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Construction of Improved PCR Primer Set for the Detection of Human Enteric Adenovirus 41
Biomed Sci Letters 2018;24:230-238
Published online September 30, 2018;  https://doi.org/10.15616/BSL.2018.24.3.230
© 2018 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
Received August 17, 2018; Revised September 10, 2018; Accepted September 11, 2018.
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 enteric Adenovirus-41 (HuEAdV-41) causes gastroenteritis, which detected by the polymerase chain reaction (PCR) base diagnostic system for clinical, food, environmental, fish and shellfish samples. We developed improved PCR and nested PCR primer set which had high specificity, sensitivity and reduced times. In this study, we compared seventeen conditions reported in the previous study that was using the PCR based HuEAdV-41 detection system, and non-enteric Adenovirus were detected in nine conditions. The most sensitive detection condition was up to 25 copies however it took 184 minutes of PCR reaction time. In this study, the PCR primer set developed had same level of sensitivity, it reduced the time of detection for clinical, food and seafood samples to 112 minutes. Developed nested PCR primer set needed 112 minutes but detected up to approximately 1 copy. In addition, developed PCR and nested PCR primer set was validated with twenty samples of underground water at random, of which ten samples showed specific band without non-specific reaction. We expect this study will be used to diagnose HuEAdV-41 from various samples.

Keywords : Adenovirus-41, HuEAdV-41, Polymerase chain reaction, Nested PCR
꽌濡

궗엺 옣븘뜲끂諛붿씠윭뒪(Human Enteric Adenovirus; HuEAdV)뒗 group I쓽 dsDNA virus濡쒖뜥 븘뜲끂諛붿씠윭뒪怨쇰줈 遺꾨쪟맂떎(Rowe et al., 1953). HuEAdV뒗 利앹긽뿉 뵲씪 subgroup A-G濡 굹돇硫, 쐞옣뿼쓣 씪쑝궎뒗 洹몃9 F G濡 씠 以 F삎 냼옣怨 옣궡 깮議 媛뒫꽦씠 蹂닿퀬릺뿀떎(Uhnoo et al., 1984; Grimwood et al., 1995). 洹몃9 F삎 삁泥삎 40怨 41濡 援ъ꽦릺硫, 듅엳 냼븘 벑 끂빟옄뿉寃 쐞옣뿼쓣 씪쑝궎뒗 二쇱슂 삁泥삎 41삎씠떎(Johansson et al., 1985). HuEAdV-41쓣 寃異쒗븯湲 쐞븳 諛⑸쾿쑝濡쒕뒗 듅씠쟻 빑궛쓣 利앺룺븯뒗 以묓빀슚냼뿰뇙諛섏쓳(polymerase chain reaction; PCR)씠 二쇰줈 솢슜릺怨 엳쑝硫, 엫긽, 떇뭹, 뼱뙣瑜, 솚寃 벑뿉꽌 二쇰줈 궗슜릺뒗 寃궗諛⑸쾿씠떎(Girones et al., 1995; Echavarria et al., 1998). 洹몃윭굹 HuEAdV-41쓣 寃異쒗븯湲 쐞븳 吏꾨떒 湲곗닠 떎닔쓽 뿰援ъ옄뱾뿉 쓽빐 媛쒕컻릺뼱 寃궗議곌굔씠 紐⑤몢 떎瑜대ʼn, 듅씠룄 諛 寃異 誘쇨컧룄 벑 媛곴컖쓽 寃궗諛⑸쾿뿉 븳 鍮꾧탳 뿰援ш 긽쟻쑝濡 誘명씉븳 떎젙씠떎. 삉븳 떎뼇븳 떆猷뚮뱾 以 吏븯닔 벑怨 媛숈 鍮 냼룆닔泥섎읆 洹밸몃웾쑝濡 삤뿼맂 諛붿씠윭뒪瑜 寃異쒗븷 닔 엳쓣 젙룄쓽 寃異쒓컧룄媛 븘슂븯떎. 뵲씪꽌 씠踰 뿰援ъ뿉꽌뒗 쁽옱源뚯 媛쒕컻맂 湲곗〈쓽 PCR 諛⑸쾿蹂대떎 HuEAdV-41쓣 넂 듅씠룄 諛 誘쇨컧룄濡 寃異쒗븷 닔 엳뒗 PCR 諛 nested PCR 봽씪씠癒 議고빀쓣 媛쒕컻븯怨좎옄 蹂 뿰援щ 닔뻾븯떎.

