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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
Published online December 31, 2019;  https://doi.org/10.15616/BSL.2019.25.4.331
© 2019 The Korean Society For Biomedical Laboratory Sciences.

Siwon Lee* and 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 September 2, 2019; Revised October 18, 2019; Accepted November 25, 2019.
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 Aichivirus A1 (HuAiV-A1) is a waterborne human pathogenic virus classified as Picornaviridae and Kobuvirus. In this study, we developed a method that can detect about 35 minutes faster with the same detection sensitivity level than the previously reported HuAiV-A1 diagnostic RT-PCR primer. The RT-PCR primer sets developed in this study are capable of detecting HuAiV-A1 at a level of about 100 ag and formed 563 bp amplification product. In addition, the RT-nested PCR method was able to amplify 410 bp using the RT-PCR product as a template. The detection sensitivity of our method was 10 times higher than the method with the highest detection sensitivity to date. Therefore, the detection method of HuAiV-A1 developed in this study is expected to be used in the water environment in which a small amount of virus exists. Also, this detection method is expected to be used as HuAiV-A1 diagnostic technology in both clinical and non-clinical field.

Keywords : HuAiV-A1, Human aichivirus A1, Kobuvirus, RT-nested PCR, Waterborn human pathogenic virus
꽌 濡

궗엺 븘씠移섎컮씠윭뒪[Human Aichivirus 1; HuAiV-A1]뒗 1989뀈 씪蹂 븘씠移섑쁽 鍮 꽭洹좎꽦 쐞옣뿼 솚옄쓽 蹂 寃泥댁뿉꽌 理쒖큹濡 蹂닿퀬릺뿀떎(Reuter al., 2009). HuAiV-A1쓽 엯옄뒗 빟 30 nm濡 몴硫댁씠 뫁湲怨 슱뎮遺덊뎮븯硫, 遺꾨쪟븰쟻쑝濡 諛붿씠윭뒪 group IV, Picornaviridae, Kobuvirus (Aichi-virus A)뿉 냽븳떎. 誘멸뎅 援由쎌깮臾쇱젙蹂댁꽱꽣뿉 뵲瑜대㈃, 쟾泥 빑궛쓽 湲몄씠뒗 빟 8,251 nucleotide (nt)濡, 5' non-translated region (NTR) 712 nt, 3' NTR 240 nt 諛 以묒븰쓽 7,302 nt뒗 polyprotein쓣 븫샇솕븯硫, 빟 2,433媛 븘誘몃끂궛쓣 吏떆븳떎. HuAiV-A1 씤泥 蹂묒썝꽦 諛붿씠윭뒪濡 궗엺뿉寃 媛먯뿼 떆 꽕궗, 蹂듯넻, 援ы넗, 援ъ뿭吏, 諛쒖뿴 벑쓽 利앹긽씠 굹궃떎(Khamrin and Maneekarn, 2014; Khamrin et al., 2011). 쁽옱源뚯 蹂닿퀬릺怨 엳뒗 HuAiV-A1 꽕궗 솚옄뿉寃 빟 0~4.1% 닔以쑝濡 寃異쒕릺怨 엳뼱 떎瑜 닔씤꽦 옣諛붿씠윭뒪뿉 鍮꾪빐 긽쟻쑝濡 궙 異쒗쁽鍮덈룄瑜 蹂댁씠硫(Saikruang et al., 2014) 떒룆 媛먯뿼蹂대떎뒗 떎瑜 諛붿씠윭뒪 벑쓽 蹂묒썝泥댁 샎빀 媛먯뿼삎깭濡 二쇰줈 굹굹怨 엳떎.

