퀬 諛붿씠윭뒪 15醫낆쓽 빑궛뿉 鍮 듅씠쟻 諛섏쓳쓣 遺꾩꽍븳 寃곌낵, 議고빀 #2뿉꽌 4醫(SaV, HAV, PV 諛 EV-71)씠 諛섏쓳븯쑝硫 Cq 媛믪 24.06~32.14 닔以씠뿀떎. 議고빀 #4뿉꽌뒗 7醫[AiV (A, B 諛 C), AstV, EV-71, EV-68 諛 HAV]씠 諛섏쓳븯쑝硫, Cq 媛믪 29.44~32.01濡 굹궗떎. 諛섎㈃ 議고빀 #3뿉꽌뒗 李멸퀬 諛붿씠윭뒪 빑궛쑝濡쒕꽣 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡븘 HuCoxV-B5瑜 듅씠쟻쑝濡 利앺룺븷 닔 엳뒗 봽씪씠癒 議고빀쑝濡 異붿젙븯쑝硫 理쒖쥌 꽑諛쒗븯떎(Fig. 3).

Fig. 2. Specific reaction and sensitivity of four real-time PCR primer sets based on HuCoxV-B5 plasmid and cDNA.
Fig. 3. Non-specific reaction of three real-time PCR primer sets based on fifteen reference virus genes.

湲곗〈 蹂닿퀬맂 HuCoxV-B5 寃異쒖슜 real-time PCR Ref.#1怨 #2뒗 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡븯쑝硫, cDNA 湲곗 빟 500 copies源뚯 寃異 誘쇨컧룄媛 굹궗쑝굹, 꽑諛 봽씪씠癒멸 cDNA 500 copies 二쇳삎뿉꽌 1,000 relative fluorescence units (RFU) 씠긽씠 遺꾩꽍맂 諛섎㈃ ref.#1怨 #2뒗 RFU 媛믪씠 빟 87.7~105.0 닔以쑝濡 궙寃 굹굹(Data not shown), 꽑諛 봽씪씠癒멸 룷븿맂 議곗꽦臾쇰줈 諛섏쓳븳 議곌굔뿉 鍮꾪빐 긽쟻쑝濡 궙 寃異 誘쇨컧룄媛 異붿젙릺뿀떎. 삉븳 꽑諛쒗븳 봽씪씠癒 議고빀 #3쑝濡 닔怨 솚寃 떆猷 10媛쒖뿉꽌 異붿텧븳 total RNA (12.3~74.4 ng/μL)뿉꽌 HuCoxV-B5瑜 寃異쒗븳 寃곌낵, single threshold 1,000 RFU瑜 湲곗쑝濡 10媛 以 8媛 떆猷뚯뿉꽌 뼇꽦씠 異붿젙릺뿀쑝硫, Cq 媛믪 빟 31.04~33.45 닔以씠뿀떎(Fig. 4A). 꽑諛쒗븳 봽씪씠癒 議고빀쓽 씪諛 PCR 蹂삎 썑 吏븯닔 떆猷뚯뿉꽌 HuCoxV-B5瑜 寃異쒗븳 寃곌낵 紐⑤몢 쓬꽦쑝濡 굹궗떎(Fig. 4B). 삉븳 HuCoxV-B5_F452怨 HuCoxV-B5_R604 봽씪씠癒몃 씠슜 1李 利앺룺뿉꽌룄 떆猷 10媛 紐⑤몢 쓬꽦, 1李 利앺룺 궛臾쇨낵 꽑諛 봽씪씠癒 議고빀쓣 씠슜븯뿬 nested PCR쓣 닔뻾븳 寃곌낵 1李 PCR뿉꽌 10媛 떆猷뚯뿉꽌 紐⑤몢 쓬꽦씠 솗씤릺뿀怨 nested PCR 寃곌낵 3媛쒖뿉꽌 뼇꽦 異붿젙 諛대뱶媛 굹궗떎(Fig. 4B). Nested PCR濡 利앺룺맂 異붿젙 諛대뱶쓽 뿼湲곗꽌뿴 遺꾩꽍 寃곌낵 #5뒗 鍮 듅씠쟻 諛섏쓳, #8怨 #10 HuCoxV-B5濡 理쒖쥌 遺꾩꽍릺뼱 10媛 以 2媛쒖뿉꽌 HuCoxV-B5 뼇꽦쑝濡 굹궗떎(Data not shown). PCR怨 nested PCR濡 諛섏쓳븳 씪諛 PCR뿉꽌뒗 鍮 듅씠쟻 諛섏쓳쓣 뼲젣븯뒗 臾쇱쭏쓣 궗슜뻽쓬뿉룄 遺덇뎄븯怨 鍮 듅씠쟻 利앺룺씠 떎닔 굹궗쑝硫, 씠윭븳 臾몄젣젏 씪諛 nested PCR쓣 씠슜븯뿬 떆猷뚮 寃젙븯뒗 Lee et al. (2013) 벑 떎瑜 쑀愿 뿰援щ뱾뿉꽌 굹굹뒗 寃곌낵뱾怨 쑀궗븯떎. 씠윭븳 臾몄젣젏쓣 媛쒖꽑븯湲 쐞빐꽌뒗 鍮 듅씠쟻 諛섏쓳쓣 뼲젣븯뒗 臾쇱쭏쓽 怨좊냽룄 궗슜, 臾쇱쭏쓽 蹂솕 벑 PCR 議곗꽦 諛 議곌굔쓣 꽕젙, 뿼湲곗꽌뿴 遺꾩꽍 쟾 異붿젙 諛대뱶쓽 gel-purification 벑 씪諛 PCR 씠 썑 썑냽 끂젰씠 븘슂븳 怨쇱젙쓣 룷븿븳떎.

