Search for


TEXT SIZE

search for



CrossRef (0)
Development of Reverse Transcriptase Polymerase Chain Reaction Primer Sets and Standard Positive Control Capable of Verifying False Positive for the Detection of Severe acute respiratory syndrome coronavirus 2
Biomed Sci Letters 2021;27:283-290
Published online December 31, 2021;  https://doi.org/10.15616/BSL.2021.27.4.283
© 2021 The Korean Society For Biomedical Laboratory Sciences.

Kyu Bong Cho†,*

Department of Biomedical Laboratory Science, Shinhan University, Uijeongbu 11644, Korea
Correspondence to: *Professor.
Corresponding author: Kyu Bong Cho. Department of Biomedical Laboratory Science, Shinhan University, 95, Hoam-ro, Uijeongbu-si, Gyeonggi-do 11644, Korea.
Tel: +82-31-870-3712, Fax: +82-31-870-3719, e-mail: kbcho@shinhan.ac.kr
Received October 21, 2021; Revised November 22, 2021; Accepted November 22, 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
Severe acute respiratory syndrome coronavirus (SARS-CoV2) is a major coronavirus that infects humans with human Coronavirus (HuCoV)-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle east respiratory syndrome coronavirus (MERS-CoV). SARS-CoV2 is currently a global pandemic pathogen. In this study, we developed conventional RT-PCR based diagnostic system for the detection of SARS-CoV2 which is relatively inexpensive but has high stability and a wide range of users. Three conventional RT-PCR primer sets capable of forming specific band sizes by targeting the ORF1ab [232 nucleotide (nt)], E (200 nt) and N (288 nt) genes of SARS-CoV2 were developed, respectively, and it were confirmed to be about 10~100 times higher detection sensitivity than the previously reported methods. In addition, a standard positive control that can generate specific amplicons by reacting with the developed RT-PCR primers and verify the false-positiv from contamination of the laboratory was produced. Therefore, the diagnostic system that uses the RT-PCR method is expected to be used to detect SARS-CoV2.
Keywords : Conventional RT-PCR, SARS-CoV2, Severe acute respiratory syndrome coronavirus, Standard positive control
꽌 濡