삉븳 媛쒕컻룄긽援怨 꽑吏꾧뎅뿉 愿怨꾩뾾씠 븘떆븘, 쑀읇, 궓誘, 遺곷 벑 쟾꽭怨꾩쟻쑝濡 諛쒖깮릺怨 엳쑝硫, 깮援, 젗媛 벑 닔궛臾, 긽닔썝닔, 媛 벑 솚寃쎌뿉룄 꼻 踰붿쐞濡 삤뿼씠 릺뼱 엳뒗 寃껋쑝濡 蹂닿퀬릺怨 엳떎(Yamashita et al., 2000; Oh et al., 2006; Guyader et al., 2008; Pham, 2011). 뵲씪꽌 援궡뿉꽌룄 씠윭븳 臾몄젣젏쓣 吏곸떆븯뿬 援由쏀솚寃쎄낵븰썝 벑 援媛湲곌쓣 以묒떖쑝濡 HuAiV-A1 벑怨 媛숈 誘 洹쒖젣 蹂묒썝泥댁뿉 븳 紐⑤땲꽣留곸쓣 쐞븳 뿰援ъ궗뾽, 몴以寃궗踰 媛쒕컻 벑쓽 쓳씠 씠猷⑥뼱吏怨 엳떎. 쁽옱 닔씤꽦 諛붿씠윭뒪쓽 몴以寃궗踰뺤쑝濡 떆猷뚯뿉꽌 異붿텧븳 珥 빑궛뿉꽌 conventional polymerase chain reaction (C-PCR)쑝濡 諛붿씠윭뒪쓽 듅젙 떒렪쓣 利앺룺븯뒗 諛⑸쾿씠 솢슜릺怨 엳떎. C-PCR real-time qPCR怨 벑삩利앺룺踰(loop mediated isothermal amplification; LAMP)뿉 鍮꾪빐 삤옖 湲곌컙 뿰援щ릺뼱 븞젙꽦씠 엯利앸릺뿀怨 뵲씪꽌, 꼻 踰붿쐞쓽 궗슜옄瑜 媛吏怨 엳쑝硫, 븘슱윭 寃곌낵 궛臾쇱쓣 異붽 諛섏쓳 뾾씠 sequencing 븷 닔 엳뒗 옣젏씠 엳뼱 엫긽 諛 鍮 엫긽 떆猷뚯뿉꽌 蹂묒썝泥대 吏꾨떒븯뒗 몴以솕 諛⑸쾿쑝濡 솢슜씠 쟻빀븯떎(Yamashita et al., 2003; Reuter et al., 2009; Santos et al., 2015). 洹몃윭굹 HuAiV-A1 吏꾨떒슜 C-PCR 湲곗닠 떎닔쓽 뿰援ъ옄뱾뿉 쓽븳 留롮 蹂닿퀬媛 엳吏留 냽룄, 寃異 誘쇨컧룄 벑뿉 븳 鍮꾧탳 뿰援щ뒗 遺議깊븯떎. 듅엳 臾 솚寃 떆猷뚯쓽 寃쎌슦 誘몃웾 삤뿼 諛 떎닔쓽 PCR 빐 臾쇱쭏씠 룷븿릺뼱 엳뼱 넂 듅씠꽦 諛 寃異 誘쇨컧룄媛 슂援щ릺怨 엳떎. 삉븳 湲곗〈쓽 뿰援щ뱾 PCR 諛섏쓳 議곌굔씠 떖씪 媛숈 떆猷뚯뿉꽌 異붿텧븳 珥 빑궛뿉꽌 Rotavirus-A, Norovirus 벑쓽 떎瑜 닔씤꽦 諛붿씠윭뒪뱾怨 룞떆뿉 PCR쓣 닔뻾븷 닔 뾾뼱 떎웾쓽 遺꾩꽍씠 븘슂븳 寃궗湲곌뿉꽌 솢슜뿉 留롮 븷濡쒓 뵲옄떎. 뵲씪꽌 씠윭븳 臾몄젣젏쓣 빐寃고븯湲 쐞븳 寃異 誘쇨컧룄 諛 듅씠꽦씠 넂怨 쁽옣뿉꽌쓽 솢슜꽦씠 솗蹂대맂 깉濡쒖슫 吏꾨떒 湲곗닠쓽 媛쒕컻씠 븘슂븳 떎젙씠떎.

뵲씪꽌 蹂 뿰援ъ뿉꽌뒗 쐞 議곌굔뿉 遺빀븯뒗 reverse transcription nested (RT-nested) PCR 湲곕컲쓽 HuAiV-A1 吏꾨떒슜 PCR 봽씪씠癒 議고빀쓣 媛쒕컻븯怨, 湲곗〈뿉 蹂닿퀬맂 C-PCR 諛⑸쾿뱾怨 誘쇨컧룄, 듅씠꽦, 쁽옣 솢슜꽦 벑씠 슦닔븳 C-PCR 떆뒪뀥쓣 媛쒕컻븯怨좎옄 븯떎.

옱猷 諛 諛⑸쾿

듅씠쟻 PCR 봽씪씠癒 젣옉 諛 몴以洹좎< 닔吏

HuAiV-A1 듅씠쟻 봽씪씠癒 젣옉쓣 쐞븯뿬 DNAMAN sof tware package version 6.0 (Lynnon Biosof t, Quebec, Canada)瑜 궗슜븯떎. 깘깋 議곌굔 HuAiV-A1 NCBI accession NC_001918 2,740~3,918 (1,179 nt)瑜 湲곗쑝濡 븯쑝硫, 寃궗옄쓽 렪쓽꽦쓣 쐞빐 湲곗〈 닔씤꽦 諛붿씠윭뒪쓽 PCR 議곌굔쑝濡 솢슜릺怨 엳뒗 annealing 삩룄 51~59℃(理쒖쟻 55℃)濡 븯떎(NIER, 2016). HuAiV-A1뿉 듅씠쟻 寃고빀쓣 븷 寃껋쑝濡 異붿젙릺뒗 젙諛⑺뼢 諛 뿭諛⑺뼢 봽씪씠癒몃뱾쓣 긽쑝濡 Oligo Calculator version 3.27瑜 넻빐 봽씪씠癒몄쓽 듅씠꽦, 뒪뒪濡 遺李⑸릺뼱 슚쑉씠 븯맆 닔 엳뒗吏뿉 븳 媛뒫꽦 벑쓣 遺꾩꽍븯뿬 理쒖쥌 썑蹂 봽씪씠癒 議고빀뱾쓣 젣옉븯떎.

듅씠쟻 諛섏쓳뿉 궗슜븷 HuAiV-A1 二쇳삎 빑궛 NC_-001918.1 VP3-VP1 [2,740~3,918 (1,179 nt)]쓣 留덊겕濡쒖젨(Seoul, Korea)뿉 쓽猶고븯뿬 linear 긽깭濡 gene쓣 빀꽦븯떎. 鍮 듅씠쟻 諛섏쓳뿉 솢슜븷 李멸퀬 諛붿씠윭뒪二 빑궛[RNA, DNA or cDNA; (Rotavirus A, Parechovirus A, Norovirus GII, Corona-virus, Enterovirus-68, Enterovirus-71, Coxakievirus-A6, Coxakie-virus-B1, Coxakievirus-B5, non-enteric Adenovirus C 諛 enteric Adenovirus 41)] linear type gene 빀꽦 [Aichivirus-B (NC_-004421; 4,571~5,370), Aichivirus-C (NC_011829; 4,417~5,216), Parvovirus-B19 (NC_000883.2; 3,001~5,000), Hepatitisvirus-A (K02990.1; 2,014~4,013) 諛 Sapovirus (KP298674.1; 4,440~ 6,439)]쓣 닔吏묓븯떎. RNA 삎깭濡 닔吏묐맂 寃껋 ReverTra Ace-α-® (TOYOBO, Osaka, Japan)쑝濡 cDNA濡 빀꽦븯뿬 궗슜븯떎. 븳렪, HuAiV-A1 李멸퀬 봽씪씠癒몄쓽 諛섏쓳쓣 쐞빐 3CD [NC_001918.1; 6,038~8,011 (1,974 nt)]瑜 linear type쑝濡 gene 빀꽦븯떎.