옱猷 諛 諛⑸쾿

봽씪씠癒 꽕怨

HuEAdV쓽 삁泥삎 寃곗젙 쑀쟾옄 遺쐞씤 HuEAdV-41 hexon gene쓣 긽쑝濡 듅씠쟻 봽씪씠癒몃 꽕怨꾪븯떎(Fig. 1). 誘쇨컧룄 듅씠룄瑜 寃利앺븯湲 쐞븯뿬 誘멸뎅 援由쎌깮臾쇱젙蹂댁꽱꽣(National Center for Biotechnology Information)뿉꽌 Human Adenovirus 41 isolate Tak (AB610519.1)쓣 鍮꾨’븳 20媛 李멸퀬 닔씤꽦 · 떇뭹留ㅺ컻 諛붿씠윭뒪 뿼湲곗꽌뿴쓣 닔吏묓븯떎. 뿼湲곗꽌뿴뱾 BioEdit version 7.1.3쓣 궗슜(Hall, 1999)븯뿬 떎以묒뿼湲곗꽌뿴 젙젹 썑, DNAMAN software package version 6.0 (Lynnon Biosoft, Quebec, Canada)쓣 궗슜븯뿬 annealing 議곌굔 51~59°C 踰붿쐞뿉꽌 봽씪씠癒 醫낅쪟뿉 뵲씪 媛곴컖 2°C 李⑥씠濡 醫 듅씠쟻씤 뿼湲곗꽌뿴쓣 깘깋븯떎. 깘깋븳 듅씠쟻 봽씪씠癒몃뱾 Oligo Calculator version 3.27쓣 궗슜븯뿬 듅꽦 솗씤 諛 potential hairpin formation 벑쓣 寃젙븯떎(Kibbe, 2007).

Fig. 1.

Specific PCR primer map for the detection of human enteric Adenovirus 41


諛붿씠윭뒪 빑궛 닔吏

HuEAdV-41 봽씪씠癒 議고빀뱾쓽 듅씠꽦쓣 遺꾩꽍븯湲 쐞븯뿬 긽 諛붿씠윭뒪씤 HuEAdV-41 [AMPLIRUN® ADENOVIRUS 41 DNA CONTROL MBC001 (Vircell, Spain)]怨 李멸퀬 諛붿씠윭뒪 11醫[Rotavirus A (RV-A), Parechovirus A (PechoV), Norovirus GII (NoV-GII), Coronavirus 229E (CorV-229E), Enterovirus 68, 71 (EV-68, 71), Coxsackievirus A6, B1, B5 (CoxV-A6, B1, B5), non-enteric Adenovirus (NeAdV) 諛 Parvovirus B19 (PaV-B19)]쓽 빑궛쓣 닔吏묓븯떎.