以묒쬆湲됱꽦샇씉湲곗쬆썑援 肄붾줈굹諛붿씠윭뒪2(Severe acute respiratory syndrome coronavirus 2; SARS-CoV2)뒗 2019뀈 궗엺뿉寃 諛쒖깮씠 蹂닿퀬맂 썑, 꽭怨꾨낫嫄닿린援ъ뿉꽌 2020뀈 1썡 援젣쟻 怨듭쨷蹂닿굔 鍮꾩긽궗깭瑜 꽑뼵븯쑝硫 3썡뿉뒗 꽭怨꾩쟻 쑀뻾쓣 쓽誘명븯뒗 뙩뜲誘뱀씠 꽑뼵릺뼱 쁽옱源뚯룄 쟾 꽭怨꾩쟻쑝濡 臾몄젣媛 릺怨 엳떎(Gorbalenya et al., 2020; van Doremalen et al., 2020). SARS-CoV2뒗 뿉뼱濡쒖「, 샇씉湲 鍮꾨쭚 벑쓣 넻빐 媛먯뿼맆 닔 엳怨, 湲곌吏 룓뿉꽌 利앹떇븯硫, 怨좎뿴, 留덈Ⅸ湲곗묠, 샇씉怨ㅻ, 룓졃, 떖遺쟾 벑쓽 利앹긽 삉뒗 吏덊솚쓣 쑀諛쒗븳떎(Andersen et al., 2020; Zhou et al., 2020). SARS-CoV2 吏꾨떒 빆썝-빆泥 諛섏쓳쓣 씠슜븳 諛⑸쾿怨 듅젙 빑궛 떒렪쓣 利앺룺븯뒗 以묓빀슚냼뿰뇙諛섏쓳(polymerase chain reaction; PCR) 湲곕컲쓽 諛⑸쾿씠 샎슜릺怨 엳쑝굹(Carroll and McNamara, 2021; Corman et al., 2020; Domenico et al., 2021), 슦닔븳 寃異 誘쇨컧룄瑜 諛뷀깢쑝濡 븯뒗 PCR 湲곕컲쓽 諛⑸쾿씠 二쇱슂 湲곗닠濡 솢슜릺怨 엳쑝硫 씠 以묒뿉꽌룄 떎떆媛(real-time) 뿭쟾궗 젙웾 PCR [reverse transcription quantitative PCR; RT-qPCR]씠 二쇰줈 솢슜릺怨 엳떎(Corman et al., 2020; Carroll and McNamara, 2021). 洹몃윭굹 RT-qPCR뿉 룷븿릺뒗 봽濡쒕툕 떆빟 씪諛 RT-PCR뿉 鍮꾪빐 긽쟻쑝濡 怨좉濡 媛쒕컻룄긽援 벑뿉꽌쓽 吏냽쟻 솢슜뿉 븳怨꾧 엳怨, 삉븳 뿼湲곗꽌뿴쓣 諛뷀깢쑝濡 븳 쑀쟾삎 遺꾩꽍뿉 젣븳씠 엳뼱 紐⑤땲꽣留 씠 썑쓽 뿰援ъ뿉룄 븳怨꾧 엳떎(Lee and Cho, 2019). 諛섎㈃ 씪諛 RT-PCR 삤옖 뿰援щ줈 븞젙꽦 寃利 諛 궗슜옄쓽 踰붿쐞媛 꼻怨, 媛濡 媛쒕컻룄긽援 벑뿉꽌룄 솢슜맆 닔 엳쑝硫, 뿼湲곗꽌뿴 遺꾩꽍씠 媛뒫븯뿬 쑀쟾삎 遺꾩꽍 벑 썑냽 뿰援ъ쓽 媛뒫꽦씠 넂 벑쓽 吏꾨떒 湲곗닠濡쒖쓽 옣젏쓣 媛吏怨 엳떎(Yamashita et al., 2003; Reuter et al., 2009; Santos et al., 2015; Lee and Cho, 2019). SARS-CoV2뒗 떒씪 꽑삎 RNA 遺꾩젅쓽 빑궛 以 二쇰줈 Orf1ab (RdRp), N 諛 E gene쓣 긽쑝濡 PCR 吏꾨떒 湲곗닠씠 援ъ텞릺怨 엳떎(Corman et al., 2020). 洹몃윭굹 SARS-CoV2 吏꾨떒쓣 쐞빐 蹂닿퀬맂 紐뉖챺 諛⑸쾿뱾 泥댁쟻쑝濡 븳 쑀쟾옄瑜 긽쑝濡 븯怨 엳쑝硫 媛 諛⑸쾿 媛꾩쓽 鍮꾧탳 뿰援ш 誘명씉븯떎. 븳렪, PCR 湲곕컲쓽 吏꾨떒湲곕쾿 寃곌낵쓽 솗떎꽦쓣 쐞빐 떎뿕 寃곌낵뿉 빆긽 솢꽦 諛 鍮꾪솢꽦쓣 媛吏뒗 뼇꽦 諛 쓬꽦 議곕Ъ吏덉씠 슂援щ릺硫, 씠 以 뼇꽦議곕Ъ吏덉 蹂묒썝泥 寃궗쓽 젙솗꽦, 젙諛꽦 諛 옱쁽꽦뿉 븳 蹂댁옣쓣 쐞빐 諛섎뱶떆 븘슂븯떎(Jung et al., 2018). 洹몃윭굹 蹂묒썝泥댁뿉 媛먯뿼맂 떆猷 삉뒗 빑궛쓣 솢슜븯湲곗뿉뒗 寃궗옄쓽 媛먯뿼 쐞뿕꽦 벑쓽 臾몄젣媛 엳怨, 蹂닿퀬맂 뿼湲곗꽌뿴 씪遺瑜 빀꽦븯뿬 plasmid 삎깭濡 궗슜븯뒗 寃껋 뼇꽦議곌뎔쑝濡쒕꽣 삤뿼쑝濡 씤븳 쐞뼇꽦 諛섏쓳쓣 寃利앺븷 닔 뾾떎뒗 븳怨꾩젏씠 蹂닿퀬맂 諛 엳떎(Lee, 2013; Cho, 2018). 뵲씪꽌 蹂 뿰援ъ뿉꽌뒗 씪諛 RT-PCR 湲곕컲쑝濡 3醫낅쪟쓽 쑀쟾옄뿉 빐 SARS-CoV2瑜 吏꾨떒븷 닔 엳뒗 봽씪씠癒 議고빀쓣 媛쒕컻븯뿬 湲곗〈 蹂닿퀬맂 諛⑸쾿뱾怨 鍮꾧탳븯쑝硫, 媛쒕컻븳 봽씪씠癒 議고빀뱾쓽 듅씠寃고빀 諛 쐞뼇꽦 諛섏쓳쓣 寃젙븷 닔 엳뒗 몴以뼇꽦議곌뎔쓣 媛쒕컻븯떎.