봽씪씠癒 꽑諛

HuAiV-A1뿉 듅씠쟻쑝濡 利앺룺씠 媛뒫븷 寃껋쑝濡 異붿젙릺뒗 PCR 봽씪씠癒 議고빀쓣 씠슜븯뿬, 듅씠쟻, 鍮 듅씠쟻 諛 寃異 誘쇨컧룄 遺꾩꽍쓣 닔뻾븯떎. 듅씠쟻 諛섏쓳 二쇳삎 빑궛씤 HuAiV-A1 빑궛쓣 100 fg/μL 냽룄濡 궗슜븯뿬 援ъ꽦맂 PCR 봽씪씠癒 議고빀뿉 븳 듅씠쟻 諛대뱶쓽 삎꽦 뿬遺瑜 솗씤븳 썑, 諛섏쓳씠 媛뺥븯怨 利앺룺궛臾쇱쓽 겕湲곌 쟻빀븳 議고빀쓣 꽑諛쒗븯떎. 듅씠쟻 諛섏쓳뿉꽌 꽑諛쒕맂 PCR 봽씪씠癒 議고빀뱾쓣 씠슜븯뿬 李멸퀬 諛붿씠윭뒪二 15醫낆뿉 븳 鍮 듅씠쟻 諛섏쓳쓣 寃젙븯떎. 씠썑 李멸퀬 諛붿씠윭뒪二쇰뱾뿉 븯뿬 빑궛뿉 諛섏쓳씠 굹굹吏 븡 PCR 봽씪씠癒 議고빀뱾쓣 HuAiV-A1 吏꾨떒뿉 쟻빀븯떎怨 異붿젙븯떎. 삉븳 듅씠쟻 諛 鍮 듅씠쟻 떆뿕뿉꽌 꽑諛쒕맂 PCR 봽씪씠癒 議고빀뱾쓣 긽쑝濡 寃異 誘쇨컧룄瑜 遺꾩꽍븯떎. HuAiV-A1 빑궛 1 ng/μL 湲곗쑝濡 10諛 떒怨 씗꽍븯뿬 10-4 (100 fg/μL) ~ 10-9 (1 ag/μL)쓽 냽룄濡 씗꽍맂 HuAiV-A1 빑궛뱾쓣 二쇳삎쑝濡 誘쇨컧룄 遺꾩꽍쓣 떆뻾븯떎. 蹂 뿰援ъ뿉꽌 궗슜릺뒗 二쇳삎 linear type쑝濡 빀꽦맂 DNA吏留, 떎젣 떆猷뚯뿉꽌뒗 total RNA 異붿텧 썑 RT-PCR濡 諛섏쓳빐빞 븯뒗 寃껋쓣 媛먯븞븯뿬 one step RT-PCR쓣 닔뻾븯떎. RT-PCR 議곗꽦 AccuPower® RT/PCR PreMix (Bioneer, Daejeon, Korea), PCR 봽씪씠癒(젙諛⑺뼢 諛 뿭諛⑺뼢) 媛곴컖 25 pmol 냽룄濡 2 μL, 二쇳삎 빑궛 1 μL 諛 硫멸퇏利앸쪟닔 17 μL濡 븯뿬 珥 20 μL濡 諛섏쓳븯떎. RT-PCR 諛섏쓳 議곌굔 42℃뿉꽌 60遺 룞븞 뿭쟾궗, 95℃뿉꽌 5遺 룞븞 珥덇린 蹂꽦떒怨꾨 嫄곗퀜 95℃뿉꽌 45珥덇컙 蹂꽦, 55℃뿉꽌 1遺꾧컙 寃고빀, 72℃뿉꽌 1遺꾧컙 떊옣븯뒗 떒怨꾨 35쉶 諛섎났븯쑝硫 洹 썑 72℃뿉꽌 5遺꾧컙 理쒖쥌 떊옣븯떎(NIER, 2016).

븳렪, nested PCR 봽씪씠癒 議고빀쓣 꽑諛쒗븯湲 쐞빐 理쒖쥌 꽑諛쒕맂 RT-PCR 봽씪씠癒 議고빀뿉꽌 nested PCR 利앺룺씠 媛뒫븳 議고빀쓣 꽕怨 諛 젣옉븯떎. RT-PCR쓣 넻빐 깮꽦맂 궛臾쇱쓣 二쇳삎쑝濡 寃異 誘쇨컧룄 떆뿕쓣 쐞븳 理쒖쥌 nested PCR 봽씪씠癒 議고빀쓣 꽑諛쒗븯떎. Nested PCR쓽 議곗꽦 AccuPower® HotStart PCR PreMix (Bioneer), PCR 봽씪씠癒(젙諛⑺뼢 諛 뿭諛⑺뼢) 媛곴컖 25 pmol 냽룄濡 2 μL, 二쇳삎 DNA (RT-PCR 궛臾) 1 μL 諛 硫멸퇏利앸쪟닔 17 μL濡 븯뿬 珥 20 μL濡 븯떎. Nested PCR 議곌굔 RT-PCR 怨쇱젙 以 뿭쟾궗 怨쇱젙쓣 젣쇅븯怨 룞씪븯寃 븯떎. RT-PCR 諛 nested PCR 궛臾쇰뱾 1.2% agarose gel쓣 궗슜븯뿬 100 V뿉꽌 30遺 룞븞 쟾湲곗쁺룞 썑 gel documentation system (Bio Rad, California, USA)뿉꽌 솗씤븯떎.