PCR 諛 씠 以 以묓빀슚냼뿰뇙諛섏쓳(nested PCR) 봽씪씠癒 꽑諛

꽕怨꾨맂 PCR 봽씪씠癒몃 議고빀븯뿬 利앺룺씠 媛뒫븳 50媛 議고빀쓣 援ъ꽦븯떎. 援ъ꽦븳 議고빀뱾쓣 긽쑝濡 HuEAdV-41 DNA뿉 듅씠쟻쑝濡 諛섏쓳쓣 굹궡怨, 李멸퀬 諛붿씠윭뒪 11醫낆쓽 빑궛뿉 鍮 듅씠쟻쑝濡 諛섏쓳븯뒗 봽씪씠癒 議고빀쓣 꽑諛쒗븯떎. 씠썑 듅씠쟻 諛 鍮 듅씠쟻 諛섏쓳뿉꽌 꽑諛쒗븳 봽씪씠癒 議고빀뱾쓣 긽쑝濡 HuEAdV-41 DNA瑜 10諛 怨꾨떒 씗꽍븳 썑 二쇳삎怨쇱쓽 寃異쒓컧룄 鍮꾧탳瑜 넻빐 봽씪씠癒몃 꽑諛쒗븯떎. 삉븳, 洹밸몃웾 삤뿼맂 떆猷 벑뿉꽌룄 솢슜씠 媛뒫븯룄濡 nested PCR 봽씪씠癒 議고빀쓣 媛쒕컻븯떎. 寃異쒓컧룄 떆뿕뿉꽌 꽑諛쒕맂 봽씪씠癒 議고빀쓣 씠슜븯뿬 nested PCR 議고빀뱾쓣 꽕怨꾪븳 썑, 媛 PCR 궛臾쇱쓣 二쇳삎쑝濡 寃異쒓컧룄瑜 鍮꾧탳븯떎. 븳렪, PCR 諛섏쓳븸 AccuPower® HotStart PCR PreMix (Bioneer, Korea), 젙諛⑺뼢 諛 뿭諛⑺뼢 봽씪씠癒(25 pmol) 媛 1 μL, 二쇳삎 DNA 1 μL 諛 nucleic acid free water 17 μL濡 理쒖쥌 20 μL濡 닔뻾븯떎. PCR 諛섏쓳 삩룄 議곌굔 95°C뿉꽌 10遺 룞븞 珥덇린 蹂꽦떒怨꾨 嫄곗퀜 75°C뿉꽌 45珥덇컙 蹂꽦, 55°C뿉꽌 1遺꾧컙 寃고빀, 72°C뿉꽌 1遺꾧컙 떊옣븯뒗 떒怨꾨 35쉶 諛섎났븯쑝硫 洹 썑 72°C뿉꽌 5遺꾧컙 理쒖쥌 떊옣븯떎. PCR 궛臾쇰뱾 1.2% agarose gel뿉꽌 100 V, 30遺꾧컙 쟾湲곗쁺룞 썑 gel documentation system (BioRad, USA)뿉꽌 솗씤븯떎.

湲곗〈 떆뿕踰 鍮꾧탳

HuEAdV-41쓽 듅씠 쑀쟾옄瑜 利앺룺븯뒗 PCR 諛 nested PCR 17媛 議곌굔씠 寃깋릺뿀쑝硫, 씠뱾 諛⑸쾿뱾쓽 諛섏쓳떆媛, 寃異 誘쇨컧룄 諛 듅씠룄瑜 遺꾩꽍븯떎. PCR 議곌굔 媛곴컖쓽 李멸퀬臾명뿄뱾怨 룞씪븯寃 닔뻾븯떎(Allard et al., 1992; Flomengerg et al., 1995; Kidd et al., 1996; Akalu et al., 1998; Castignolles et al., 1998; Echavarría et al., 1998; Xu et al., 2000; Allard et al., 2001; Oh et al., 2003; Hansman et al., 2008; Tai, 2008; Kuo et al., 2009; KMFDS, 2013; Arcangeletti et al., 2014; Jang et al., 2017). 諛섏쓳떆媛꾩 湲곗〈 諛⑸쾿뱾쓽 諛섏쓳삩룄 議곌굔쓣 쟻슜븯怨, HuEAdV-41 DNA瑜 10諛 怨꾨떒 씗꽍븳 썑 二쇳삎쑝濡 寃異 誘쇨컧룄瑜 솗씤븯쑝硫, NeAdV DNA瑜 二쇳삎쑝濡 듅씠룄瑜 遺꾩꽍븯떎.

떆猷 쟻슜꽦 떆뿕 諛 理쒖쥌 꽑諛

HuEAdV-41 寃異쒖슜 봽씪씠癒 議고빀뱾쓽 떆猷 쟻슜꽦 떆뿕쓣 쐞븯뿬 誘몃웾 삤뿼 삉뒗 쓬꽦씠 삁긽릺뒗 吏븯닔 떆猷뚮 臾댁옉쐞濡 20媛 梨꾩랬븯쑝硫, 援由쏀솚寃쎄낵븰썝 怨좎떆젣 2017-50샇뿉 쓽嫄고븯뿬 吏븯닔 떆猷뚮뒗 깉由 諛 냽異뺤쓣 닔뻾븯떎. 냽異 떆猷뚮뒗 DNeasy® PowerWater® Kit (Qiagen, Germany)瑜 궗슜븯뿬 젣뭹 留ㅻ돱뼹뿉 뵲씪 珥 빑궛(total DNA)쓣 異붿텧븯떎. 異붿텧븳 빑궛쓣 二쇳삎쑝濡 깉濡쒖씠 媛쒕컻븳 봽씪씠癒 議고빀뱾怨 湲곗〈 PCR 諛⑸쾿뱾 以 듅씠룄 諛 寃異 誘쇨컧룄媛 媛옣 넂 봽씪씠癒 議고빀쓣 긽쑝濡 鍮꾧탳븯떎.