옱猷 諛 諛⑸쾿

빑궛 닔吏 諛 봽씪씠癒 꽕怨

SARS-CoV2[誘멸뎅援由쎌깮臾쇱젙蹂댁꽱꽣(National center for biotechnology information (NCBI) accession number NC_045512.2 湲곗 Orf1ab (15,347~15,614), E (26,246~26,471) 諛 N (28,622~28,917)], SARS [NC_004718.3 湲곗 Orf1ab (15,277~15,544), E (26,117~26,347), N (28,471~28,766)] 諛 Middle east respiratory syndrome coronavirus (MERS-CoV) NC_019843.3 湲곗 Orf1ab (15,301~15,600), E (27,590~ 27,838), N (28,585~28,836)]瑜 긽쑝濡 븯怨, vector뿉 젒빀릺뒗 遺쐞濡 젣븳슚냼 NruI (TCGCGA) 諛 PstI (CTGCAG)쑝濡 븯뿬 (二)Marcrogen (Seoul, Korea)뿉꽌 빑궛쓣 빀꽦븯떎. 삉븳 human coronavirus (HCoV)-229E 샇씉湲 븘뜲끂諛붿씠윭뒪(respiratory Adenovirus; R-AdV)뒗 vircell (Granada, Spain)쓣 넻빐 RNA瑜 닔吏묓븳 썑 ReverTra Ace-α-TM (Toyobo, Osaka, Japan)쓣 궗슜븯뿬 cDNA濡 빀꽦븯떎. 븳렪, 봽씪씠癒몄쓽 꽕怨꾨 쐞븯뿬 NCBI뿉꽌 SARS-CoV2 (NC_045512.2) 諛 궗엺뿉寃 媛먯뿼릺뒗 洹 諛뽰쓽 肄붾줈굹諛붿씠윭뒪 6醫[SARS-CoV (NC_004718.3), MERS-CoV (NC_019843.3), HCoV-OC43 (NC_006213.1), HCoV-229E (NC_002645.1), HCoV-NL63 (NC_0065831.2) 諛 HCoV-HKU1 (NC_006577.2)] 뿼湲곗꽌뿴쓣 닔吏묓븯떎. Primer 3 ver.0.4.0.쓣 씠슜븯뿬 뵿궧븳 봽씪씠癒몃 BioEdit version 7.2.6 (Hall, 1999) software package뿉꽌 닔吏묓븳 뿼湲곗꽌뿴뱾怨 븿猿 떎以 뿼湲곗꽌뿴 젙젹븯뿬 듅씠꽦쓣 寃利앺븯怨, Oligo Calculator version 3.27뿉꽌 옞옱쟻 뿤뼱 援ъ“ 삎꽦 벑 씠濡좎쟻 臾몄젣젏쓣 寃넗 썑 媛 쑀쟾옄 蹂 썑蹂 봽씪씠癒몃 꽑젙븯떎.