湲곗〈 떆뿕踰 鍮꾧탳

湲곗〈 떆뿕踰뺢낵 寃異 誘쇨컧룄瑜 鍮꾧탳븯湲 쐞븯뿬 HuAiV-A1쓽 VP3-1 遺遺꾩쓣 빀꽦븳 1,179 nt 3CD 遺遺꾩쓣 빀꽦븳 1,974 nt瑜 샎빀븳 移듯뀒씪쓣 젣옉븯떎. Oligo Calculator version 3.27 꽌踰꾩뿉 媛 뿼湲곗꽌뿴쓣 엯젰븳 썑 遺꾩옄웾쓣 怨꾩궛븳 寃곌낵 빀꽦븳 3CD 遺遺꾩씠 VP3-1 遺遺꾩쓽 빟 1.68諛곗씤 寃껋쓣 怨좊젮븯뿬 샎빀 떆 VP3-1怨 3CD쓽 鍮꾩쑉쓣 1.68: 1.00쑝濡 議곗젅븯뿬 copy 닔瑜 留욎텛뿀떎. 샎빀븳 移듯뀒씪쓣 10諛 떒怨 씗꽍븯쑝硫, 씠寃껊뱾쓣 二쇳삎쑝濡 蹂 뿰援ъ뿉꽌 꽑諛쒗븳 RT-nested PCR 봽씪씠癒 議고빀 諛 湲곗〈 conventional PCR 봽씪씠癒 議고빀뱾쓽 寃異 誘쇨컧룄瑜 遺꾩꽍븯떎. 삉븳, 議곌굔 蹂 諛섏쓳 떆媛 鍮꾧탳瑜 쐞븯뿬 뿭쟾궗 怨쇱젙 KIT쓽 봽濡쒗넗肄쒖뿉 뵲씪 30遺~1떆媛 닔以쑝濡 긽씠븯吏留 紐⑤몢 60遺꾩쑝濡 넻빀 怨꾩궛븯쑝硫, PCR씠 떆옉맂 썑 醫낅즺 떆젏源뚯쓽 떆媛꾩쓣 鍮꾧탳븯떎.

寃 怨

봽씪씠癒 젣옉

HuAiV-A1뿉 듅씠쟻씠怨, 뒪뒪濡 寃고빀릺뒗(self annealing) 벑쓽 臾몄젣媛 굹굹吏 븡뒗 9媛쒖쓽 PCR 봽씪씠癒(젙諛⑺뼢 5媛 諛 뿭諛⑺뼢 4媛)媛 깘깋릺뿀떎(Fig. 1, Table 1). PCR 봽씪씠癒몃 議고빀븯뿬 PCR 利앺룺씠 媛뒫븳 20媛 議고빀쓣 援ъ꽦븯쑝硫, 빟 248~874 base pair (bp)쓽 湲몄씠뿀떎(Data not shown).

Information of RT-PCR and nested PCR primer sets for the detection of Human Aichivirus 1 (HuAiV-A1)

# PCR type Target gene Primer Product size (nt) References

Name Sequence (5'-3')
PCR primer pairs #01~20 RT-PCR & nested PCR VP3-VP1 HuAiV-A1_VP3-1_F10 ATCTCCGCAGGTGAATCC 265~874 This study
HuAiV-A1_VP3-1_F20 CTCACTTCCCAAACCCTC
HuAiV-A1_VP3-1_F30 CACCAAACTGGAAAACTTCTT
HuAiV-A1_VP3-1_F40 GCYRACGAAGGCACAATC
HuAiV-A1_VP3-1_F50 GATGCTRTCGTGCTTCAC
HuAiV-A1_VP3-1_R10 GCACGATGTAGACCCGRT
HuAiV-A1_VP3-1_R20 GGACCCAGGCTTTGAAGT
HuAiV-A1_VP3-1_R30 CTGGAAGTCRARGGGRATRG
HuAiV-A1_VP3-1_R40 GTCCTCCCAWCCRAAGTAY

Ref. 01 RT-PCR VP1 Yip-VP1_F TCTTCTCCTTCTACCGCTTG 357 Yip et al. (2014)
Yip-VP1_R GAGGTGTAGGGGATGGAGAA

Ref. 02 RT-nested PCR VP3-VP1 KBP1 CTCYTTCATCTCYMAMTCCTACTGG 1,245 Yamashita et al. (2014)
KBN1 AAGTGGGTRCAGTTGTTGG

KbP1P CAGCCMCGCACCACYTTCSASTAC 453
KbP1N GAAGTAKGARGTGGGRATRGCAGA

Ref. 03 RT-nested PCR VP3-VP1 AiV-VP3-F1 CACACCGCCCCTGCGTCRGCCCTCGT 721 Lodder et al. (2013)
AiV-VP1-R1 GAGAGCTGGAAGTCRAAGGG