寃곌낵

PCR 봽씪씠癒 꽑諛

HuEAdV-41 쑀쟾옄뿉 븳 듅씠쟻 諛섏쓳뿉꽌 50媛 議고빀 紐⑤몢뿉꽌 126~1,519 bp쓽 듅젙 諛대뱶媛 삎꽦릺뿀쑝硫 諛대뱶쓽 겕湲, 媛뺣룄 諛 쐞移섎 怨좊젮븯뿬 11媛 議고빀쓣 꽑諛쒗븯떎(Fig. 2). 꽑諛쒕맂 11媛 議고빀쓽 듅씠 봽씪씠癒몃 씠슜븯뿬 李멸퀬 諛붿씠윭뒪 11醫낆뿉 븯뿬 PCR瑜 떆뻾븳 寃곌낵 紐⑤뱺 議고빀뿉꽌 鍮 듅씠쟻 諛섏쓳 굹굹吏 븡븯떎(Data not shown). HuEAdV-41 DNA瑜 100, 50, 25, 10, 5 諛 1 copy濡 씗꽍븯뿬 11媛 議고빀뿉 븳 寃異 誘쇨컧룄瑜 遺꾩꽍븳 寃곌낵, 긽쟻쑝濡 넂 寃異 誘쇨컧룄瑜 굹궦 議고빀 6媛쒖떎. 씠 以 nested PCR 봽씪씠癒 꽑諛쒖쓣 怨좊젮븯뿬 600~850 bp瑜 利앺룺븷 닔 엳뒗 5媛 議고빀(#12, #22, #32, #41 諛 #47)쓣 꽑諛쒗븯쑝硫, 寃異 誘쇨컧룄뒗 10~25 copies 닔以씠뿀떎(Table 1).

Fig. 2.

Specific reaction of primer sets for detection of HEAdV-41. Lane M, 100 bp Ladder marker; lane number, primer sets number (1~50); N, negative control; dot, selective primer sets.


Sensitive test of PCR and nested PCR primer sets for detection of HEAdV-41

PCR primer setNested PCR primer set


Number (forward/reverse primerLength (bp)Sensitivity (copies)Number of candidatesaSelective primer set (size, bp)Sensitivity (copies)
#06 (AdV41_F10/R60)74325-NDb
#12 (AdV41_F20/R60)600102AdV41_F40/R60 (486)1
#16 (AdV41_F30/R40)81450-ND
#22 (AdV41_F40/R40)759105AdV41_F90/R30 (320)1
#24 (AdV41_F40/R60)48625-ND
#32 (AdV41_F60/R20)824104AdV41_F90/R20 (434)1
#37 (AdV41_F70/R10)845100-ND
#41 (AdV41_F80/R10)773103AdV41_F90/R20 (434)1
#47 (AdV41_F90/R10)631252AdV41_F90/R20 (434)1
#48 (AdV41_F90/R20)43450-ND
#49 (AdV41_F90/R30)320100-ND

aNumber of nested PCR primer sets of selecteved PCR primer set;

bNo data.


Nested PCR 봽씪씠癒 꽑諛

1李 PCR 寃異 誘쇨컧룄 떆뿕뿉꽌 꽑諛쒕맂 5媛 PCR 봽씪씠癒 議고빀뿉 븳 nested PCR 봽씪씠癒 議고빀쓣 꽕怨꾪븯떎. 洹 寃곌낵 議고빀 #12(2媛), #22(5媛), #32(4媛), #41(3媛) 諛 #47(2媛)쓽 nested PCR 봽씪씠癒 議고빀씠 媛곴컖 꽕怨꾨릺뿀떎. Nested PCR쓽 寃異 誘쇨컧룄뒗 1~5 copies 닔以씠뿀쑝硫, 諛대뱶쓽 媛뺣룄, 겕湲 벑쓣 怨좊젮븯뿬 媛곴컖쓽 PCR 봽씪씠癒 議고빀留덈떎 1媛쒖쓽 nested PCR 봽씪씠癒 議고빀쓣 理쒖쥌 꽑諛쒗븯떎(Table 1).