봽씪씠癒 꽑諛 諛 鍮꾧탳

PCR 利앺룺씠 媛뒫븳 봽씪씠癒 議고빀쓽 援ъ꽦, SARS-CoV2뿉 듅씠쟻 諛섏쓳, 李멸퀬 諛붿씠윭뒪 4醫 빑궛(SARS-CoV, MERS-CoV, HuCoV-229E 諛 R-AdV)뿉 鍮꾪듅씠쟻 諛섏쓳 諛 寃異 誘쇨컧룄 떆뿕쓣 씠슜븳 봽씪씠癒 議고빀 꽑諛 怨쇱젙, PCR 議곗꽦 諛 議곌굔 湲곗〈 蹂닿퀬뻽뜕 Lee and Cho (2019) 룞씪븯寃 닔뻾븯떎. 듅씠쟻 諛섏쓳뿉 솢슜븳 媛 빑궛쓽 냽룄뒗 紐⑤몢 1 pg/μL씠뿀쑝硫, 鍮 듅씠쟻 諛섏쓳뿉 궗슜맂 빑궛쓽 냽룄뒗 SARS-CoV MERS-CoV뒗1 pg/μL, R-AdV뒗 100 copies濡 븯떎. 삉븳 씤쐞쟻 媛먯뿼쓣 넻븳 寃異 誘쇨컧룄瑜 寃젙븯湲 쐞븯뿬 嫄닿컯븳 궗엺쓽 샇씉湲 떆猷뚮 梨꾩랬븯怨, GeneAll® RibospinTM vRD (GeneAll, Korea)濡 珥 RNA瑜 異붿텧 썑 ReverTra Ace-α-TM (Toyobo)濡 cDNA 빀꽦븯쑝硫, 빀꽦븳 떆猷 total cDNA瑜 슜留ㅻ줈 븯뿬 뒠釉 궡뿉꽌 SARS-CoV2 빑궛쓣 냽룄 蹂꾨줈 씤쐞쟻 媛먯뿼떆耳곕떎. 븳렪, 湲곗〈 蹂닿퀬맂 SARS-CoV2 RT-PCR 諛⑸쾿 以 蹂 뿰援ъ뿉꽌 빀꽦븳 빑궛뿉 寃고빀븷 닔 엳뒗 4媛 봽씪씠癒(Corman et al., 2020; Mollaei et al., 2020)瑜 긽쑝濡 븯뿬 鍮꾪듅씠쟻 諛섏쓳 諛 寃異 誘쇨컧룄 벑쓣 鍮꾧탳븯떎.

몴以뼇꽦議곌뎔 꽕怨, 젣옉 諛 룊媛

젣옉 諛 빀꽦븳 뼇꽦 빑궛쓣 諛뷀깢쑝濡 EZchangeTM site-directed mutagenesis kit (Enzynomics, Korea)瑜 씠슜븯뿬 봽濡쒗넗肄쒖뿉 뵲씪 뿼湲곗꽌뿴쓣 궫엯븯쑝硫, ORF1ab, E 諛 N gene쓽 봽씪씠癒 꽌뿴씠 寃고빀븯뒗 궛臾 븞履쎌쑝濡 젣븳슚냼 EcoR V (GATATC)媛 룷븿맆 닔 엳룄濡 븯떎(Lee et al., 2015). 젣옉븳 빑궛쓣 1 pg/μL濡 븯뿬 꽑諛쒗븳 3醫낅쪟쓽 봽씪씠癒 議고빀쑝濡 RT-PCR 븯쑝硫, 利앺룺궛臾쇱쓣 MEGA quick-spin Plus Fragment DNA Purification Kit (iNtRON, Daejeon, Korea)濡 젙젣 썑 sequencing쓣 넻빐 뿼湲곗꽌뿴 궫엯 뿬遺瑜 솗씤븯떎. 븳렪, 利앺룺 궛臾쇱 EcoR V (Enzynomics) 37℃뿉꽌 1 hr 諛섏쓳븯떎. 理쒖쥌 궛臾 5 μL瑜 6X loading dye (Enzynomics) 1 μL 꽎뼱 2% agarose gel뿉꽌 쟾湲곗쁺룞 썑 UV 븯뿉꽌 寃곌낵瑜 愿李고븯떎. 젣옉맂 뿼湲곗꽌뿴 뼢썑 몴以뼇꽦議곌뎔쓽 븞젙쟻 蹂댁〈 諛 솢슜쓣 쐞븯뿬 겢濡쒕떇븯떎.

寃곌낵 諛 怨좎같

봽씪씠癒 꽕怨 諛 PCR 議고빀 援ъ꽦

SARS-CoV2 吏꾨떒쓣 쐞븯뿬 3媛 쑀쟾옄濡쒕꽣 媛곴컖 寃넗븳 寃곌낵, ORF1ab 쑀쟾옄 7媛(젙諛⑺뼢 3媛 諛 뿭諛⑺뼢 4媛), E 쑀쟾옄 6媛(젙諛⑺뼢 2媛 諛 뿭諛⑺뼢 4媛), N 쑀쟾옄 6媛(젙諛⑺뼢 2媛 諛 뿭諛⑺뼢 4媛)媛 썑蹂 봽씪씠癒몃줈 꽑깮릺뿀떎(Fig. 1). 삉븳 媛곴컖쓽 쑀쟾옄 蹂 PCR 議고빀 닔뒗 ORF1ab 쑀쟾옄 11媛, E 쑀쟾옄 7媛 諛 N 쑀쟾옄 12媛쒖떎.