AiV-VP1-F2 CTCGATGCRCCMCAAGACACCGG 505
AiV-VP1-R2 CCTGACCAGTCCTCCCAWCCGAAGTA

Ref. 04 RT-nested PCR VP3-VP1 AiV-VP3-F1 CACACCGCCCCTGCGTCRGCCCTCGT 721
AiV-VP1-R1 GAGAGCTGGAAGTCRAAGGG

AiV-VP1-F3 GTGCTTCACRTACATCGCYGCGG 239
AiV-VP1-R2 CCTGACCAGTCCTCCCAWCCGAAGTA

Ref. 05 RT-PCR 3CD C94b GACTTCCCCGGAGTCGTCGTCT 158 Khamrin and Maneekarn (2014)
AiMP-R GCRGAGAATCCRCTCGTRCC

Ref. 06 RT-nested PCR 3CD 6261 (RT-PCR) ACACTCCCACCTCCCGCCAGTA 519 Saikruang et al. (2014)
6779 (RT-PCR) GGAAGAGCTGGGTGTCAAGA

C94b (Nested PCR) GACTTCCCCGGAGTCGTCGTCT 223
246k (Nested PCR) GACATCCGGTTGACGTTGAC

Ref. 07 RT-PCR 3CD C94b GACTTCCCCGGAGTCGTCGTCT 158 Pham (2011)
AiMP-R GCRGAGAATCCRCTCGTRCC

Ref. 08 RT-PCR 3CD 10f (7357) GATGCTCCTCGGTGGTCTCA 631 Santos et al. (2015)
10r (7987) GTCGGGGTCCATCACAGGGT

Ref. 09 RT-PCR 3CD UNIV-kobu-F TGGAYTACAARTGTTTTGATGC 216 Reuter et al. (2009)
UNIV-kobu-R ATGTTGTTRATGATGGTGTTGA

Ref. 10 RT-nested PCR 3CD 6261 (RT-PCR) ACACTCCCACCTCCCGCCAGTA 519 Alcalá et al. (2010)
6779 (RT-PCR) GGAAGAGCTGGGTGTCAAGA

AiF2 (6283~6305) CAAGGACTTGCGGCGCTTCATCG 412
AiR2b (6672~6694) GCACCCYTCYCCCGCCTACGGTG

Fig. 1.

Specific RT-nested PCR primers map for the detection of Human Aichivirus A1 (HuAiV-A1).


RT-PCR 봽씪씠癒 꽑諛

珥 20媛 PCR 봽씪씠癒 議고빀쓣 긽쑝濡 HuAiV-A1뿉 븳 듅씠쟻 諛섏쓳쓣 遺꾩꽍븳 寃곌낵, 20媛 以 15媛 議고빀(議고빀 #02, #03, #04, #06, #07, #08, #10, #11, #12, #14, #15, #16, #18, #19 諛 #20)뿉꽌 듅씠쟻 諛섏쓳씠 굹궗쑝硫 諛대뱶쓽 媛뺣룄, 利앺룺븳 PCR 궛臾쇱쓽 湲몄씠, 利앺룺씠 맂 쐞移 벑쓣 怨좊젮븯뿬 14媛 以 5媛 議고빀(#02, #06, #08, #10 諛 #18)쓣 꽑諛쒗븯떎(Fig. 2). 듅씠쟻 諛섏쓳뿉꽌 꽑諛쒕맂 5媛쒖쓽 PCR 봽씪씠癒 議고빀쓣 긽쑝濡 15媛 李멸퀬 諛붿씠윭뒪二 빑궛뿉 鍮 듅씠쟻 諛섏쓳쓣 遺꾩꽍븳 寃곌낵, 5媛 PCR 봽씪씠癒 議고빀 紐⑤몢뿉꽌 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡븘(Fig. 3), 꽑諛쒕맂 5媛 PCR 봽씪씠癒 議고빀 HuAiV-A1瑜 듅씠쟻쑝濡 吏꾨떒븷 닔 엳쓣 寃껋쑝濡 異붿젙릺뿀떎. 삉븳, 5媛쒖쓽 PCR 봽씪씠癒 議고빀쓣 긽쑝濡 寃異 誘쇨컧룄瑜 遺꾩꽍븳 寃곌낵, 4媛 議고빀(#02, #06, #08 諛 #18)뿉꽌뒗 1 fg 닔以씤 10-6源뚯 諛대뱶媛 삎꽦릺뿀쑝硫, 議고빀 #10쓽 寃쎌슦 100 ag 닔以씤 10-7源뚯 寃異 誘쇨컧룄媛 굹굹, HuAiV-A1瑜 吏꾨떒븯湲곗뿉 쟻빀븳 RT-PCR 봽씪씠癒 議고빀쑝濡 꽑諛쒕릺뿀떎. 理쒖쥌 꽑諛쒕맂 HuAiV-A1 吏꾨떒슜 RT-PCR 봽씪씠癒 議고빀 HuAiV-1쓽 VP3-1뿉 븳 봽씪씠癒 F30怨 R20 議고빀쑝濡 563 bp쓽 PCR 궛臾쇱쓣 利앺룺븷 닔 엳뿀떎(Fig. 4).

Fig. 2. Specific reaction of RT-PCR primer pairs for the detecting HuAiV-A1.

Lane M, 100 bp Ladder marker; lane number, primer sets number (1~20); N, negative control; dot, selective primer sets.


Fig. 3. Non-specific reaction of five RT-PCR primer pairs.

Lane M, 100 bp Ladder marker; P, positive control; N, negative control; lane number, reference viruses [1, Aichivirus-B; 2, Aichivirus-C; 3, Rotavirus A; 4, Parechovirus A; 5, Norovirus GII; 6, Coronavirus 229E; 7, Enterovirus-68; 8, Enterovirus-71; 9. Coxakievirus-A6; 10, Coxakievirus-B1; 11, Coxakievirus-B5; 12, non-enteric Adenovirus C; 13, enteric Adenovirus 41; 14, Hepatitisvirus-A; 15, Sapovirus].