湲곗〈 떆뿕踰 鍮꾧탳

HuEAdV-41쓽 듅씠쟻 빑궛 利앺룺씠 媛뒫븳 湲곗〈쓽 17媛 議곌굔쓣 긽쑝濡 寃異 誘쇨컧룄, 듅씠룄, 湲고 鍮 듅씠쟻 利앺룺, 諛섏쓳떆媛꾩쓣 遺꾩꽍븯떎. 씠 以 9媛 議고빀 NeAdV 빑궛 寃異쒕릺뿀吏留 듅씠룄媛 醫뗭 븡븯쑝硫, 듅씠룄媛 醫뗭븯뜕 8媛 議곌굔 以, 6媛 議곌굔 100 copies 씠긽씪 븣 寃異쒕릺뿀怨, 굹癒몄 2媛 議고빀 媛곴컖 50怨 25 copies 닔以쑝濡 寃異쒕릺뿀떎(Table 2). 洹몃윭굹 媛옣 醫뗭 듅씠꽦 諛 寃異 誘쇨컧룄瑜 媛吏 議고빀 184遺꾩쓽 PCR 諛섏쓳씠 븘슂븯뿬 떎瑜 議곌굔뱾뿉 鍮꾪빐 諛섏쓳떆媛꾩씠 湲몄뿀떎.

Comparision of reference primer sets for the detection of HEAdV-41

Reference primer setProduct size (bp)Sensitivity (copies)NEAdV reactionNon-specific bandTime (min)Reference

#PCR type*
01PCRF482100+-78[10, 11]
R

02PCRF26025--184[12]
R

03PCRF30150+-78[12, 13]
R

Nested PCRNF17125+-78
NR

04PCRF300100--71[13]
R

Nested PCRNF14250+-71
NR

05PCRF301> 100--56
R

Nested PCRNF12050-+71
NR

06PCRF371> 100+-157[14]
R

Nested PCRNF172> 100++122
NR

07PCRF30650+-157
R

Nested PCRNF1325+-122
NR

08PCRF475> 100--90[15]
R

Nested PCRNF256> 100--170
NR

09PCRF482> 100--88[16-18]
R

Nested PCRNF464100--88
NR

10PCRF300100+-100[19-20]
R

Nested PCRNF22050+-100
NR

11PCRF401100--116[21]
R

12PCRF401100--116
R

13PCRF30850+-154[13, 22]
R

Nested PCRNF13925+-154
NR

14PCRF482100--123[23]
R

15PCRF541100--123
R

16PCRF24125+-137[24]
R

17PCRF329100--80[25]
R

Nested PCRNF25325+-72
NR

*F, forward primer; R, reverse primer; NF, nested PCR forward primer; NR, nested PCR reverse primer.