Fig. 1. Map of specific candidate RT-PCR primers capable of diagnosing SARS-CoV2 ORF1ab, E and N genes, respectively.

봽씪씠癒 꽑諛 諛 鍮꾧탳

SARS-CoV2쓽 ORF1ab 쑀쟾옄 遺遺꾩쓣 寃異쒗븷 닔 엳뒗 RT-PCR 봽씪씠癒 11媛 議고빀 以 諛섏쓳쓽 꽭湲곗 궛臾쇱쓽 겕湲곗뿉 뵲씪 議고빀 ORF1ab_#06쓣 꽑諛쒗븯떎. RT-PCR 봽씪씠癒 議고빀 ORF1ab_#06 李멸퀬諛붿씠윭뒪 빑궛뱾怨 諛섏쓳븯吏 븡븘 SARS-CoV2 듅씠쟻 利앺룺씠 異붿젙릺뿀쑝硫, plasmid 湲곕컲 寃異 誘쇨컧룄 10-7(= 100 ag/μL 닔以) 諛 씤쐞쟻 媛먯뿼뿉 뵲瑜 寃異 誘쇨컧룄 10-5 닔以(= 10 fg/μL)쑝濡 굹궓뿉 뵲씪 SARS-CoV 寃異쒖뿉 쟻빀븳 봽씪씠癒 議고빀쑝濡 솗씤릺뿀떎(Fig. 2A). E 쑀쟾옄 遺遺꾩쓣 寃異쒗븷 닔 엳뒗 RT-PCR 봽씪씠癒 7媛 議고빀 以 諛섏쓳쓽 꽭湲곌 媛옣 슦닔븯寃 굹궃 議고빀 E_#02瑜 꽑諛쒗븯떎. RT-PCR 봽씪씠癒 議고빀 E_#02뒗 李멸퀬諛붿씠윭뒪 빑궛뿉 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡븯쑝硫, plasmid 湲곕컲 寃異 誘쇨컧룄 諛 씤쐞쟻 媛먯뿼뿉 뵲瑜 寃異 誘쇨컧룄 紐⑤몢 10-6 닔以(= 1 fg/μL)쑝濡 굹궓뿉 뵲씪 SARS-CoV2 寃異쒖슜쑝濡 슦닔븳 봽씪씠癒 議고빀쑝濡 굹궗떎(Fig. 2B). N 쑀쟾옄 遺遺꾩쓣 寃異쒗븷 닔 엳뒗 RT-PCR 봽씪씠癒 12媛 議고빀 以 諛섏쓳쓽 꽭湲곗 궛臾쇱쓽 겕湲 以 媛옣 쟻빀븳 4媛 議고빀(N_#01, N_#02, N_#06 諛 N_#07)씠 꽑諛쒕릺뿀떎. 紐⑤뱺 議고빀뱾 李멸퀬諛붿씠윭뒪 빑궛뿉 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡븘 SARS-CoV留뚯쓣 듅씠쟻쑝濡 寃異쒗븷 닔 엳뒗 봽씪씠癒 議고빀쑝濡 異붿젙맖뿉 뵲씪 plasmid 湲곕컲 寃異 誘쇨컧룄瑜 鍮꾧탳븯뿬 긽쟻쑝濡 媛옣 슦닔븳 N_#02瑜 꽑諛쒗븯쑝硫, 씤쐞쟻 媛먯뿼뿉 뵲瑜 寃異 誘쇨컧룄媛 10-6 닔以(= 1 fg/μL)쑝濡 굹굹 SARS-CoV2 吏꾨떒뿉 쟻빀븳 봽씪씠癒 議고빀쑝濡 솗씤릺뿀떎(Fig. 2C). 븳렪, 湲곗〈 蹂닿퀬맂 SARS-CoV2 RT-PCR 諛⑸쾿 以 4媛쒖쓽 諛⑸쾿쓣 긽쑝濡 鍮꾧탳븳 寃곌낵, 3醫낅쪟쓽 諛⑸쾿 (Ref. #1, #3 諛 #4)뿉꽌뒗 빑궛뿉 諛섏쓳 諛 plasmid 湲곗 10-4~10-5 닔以(= 10~1 fg/μL)쓽 寃異 誘쇨컧룄媛 솗씤릺뿀쑝굹, ref. #1 SARS-CoV HuCoV-229E 빑궛뿉 諛섏쓳, ref. #3 SARS-CoV, MERS-CoV 諛 RAdV 빑궛뿉 諛섏쓳 諛 ref. #4뒗 SARS-CoV, MERS-CoV 諛 HuCoV-229E뿉 媛곴컖 鍮꾪듅씠쟻쑝濡 諛섏쓳븿뿉 뵲씪 SARS-CoV2留뚯쓣 듅씠쟻쑝濡 寃異쒗븷 닔 뾾뿀떎. 諛섎㈃ E 쑀쟾옄瑜 긽쑝濡 븯뒗 ref.#2뒗 李멸퀬諛붿씠윭뒪 빑궛뿉꽌 鍮꾪듅씠쟻 諛섏쓳씠 굹굹吏 븡븯쑝硫, plasmid 湲곗 寃異 誘쇨컧룄 10-5 닔以(= 10 fg/μL)쑝濡 굹궓뿉 뵲씪 蹂닿퀬 諛 寃넗븳 諛⑸쾿 以묒뿉꽌뒗 媛옣 슦닔븳 봽씪씠癒 議고빀쑝濡 룊媛릺뿀떎. 洹몃윭굹 蹂 뿰援ъ뿉꽌 媛쒕컻븳 諛⑸쾿씤 ORF1ab_#06뒗 10-7 (= 100 ag/μL), E_#02 諛 N_#02뒗 10-6 (= 1 fg/μL) 닔以쑝濡 굹궓뿉 뵲씪 湲곗〈 諛⑸쾿 鍮 寃異 誘쇨컧룄媛 빟 10~100諛 슦닔븯떎(Table 1).