Fig. 4. Sensitivity tests of five specific RT-PCR primer pairs.

Lane M, 100 bp Ladder marker; lane number, diluted rates from 1 ng/μL; N, negative control; dot, selective primer sets.


Nested PCR 봽씪씠癒 꽑諛

RT-PCR 봽씪씠癒 議고빀 #10 (F30/R20)쓽 궛臾쇱쓣 二쇳삎쑝 濡 nested PCR 利앺룺씠 媛뒫븳 2媛 봽씪씠癒 議고빀[#10-1 (F40/R30; 441 bp) 諛 #10-2 (F50/R20; 410 bp)]씠 꽕怨꾨릺뿀떎. 寃異 誘쇨컧룄 遺꾩꽍 寃곌낵, 紐⑤몢 10 ag 닔以씤 10-8源뚯 룞씪븳 寃異 誘쇨컧룄媛 遺꾩꽍릺뿀쑝硫, 씠 以 李멸퀬 諛붿씠윭뒪뿉 鍮 듅씠쟻 諛섏쓳씠 寃젙맂 #10-2 (F50/R20; 410 bp)瑜 理쒖쥌 꽑諛쒗븯떎(Fig. 5).

Fig. 5. Selection of nested PCR primer pair for the detecting HuAiV-A1.

Lane M, 100 bp Ladder marker; lane number, diluted rates from 1 ng/μL; N, negative control PN, PCR-negative control; dot, selective primer sets


湲곗〈 떆뿕踰 鍮꾧탳

湲곗〈 蹂닿퀬맂 HuAiV-A1 吏꾨떒쓣 쐞븳 RT-PCR 삉뒗 RT-nested PCR 10媛쒕 긽(Table 2)쑝濡 寃異 誘쇨컧룄瑜 遺꾩꽍븳 寃곌낵, RT-PCR뿉꽌뒗 Yip et al. (2014)Pham (2011)씠 蹂닿퀬븳 諛⑸쾿뿉꽌뒗 빟 100 ag (10-7)쓽 寃異 誘쇨컧룄媛 굹궗쑝硫, 굹癒몄 諛⑸쾿뱾 빟 100 fg (10-4)遺꽣 1 fg (10-6) 닔以쑝濡 굹궗떎. 洹몃윭굹 湲곗〈 諛⑸쾿 媛곴컖 227遺 諛 193遺꾩쓽 諛섏쓳 떆媛꾩씠 냼슂릺뿀쑝굹, 씠踰덉뿉 媛쒕컻븳 諛⑸쾿 175遺꾩쑝濡 湲곗〈 諛섏쓳 떆媛꾩쓣 빟 18~52遺 떒異뺤떆耳곕떎. 삉븳 RT-nested PCR뿉꽌 理쒓퀬쓽 寃異 誘쇨컧룄媛 굹궃 Saikruang et al. (2014) 諛 Alcalá et al. (2010)媛 蹂닿퀬븳 湲곗〈 諛⑸쾿씠 媛곴컖 254遺 諛 257遺꾩씤뜲 諛섑빐, 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 nested PCR RT-PCR쓣 룷븿 諛 以묎컙 쟾湲곗쁺룞 諛섏쓳 떆媛꾩쓣 젣쇅븯硫 珥 290遺꾩쑝濡 湲곗〈 RT-nested PCR 諛섏쓳 떆媛꾩뿉 鍮꾪빐 빟 33~36遺 젙룄 諛섏쓳 떆媛꾩씠 湲몄뿀떎. 洹몃윭굹 湲곗〈쓽 몢 諛⑸쾿 빟 100 ag 닔以쓽 寃異 誘쇨컧룄媛 굹궃 諛섎㈃, 蹂 뿰援ъ뿉꽌 媛쒕컻븳 諛⑸쾿 10 ag쓽 寃異 誘쇨컧룄瑜 굹궡 湲곗〈뿉 鍮꾪빐 빟 10諛 醫뗭 誘쇨컧룄媛 굹궗떎. 洹몃윭굹 諛섏쓳뿉 鍮 듅씠쟻 諛대뱶룄 씪遺媛 삎꽦릺뿀떎. 븳렪, 떎瑜 RT-nested PCR 諛⑸쾿뱾쓽 寃異 誘쇨컧룄뒗 빟 1~10 fg 닔以쑝濡 굹굹 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 RT-nested PCR 諛⑸쾿뿉 鍮꾪빐 빟 10~1,000諛 궙 誘쇨컧룄瑜 굹깉떎(Fig. 5).

Comparison of reaction time and sensitivity between this study and previously reported RT-nested PCR primer sets for the detection of HuAiV-A1

Primer set Reaction time (min) Sensitivity Remark
This study RT-PCR 175 100 atto gram
Nested PCR* 290 10 atto gram

Ref. primer set #01 RT-PCR 227 100 atto gram

Ref. primer set #02 RT-PCR 155 100 femto gram
219 1 femto gram Non-specific reaction

Ref. primer set #03 RT-PCR 142 10 femto gram
Nested PCR* 214 10 femto gram Non-specific reaction

Ref. primer set #04 RT-PCR 142 10 femto gram
Nested PCR* 214 1 femto gram Non-specific reaction

Ref. primer set #05 RT-PCR 203 1 femto gram

Ref. primer set #06 RT-PCR 157 1 femto gram
Nested PCR* 254 100 atto gram Non-specific reaction