Fig. 4. Evaluation of developed method using the groundwater samples. Panel A. Demonstration test using ten groundwater samples. Panel B. Comparative evaluation of developed method and conventional PCRs. M, 100 bp DNA Ladder maker (Enzynomics, Korea); P, positive control; N, negative control 1~10, sample number.

蹂 뿰援ъ뿉꽌 媛쒕컻븳 HuCoxV-B5 寃異쒖슜 봽씪씠癒 議고빀, 議곗꽦臾 諛 議곌굔 듅씠쟻쑝濡 HuCoxV-B5瑜 寃異쒗븷 닔 엳쓣 寃껋쑝濡 蹂댁씤떎. 湲곗〈 蹂닿퀬맂 real-time PCR 諛⑸쾿 以 빟 68遺꾩씠뜕 ref.#1 (Mohamed et al., 2004)쓽 諛섏쓳쓣 빟 20遺 떒異뺥븯쑝硫, 빐떦 諛⑸쾿뿉 鍮꾪빐 긽쟻쑝濡 슦닔븳 寃異 誘쇨컧룄瑜 蹂댁떎. 湲곗〈 蹂닿퀬맂 real-time PCR 諛⑸쾿뱾 紐⑤몢 TaqMan probe 湲곕컲쓽 寃궗踰뺤쑝濡 1媛쒖쓽 봽濡쒕툕 봽씪씠癒몃 異붽濡 궗슜븿쑝濡쒖뜥 SYBR green 湲곕컲쓽 real-time PCR蹂대떎 듅씠꽦 뼢긽씠 삁긽릺뿀吏留 씠踰 뿰援ъ뿉꽌뒗 솗씤븷 닔 뾾뿀떎. 洹몃윭굹 봽濡쒕툕 봽씪씠癒몄쓽 궗슜쑝濡 듅닔 耳誘몄뺄씠 異붽릺硫 SYBR green 湲곕컲쓽 real-time PCR 삉뒗 씪諛 PCR뿉꽌 궗슜릺뒗 봽씪씠癒 議고빀 鍮 빟 25諛곗쓽 봽씪씠癒 議고빀 떒媛 李⑥씠媛 굹뒗 벑 怨좉씠硫, raw material 떒媛 뼢긽쑝濡 씤빐 씠寃껋 뼢썑 KIT 媛쒕컻 떆뿉룄 쁺뼢쓣 誘몄튌 寃껋쑝濡 蹂댁씤떎. 삉븳 꽑諛 봽씪씠癒몃 씠슜븳 씪諛 PCR 諛⑸쾿뱾(1떒怨 PCR 諛 nested PCR)뿉 鍮꾪빐 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 real-time PCR 1떒怨 PCR뿉 鍮꾪빐꽌뒗 빟 1떆媛, nested PCR뿉 鍮꾪빐꽌뒗 빟 4떆媛 닔以쓣 떒異뺥븷 닔 엳뿀쑝硫, 떆猷 쟻슜 룊媛뿉꽌 긽쟻 슦닔븳 寃곌낵媛 遺꾩꽍릺뿀떎. 븳렪, 吏븯닔 벑 닔怨 솚寃 떆猷뚮뒗 엫긽, 떇뭹, 뙣瑜 벑쓽 떆猷뚯뿉 鍮꾪빐 웾쓽 留ㅼ쭏 궡 誘몃웾쓽 諛붿씠윭뒪 삤뿼 諛 떎닔쓽 PCR 빐 臾쇱쭏씠 議댁옱븳떎(Cho, 2018; Lee and Cho, 2019). 遺꾨-援ш컯 寃쎈줈濡 媛먯뿼릺뒗 HuCoxV-B5쓽 寃쎌슦 吏븯닔 以 寃異쒖씠 媛뒫븯떎硫 떎瑜 留ㅼ쭏濡쒖쓽 솢슜꽦씠 媛뒫븷 寃껋쑝濡 蹂댁씤떎. 씠踰 뿰援ъ뿉꽌뒗 10媛 以 8媛쒖쓽 吏븯닔 떆猷뚯뿉꽌 HuCoxV-B5 뼇꽦씠 굹궗쑝硫 씪諛 PCR 寃궗踰 鍮 듅씠꽦, 諛섏쓳쓽 媛뺣룄 벑뿉꽌 슦닔븳 寃곌낵媛 遺꾩꽍릺뿀떎(Fig. 4). 뵲씪꽌 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 諛⑸쾿 뼢썑 HuCoxV-B5 愿젴 利앹긽씠 굹굹뒗 엫긽 솚옄뿉꽌 떊냽븳 遺꾩옄吏꾨떒쑝濡 썑냽 泥섎갑뿉 湲곗뿬, 닔怨 솚寃, 떇뭹, 뙣瑜 벑 鍮 엫긽뿉꽌룄 HuCoxV-B5 紐⑤땲꽣留곸쑝濡 솢슜릺뼱 怨듭쨷 蹂닿굔븰쟻 븞쟾꽦 벑 愿젴 궗怨 쓳뿉 湲곗뿬븷 닔 엳쓣 寃껋쑝濡 湲곕맂떎.

ACKNOWLEDGEMENT

This work was supported by the Shihan University Research Fund, 2020.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

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