Information of final selectived and reference RT-PCR primer sets for the detection of SARS-CoV2

Division Target gene Set # Primer information and PCR product size References Results
Name Sequence (5'→3') PCR product Length (nt) Presence of nonspecific reaction Sensitivity based on SARS-CoV2 plasmid
Development ORF1ab ORF1ab_#06 O_F10 AACGTGTTGTAGCTTGTCAC 232 This study × 10-7 (100 ag/μL)
O_R22 GACATACTTATCGGCAATTTTG
E E_#02 E_F05 CTCATTCGTTTCGGAAGAG 200 × 10-6 (1 fg/μL)
E_R75I TAGAAGAATTCAGAIIITTAACACGA
N N_#2 N_F10 GCTGGACTTCCCTATGGT 288 × 10-6 (1 fg/μL)
N_R20 GCCATTGCCAGCCATTCTA
Reference ORF1ab Ref.#1 RdRp_SARSr-F GTGARATGGTCATGTGTGGCGG 100 Corman et al., 2020 10-4 (100 fg/μL)
RdRp_SARSr-R CARATGTTAAASACACTATTAGCATA
E Ref.#2 E_Sarbeco_F ACAGGTACGTTAATAGTTAATAGCGT 146 × 10-6 (1 fg/μL)
E_Sarbeco_R ATATTGCAGCAGTACGCACACA
N Ref.#3 N_Sarbeco_F CACATTGGCACCCGCAATC 112 10-4 (100 fg/μL)
N_Sarbeco_R GAGGAACGAGAAGAGGCTTG
E Ref.#4 Forward GGAAGAGACAGGTACGTTAA 128 Mollaei et al., 2020 10-5 (10 fg/μL)
Reverse AAGGTTTTACAAGACTCACG


Fig. 2. Specific reaction and sensitivity test of candidate RT-PCR primer sets associated with three genes, respectively. Panel A, Specific reaction and sensitivity test of RT-PCR primer sets targeting ORF1ab gene. Panel B, E gene. Panel C, N gene. Lane M, 100 bp Ladder maker (Enzynomics, Korea); #01~#12, number of candidate RT-PCR primer sets each genes; N, negative control; P, positive control; SARS, SARS-CoV nucleic acid (1 pg/μL); MERS, MERS-CoV nucleic acid (1 pg/μL); 229E, HuCoV-229E nucleic acid (1 pg/μL); R-AdV, respiratory Adenovirus (100 copies).