떆猷 쟻슜꽦 떆뿕 諛 理쒖쥌 꽑諛

吏븯닔뿉꽌 異붿텧븳 20媛 珥 빑궛쓣 궗슜븯뿬 湲곗〈 떆뿕踰뺤뿉꽌 넂 듅씠꽦 諛 寃異 誘쇨컧룄瑜 蹂댁씤 봽씪씠癒 議고빀 諛 1李 PCR 봽씪씠癒 5媛 議고빀(#12, #22, #32, #41 諛 #47)쑝濡 PCR 諛섏쓳쓣 닔뻾븳 寃곌낵, 湲곗〈 떆뿕踰 議곌굔怨 꽑諛쒕맂 5媛 PCR 봽씪씠癒 議고빀 봽씪씠癒 紐⑤몢뿉꽌 쓬꽦 諛섏쓳 삉뒗 옒뜑留 벑 鍮 듅씠쟻 諛섏쓳씠 굹굹 nested PCR 諛섏쓳쓣 닔뻾븯떎. 洹 寃곌낵 PCR 議고빀 #12, #22, #32 諛 #41쓣 씠슜븳 nested PCR쓽 寃쎌슦 듅젙 떆猷뚯뿉꽌 뼇꽦쑝濡 異붿젙릺뒗 겕湲곗쓽 諛대뱶媛 삎꽦릺뿀쑝굹 鍮 듅씠쟻 諛대뱶媛 留롮씠 굹궗떎. 諛섎㈃ #47쓽 寃쎌슦 nested PCR 寃곌낵 20媛 以 10媛 떆猷(1, 4, 6, 7, 9, 10, 14, 15, 16 諛 20踰)뿉꽌 듅씠 諛대뱶媛 삎꽦릺뿀怨 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡븘 HuEAdV-41쓣 寃異쒗븯뒗뜲 理쒖쟻쓽 議고빀쑝濡 遺꾩꽍릺뿀떎(Fig. 3). 1李 PCR怨 nested PCR 봽씪씠癒몄쓽 뿼湲곗꽌뿴 Table 3怨 媛숇떎.

Fig. 3.

Environmental sample test of six specific PCR primer sets for the detection of HEAdV-41. Lane M, 100 bp Ladder marker; lane number, sample number (1~20); N, negative control.


Information of finally selective PCR and nested PCR primer set for the detection of HEAdV-41

 PCR type Primer Sequence (5’ → 3’)Mer (nt)Amplicon size (bp)
PCRHEAdV-41_F90TAC TTG GAT GGT ACC TTT TAC C22631
HEAdV-41_R10AAA GAA GTG TGG GCT CAT18

Nested PCRHEAdV-41_F90TAC TTG GAT GGT ACC TTT TAC C22434
HEAdV-41_R20ATA AGG GGG TAA GGG TAG TTA G22

怨좎같

듅젙 誘몄깮臾쇱쓽 듅씠쟻 빑궛쓣 寃異쒗븯뒗 諛⑸쾿 conventional PCR, multiplex-PCR, real-time quantitative PCR (RT-qPCR), 벑삩利앺룺(loop-mediated isothermal amplification; LAMP) 諛⑸쾿 벑씠 蹂닿퀬릺怨 엳떎(Kim et al., 2004; Wang et al., 2007; Kim et al., 2008; Munkhdelger et al., 2013). 씠 以 LAMP뒗 듅씠룄 諛 寃異 誘쇨컧룄媛 넂怨 룞씪븳 삩룄뿉꽌 떎닔쓽 떆猷뚮 諛섏쓳븷 닔 엳뒗 벑쓽 옣젏씠 엳쑝굹, 諛섎㈃ 쐞 뼇꽦 벑 鍮 듅씠쟻 諛섏쓳씠 긽쟻쑝濡 留롮씠 굹굹硫 떒媛媛 넂 떒젏씠 엳떎. RT-qPCR 以 듅엳 Taq-man probe 諛⑸쾿 듅씠룄 寃異 誘쇨컧룄媛 넂怨 떊냽븳 諛⑸쾿쑝濡 솢슜꽦씠 넂 렪씠吏留, 뿼湲곗꽌뿴 遺꾩꽍씠 뼱졄怨 conventional PCR뿉 鍮꾪빐 솢슜븷 닔 엳뒗 寃궗옄媛 쟻떎. 씠 媛숈씠 떎뼇븳 PCR쓽 寃궗湲곕쾿뿉뒗 怨좎쑀쓽 諛⑸쾿뿉 뵲瑜 옣떒젏씠 議댁옱븯湲 븣臾몄뿉 씠踰 뿰援ъ쓽 긽씤 HuEAdV-41쓽 寃異쒕갑踰뺤 conventional PCR쓣 꽑젙븯떎. Conventional PCR쓽 寃쎌슦 삤옖 湲곌컙 룞븞 留롮 뿰援ъ옄뱾뿉 쓽빐 븞젙꽦씠 寃利앸릺뿀怨, 븘슂븷 寃쎌슦 뿼湲곗꽌뿴 遺꾩꽍씠 媛뒫븯硫, 긽쟻쑝濡 돺寃 遺꾩꽍븷 닔 엳뼱 몴以 寃궗踰뺤쑝濡 쟻빀븯떎怨 蹂닿퀬(Park et al., 2016)릺怨 엳뒗 벑 슦닔븳 寃궗湲곕쾿쑝濡 씤젙諛쏆븘 솕떎. 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 HuEAdV-41 寃異쒖슜 conventional PCR 봽씪씠癒 議고빀 湲곗〈쓽 HuEAdV-41 寃異쒖슜 議곌굔뱾뿉 鍮꾪빐 룞벑 씠긽쓽 듅씠룄瑜 蹂댁쑝硫 諛섏쓳떆媛꾩 빟 50遺 떒異뺥븷 닔 엳뒗 寃껋쑝濡 굹궗떎. 肉먮쭔 븘땲씪 nested PCR 諛섏쓳쓽 寃쎌슦, 빟 25諛 젙룄쓽 寃異 誘쇨컧룄瑜 뼢긽떆궗 닔 엳뿀떎. 洹몃윭굹 떎뼇븳 떆猷뚯뿉 쟻슜븷 寃쎌슦 떆猷 궡 議댁옱븯뒗 諛⑺빐臾쇱쭏 벑쑝濡 씤빐 듅씠룄 諛 寃異 誘쇨컧룄뿉 蹂솕媛 엳쓣 닔룄 엳쑝誘濡 떎뼇븳 떆猷뚯뿉 븳 異붽쟻 떆뿕쓽 븘슂꽦씠 몢맂떎.