Ref. primer set #07 RT-PCR 193 100 atto gram Non-specific reaction

Ref. primer set #08 RT-PCR 160 1 femto gram

Ref. primer set #09 RT-PCR 193 1 femto gram Non-specific reaction

Ref. primer set #10 RT-PCR 160 1 femto gram Non-specific reaction
Nested PCR* 257 100 atto gram Non-specific reaction

怨 李

蹂 뿰援ъ뿉꽌뒗 HuAiV-A1쓽 蹂대떎 슚怨쇱쟻씤 吏꾨떒슜 RT-nested PCR 봽씪씠癒 議고빀쓣 媛쒕컻븯怨좎옄 븯쑝硫 쁽옱 HuAiV-A1쓣 鍮꾨’븳 닔씤꽦 諛붿씠윭뒪 吏꾨떒쓣 쐞븳 湲곗〈쓽 諛⑸쾿쑝濡쒕뒗 ELISA 벑쓣 씠슜븳 슚냼硫댁뿭痢≪젙踰뺤씠 엳뿀쑝굹, PCR 寃궗踰뺤뿉 鍮꾪빐 빟 1,000諛 닔以쓽 궙 寃異 誘쇨컧룄, 궙 듅씠꽦, 뙋룆 삤瑜 벑씠 굹궇 닔 엳쑝硫, 듅엳 솚寃쎄낵 媛숈씠 蹂묒썝泥댁뿉 븳 삤뿼룄 諛룄媛 긽쟻쑝濡 궙 寃쎌슦 寃異 誘쇨컧룄媛 吏꾨떒 湲곗닠뿉 以묒슂븳 泥숇룄媛 맖뿉 뵲씪 理쒓렐뿉뒗 듅씠쟻 빑궛쓣 利앺룺븯뒗 遺꾩옄 吏꾨떒 湲곗닠씠 留롮씠 솢슜릺怨 엳떎(Cho, 2018). 뵲씪꽌 솚寃쎈 벑 援媛湲곌뿉꽌뒗 C-PCR, real-time qPCR, LAMP 벑 遺꾩옄湲곕컲쓽 吏꾨떒 湲곗닠 以묒뿉꽌룄 븞젙꽦, 솢슜꽦 벑쓣 怨좊젮븯뿬 寃궗踰뺤뿉 鍮꾪빐 C-PCR쓣 湲곕컲쑝濡 븳 몴以寃궗 湲곗닠쓣 二쇰줈 솢슜븯怨 엳떎(NIER, 2016).

븳렪, 湲곗〈뿉 蹂닿퀬맂 HuAiV-A1쓽 吏꾨떒슜 C-PCR 봽씪씠癒 議고빀뱾 솚寃 벑 鍮 엫긽 떆猷뚯뿉꽌뒗 3CD gene쓣 엫긽뿉꽌뒗 二쇰줈 VP3-VP1 gene쓣 긽쑝濡 븯怨 엳떎(Table 1). 洹몃윭굹 듅젙 떆猷뚯뿉꽌 諛붿씠윭뒪 듅씠쟻 빑궛쓣 吏꾨떒븯뒗뜲 엳뼱 엫긽怨 鍮 엫긽뿉 뵲瑜 떆猷뚯쓽 援щ텇 겙 쓽誘몃뒗 뾾뼱 蹂댁씤떎. 삤엳젮 理쒓렐 젙遺쓽 踰붾泥 닔씤꽦 떇뭹留ㅺ컻 諛붿씠윭뒪 삊쓽泥댁뿉꽌뒗 Norovirus 寃궗踰 넻빀씠 끉쓽릺怨 엳뒗 벑 媛 떆猷 蹂꾨줈 諛붿씠윭뒪쓽 吏뿭 蹂 諛쒖깮쁽솴, 嫄곕룞 듅꽦 벑쓽 떎뼇븳 뿰援щ 븯湲 쐞빐꽌뒗 뼢썑 엫긽, 솚寃, 빐뼇닔궛, 냽異뺤궛 벑뿉꽌 諛붿씠윭뒪 寃궗 떆 쟾 泥섎━ 諛⑸쾿쓣 젣쇅븯怨좊뒗 蹂묒썝泥 遺꾩꽍踰뺤쓽 넻빀 異붿쭊씠 끉쓽릺怨 엳뒗 떎젙씠떎. 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 RT-PCR 봽씪씠癒 議고빀 쁽옱源뚯 蹂닿퀬릺怨 엳뒗 RT-PCR 諛⑸쾿 以 理쒓퀬 닔以씤 100 atogram(理쒖큹 빀꽦맂 냽룄瑜 1 ng/μL쑝濡 븳 寃껋쓣 湲곗쑝濡 10-7) 닔以쓽 寃異 誘쇨컧룄媛 굹궗쑝硫, 룞湲 닔以쓣 굹궦 寃궗踰뺤뿉 鍮꾪빐 寃궗 떆媛꾩쓣 룊洹 빟 35遺 떒異뺤떆耳곕떎. 뵲씪꽌 蹂 寃궗踰뺤 엫긽, 떇뭹, 빐뼇닔궛 벑쓽 떆猷뚯뿉꽌 HuAiV-A1 吏꾨떒뿉 쟻빀븷 寃껋쑝濡 궗猷뚮맂떎. 븳렪, nested PCR쓽 寃쎌슦 湲곗〈쓽 떆媛(쟾湲곗쁺룞 젣쇅 빟 4떆媛 15遺) 蹂대떎 珥 寃궗 떆媛꾩씠 빟 35遺 湲몄뼱吏 4떆媛 50遺꾩씠 냼슂릺뿀떎. 洹몃윭굹 쓬슜 諛 鍮꾩쓬슜 吏븯닔 냽뾽슜닔 벑쓽 떆猷뚯뿉꽌뒗 寃異 誘쇨컧룄媛 以묒슂븯떎. 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 nested PCR 湲곗〈 諛⑸쾿 以 슦닔븳 寃異 誘쇨컧룄瑜 蹂댁쑝誘濡 솚寃 떆猷 벑뿉꽌쓽 솢슜씠 쟻빀븷 寃껋쑝濡 蹂댁씤떎. 洹몃윭굹 뼢썑 떎吏덉쟻 떆猷뚯뿉꽌쓽 솢슜꽦 벑 留롮 궗濡媛 寃利앸릺뼱빞 븷 寃껋쑝濡 깮媛곷맂떎. 蹂 뿰援ъ뿉꽌 媛쒕컻맂 RT-nested PCR 봽씪씠癒 議고빀 엫긽怨 鍮 엫긽뿉꽌 HuAiV-A1 吏꾨떒뿉 슚쑉쟻씤 湲곗닠濡 솢슜맆 寃껋쑝濡 湲곕맂떎.