븳렪, HuEAdV-41 엫긽, 떇뭹, 뼱뙣瑜, 솚寃 벑쓽 떆猷뚯뿉꽌 二쇰줈 遺꾩꽍릺怨 엳떎. 쁽옱源뚯뒗 떆猷뚮퀎, 寃궗遺泥섎퀎, 뿰援ъ옄 諛 寃궗옄뿉 뵲씪 떖由 PCR 諛⑸쾿쓣 솢슜릺怨 엳쑝굹, 꽌濡 떎瑜 봽씪씠癒, 議곌굔, 議곗꽦 벑쓣 궗슜븯怨 엳떎. 씠濡 씤븯뿬 媛 떆猷뚯뿉꽌 寃異쒕릺怨 엳뒗 빑궛 떒렪뿉 븳 鍮꾧탳 뿰援ш 뼱졄怨, 엫긽, 떇뭹, 솚寃 벑 떆猷뚮 룞떆뿉 떎猷⑤뒗 蹂닿굔솚寃쎌뿰援ш린愿뿉꽌 떎臾대 떞떦븯뒗 寃궗옄뱾 媛꾩뿉 遺덊렪븿씠 諛쒖깮븷 닔 엳떎. 뵲씪꽌 援궡뿉꽌룄 씠윭븳 臾몄젣瑜 빐寃고븯怨좎옄 吏덈퀝愿由щ낯遺, 떇뭹쓽빟뭹븞쟾泥, 援由쏀솚寃쎄낵븰썝, 援由쎌닔궛怨쇳븰썝, 援由쎈냽뾽怨쇳븰썝, 떆·룄 蹂닿굔솚寃쎌뿰援ъ썝 벑 愿젴 遺泥섏뿉꽌 닔씤꽦떇뭹留ㅺ컻諛붿씠윭뒪 뿰援ы삊쓽泥대 援ъ텞븯뿬 몴以솕맂 conventional PCR 諛⑸쾿쓣 솢슜븳 끂濡쒕컮씠윭뒪 寃궗, 쑀쟾삎 遺꾩꽍 벑쓽 遺꾩꽍 諛⑸쾿쓣 넻빀븯湲 쐞븳 끂젰씠 吏꾪뻾릺怨 엳떎. 끂濡쒕컮씠윭뒪 쇅 HuEAdV-41 벑 떎瑜 닔씤꽦 떇뭹留ㅺ컻 諛붿씠윭뒪 뿭떆 씠윭븳 怨쇱젙씠 븘슂븷 寃껋쑝濡 쟾留앸릺硫, 寃궗踰 媛쒕컻뿉꽌룄 怨좊젮릺뼱빞 븷 寃껋쑝濡 蹂댁씤떎. 븘슱윭 슚쑉쟻씤 寃異쒖쓣 쐞븯뿬 媛먯뿼 삉뒗 삤뿼 떆 긽쟻 怨좊냽룄濡 굹굹뒗 엫긽, 떇뭹, 뼱뙣瑜 벑쓽 떆猷뚮뒗 臾쇰줎 吏븯닔 벑 鍮꾩냼룆닔 媛숈씠 긽쟻쑝濡 誘몃웾 삤뿼씠 삁긽릺뒗 떆猷뚯뿉꽌룄 寃異쒖씠 媛뒫븳 듅씠룄 誘쇨컧룄瑜 媛뽰텣 利앹쭊맂 PCR 湲곕쾿씠 븘슂븳 寃껋씠 쁽떎씠떎.