ACKNOWLEDGEMENT

This work was supported by the Technology development Program (S2661713) funded by the Ministry of SMEs and Startups (MSS, Korea).

CONFLICT OF INTEREST

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

References
  1. Alcal찼 A, Vizzi E, Rodriguez-Diaz J, Zambrano JL, Betancourt W, Liprandi F. Molecular detection and characterization of Aichiviruses in sewage-polluted waters of Venezuela. Appl Environ Microbiol 2010. 76: 4113-4115.
    Pubmed KoreaMed CrossRef
  2. Cho KB. Development of nested PCR primer set for the specific and highly sensitive detection of human Parvovirus B19. Biomed. Sci. Lett 2018. 24: 390-397.
    CrossRef
  3. Guyader FSL, Le Saux JC, Ambert-Balay K, Krol J, Serais O, Parnaudeau S, Giraudon H, Delmas G, Pommepuy M, Pothier P, Atmar RL. Aichi virus, Norovirus, Astrovirus, Enterovirus, and Rotavirus involved in clinical cases from a french oyster-related gastroenteritis outbreak. J Clin Microbiol 2008. 46: 4011-4017.
    Pubmed KoreaMed CrossRef
  4. Khamrin P, Maneekarn N. Epidemiology of human and animal kobuviruses. VirusDis 2014. 25: 195-200.
    Pubmed KoreaMed CrossRef
  5. Khamrin P, Okame M, Thongprachum A, Nantachit N, Nishimura S, Okitsu S, Maneekarn N, Ushijima H. A single-tube multiplexPCR for rapid detection in feces of10 viruses causing diarrhea. J Virol Methods 2011. 173: 390-393.
    Pubmed CrossRef
  6. Lodder WJ, Rutjes SA, Takumi K, de Roda Husman AM. Aichi virus in sewage and surface water, the Netherlands. Emerg Infect Dis 2013. 19: 1222-1230.
    Pubmed KoreaMed CrossRef
  7. NIER. Development and verification of genetically diagnostic method for the detection of non-regulated viruses from water environment (I). Incheon, Korea. 2016.
  8. Oh DY, Silva PA, Hauroeder B, Diedrich S, Cardoso DDP, SchreierE. Molecular characterization of the first Aichi viruses isolated in Europe and in South America. Arch Virol 2006. 151: 1199-1206.
    Pubmed CrossRef
  9. Pham NT. Study on Aichi virus, parechovirus, and bocavirus detec-ted in children with acute gastroenteritis in Japan, Bangladesh, Thailand, Vietnam, and Sri Lanka. Tokyo University. Masters dissertation.
  10. Reuter G, Boldizsar A, Papp G, Pankovics P. Detection of Aichi virus shedding in a child with enteric and extraintestinal symptoms in Hungary. Arch Virol 2009. 154: 1529-1532.
    Pubmed CrossRef
  11. Saikruang W, Khamrin P, Suantai B, Ushijima H, Maneekarn N. Molecular detection and characterization of Aichivirus A in adult patients with diarrhea in Thailand. J Med Virol 2014. 86: 983-987.
    Pubmed CrossRef
  12. Santos N, Mendes GS, Silva RC, Pena GA, Rojas M, Amorim AR, Lima DP. Salivirus and aichivirus A infections in children with gastroenteritis in Brazil. Clin Microbiol Infect 2015. 21: 799.e3.
    Pubmed CrossRef
  13. Yamashita T, Adachi H, Hirose E, Nakamura N, Ito M, Yasui Y, Kobayashi S, Minagawa H. Molecular detection and nucleotidesequence analysis of a new Aichi virus closely related to canine kobuvirus in sewage samples. J Med Microbiol 2014. 63: 715-720.
    Pubmed CrossRef
  14. Yamashita T, Ito M, Kabashima Y, Tsuzuki H, Fujiura A, Sakae K. Isolation and characterization of a new species of kobuvirus associated with cattle. J Gen Virol 2003. 84: 3069-3077.
    Pubmed CrossRef
  15. Yamashita T, Sugiyama M, Tsuzuki H, Sakae K, Suzuki Y, Miyazaki Y. Application of a reverse transcription-PCR for identification and differentiation of Aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans. J Clin Microbiol 2000. 38: 2955-2961.
    Pubmed KoreaMed CrossRef
  16. Yip CCY, Lo KL, Que TL, Lee RA, Chan KH, Yuen KY, Woo PCY, Lau SKP. Epidemiology of human parechovirus, Aichi virus and salivirus in fecal samples from hospitalized children with gastroenteritis in Hong Kong. Virol J 2014. 11: 182.
    Pubmed KoreaMed CrossRef