뵲씪꽌 蹂 뿰援ъ뿉꽌 媛쒕컻븳 1李 PCR 봽씪씠癒 議고빀 631 bp 겕湲곕줈 利앺룺릺硫 빟 112遺꾩쓽 PCR 諛섏쓳떆媛꾩씠 븘슂븯뿬, 怨좊냽룄 삤뿼 떆猷뚯씤 엫긽, 떇뭹, 뼱뙣瑜 벑쓽 떆猷뚯뿉꽌 HuEAdV-41쓣 寃異쒗븷 寃쎌슦 빑궛 異붿텧 썑 1李 PCR 닔以留뚯쑝濡쒕룄 쎇뼱궃 寃異 誘쇨컧룄뿉 쓽빐 HuEAdV-41瑜 異⑸텇엳 寃異쒗븷 닔 엳쓣 寃껋쑝濡 蹂댁씤떎. 븘슱윭 솚寃쎈 援由쏀솚寃쎄낵븰썝쓽 吏븯닔 以 끂濡쒕컮씠윭뒪 삤뿼떎깭議곗궗 뿰援 벑뿉 뵲瑜대㈃ 삤뿼씠 삁긽맂 吏븯닔 떆猷뚯뿉꽌룄 끂濡쒕컮씠윭뒪 듅씠쟻 빑궛 떒렪 遺遺 nested PCR 怨쇱젙뿉 쓽븯뿬 寃異쒕릺誘濡 吏븯닔 벑怨 媛숈 誘몃웾 삤뿼 떆猷뚯쓽 寃쎌슦 nested PCR쓣 솢슜븳 異붽 遺꾩꽍씠 븘슂븯떎怨 궗猷뚮맂떎. 蹂 뿰援щ 넻븯뿬 媛쒕컻맂 nested PCR 봽씪씠癒몄쓽 寃쎌슦 吏븯닔 벑 鍮 냼룆닔뿉 븳 1李 PCR 諛섏쓳 썑 諛섏쓳궛臾쇱쓣 二쇳삎쑝濡 nested PCR쓣 異붽濡 닔뻾븯硫 뼇꽦 떆猷뚯쓽 寃쎌슦 434 bp 겕湲곗쓽 HuEAdV-41 빑궛 떒렪씠 寃異쒕릺硫, 빟 112遺꾩쓽 諛섏쓳떆媛꾩씠 븘슂븯떎. 뵲씪꽌 씠踰 뿰援щ뒗 뼢썑 엫긽, 떇뭹, 뼱뙣瑜, 솚寃 벑 뿬윭 떆猷뚯뿉꽌 HuEAdV-41쓣 슚쑉쟻쑝濡 寃異쒗븷 닔 엳뒗 諛⑸쾿쑝濡 궗슜맆 닔 엳쓣 寃껋쑝濡 湲곕릺硫 쁽옱 吏꾪뻾 以묒씤 踰 遺泥 媛꾩쓽 PCR 諛 nested PCR쓽 몴以솕뿉 븳 湲곗큹옄猷뚮줈 솢슜맆 닔 엳쓣 寃껋씠씪 궗猷뚮맂떎.

ACKNOWLEDGEMENT

蹂 끉臾몄 2018뀈룄 떊븳븰援 븰닠뿰援щ퉬 吏썝쑝濡 뿰援щ릺뿀쓬.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

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