몴以뼇꽦議곌뎔 젣옉 諛 룊媛

씠踰 뿰援ъ뿉꽌 媛쒕컻븳 RT-PCR 봽씪씠癒 3媛 議고빀씠 紐⑤몢 諛섏쓳븯硫댁꽌 쐞뼇꽦 諛섏쓳쓣 寃젙븷 닔 엳뒗 몴以뼇꽦議곌뎔쓣 젣옉븯떎. 뿼湲곗꽌뿴 珥 812 nucleotide (nt) 湲몄씠濡 ORF1ab, E 諛 N gene쓽 봽씪씠癒 꽌뿴씠 寃고빀 諛 諛섏쓳븷 닔 엳뒗 뿼湲곗꽌뿴 遺遺꾩씠 룷븿릺뿀떎. 媛 쑀쟾옄 蹂 봽씪씠癒 議고빀씠 諛섏쓳븯뿬 삎꽦븯뒗 利앺룺 궛臾[ORF1ab_#06 236 nt(蹂묒썝泥댁뿉꽌 利앺룺 떆 232 nt), E_#02 202 nt(蹂묒썝泥댁뿉꽌 利앺룺 떆 20 nt), N_#2 292 nt(蹂묒썝泥댁뿉꽌 利앺룺 떆 288 nt) 삎꽦]쓣 솗씤븯쑝硫, sequencing 寃곌낵 2~4 nt쓽 뿼湲곗꽌뿴씠 媛곴컖 궫엯릺뿀떎(Fig. 3A). PCR 利앺룺궛臾쇰뱾濡쒕꽣 젣븳슚냼 EcoRV (GATATC) 諛섏쓳 뿬遺瑜 솗씤븳 寃곌낵, ORF1ab_#06 (146+90 nt), E_#02 (132+70 nt) 諛 N_#2 (190+102 nt)濡 紐⑤몢 몢 媛쒖쓽 諛대뱶媛 굹궗떎(Fig. 3B). 씠뿉 뵲씪 蹂 뿰援ъ뿉꽌 媛쒕컻븳 SARS-CoV2 몴以뼇꽦議곌뎔쓣 吏꾨떒뿉 솢슜 떆, 쟾湲곗쁺룞 긽뿉꽌뒗 쑁븞쑝濡 援щ텇씠 뼱졄吏留, 利앺룺븳 궛臾쇱쓣 二쇳삎쑝濡 젣븳슚냼瑜 泥섎━븯硫 諛대뱶쓽 젅떒 쑀臾댁뿉 뵲씪 議곌뎔쑝濡 遺꽣 삤뿼쑝濡 씤븳 쐞뼇꽦쓣 援щ텇븷 닔 엳떎. 븳렪, PCR 湲곕컲쓽 遺꾩옄 吏꾨떒뿉꽌 궗슜븯뒗 뼇꽦議곌뎔 삁뒗 援궡 吏덈퀝愿由щ낯遺뿉꽌 젣옉븳 끂濡쒕컮씠윭뒪 뼇꽦議곌뎔, Virology Journal뿉 蹂닿퀬맂 Lee et al. (2011) 벑씠 蹂닿퀬릺怨 엳떎. 빐떦 蹂닿퀬뱾뿉꽌뒗 洹몃윭굹 뼇꽦議곌뎔쑝濡쒕꽣 떎뿕떎 삤뿼씠 씪뼱궇 寃쎌슦媛 엳쑝誘濡 씠瑜 寃젙븷 닔 엳뒗 옣移섍 怨좎븞릺뼱빞 븯硫, 빐떦 諛⑸쾿쑝濡쒕뒗 利앺룺 겕湲 蹂寃(Lee et al., 2011), 듅젙 뿼湲곗꽌뿴 궫엯(Lee et al., 2021) 벑쓽 諛⑸쾿씠 蹂닿퀬릺怨 엳떎. 씠踰 뿰援ъ뿉꽌뒗 Lee et al. (2021)怨 룞씪븳 諛⑸쾿씤 듅젙 뿼湲곗꽌뿴 궫엯쓣 꽑깮븯怨, 븯굹쓽 몴以뼇꽦議곌뎔쑝濡쒕꽣 SARS-CoV2 3媛 쑀쟾옄 寃異쒖씠 紐⑤몢 媛뒫븷 닔 엳룄濡 꽦뒫쓣 뼢긽븯떎. 삉븳 蹂 뿰援ъ뿉꽌 媛쒕컻븳 봽씪씠癒 議고빀 쇅뿉룄 Corman et al. (2020), Mollaei et al. (2020) 벑 봽씪씠癒 寃고빀 쐞移섍 씪移섑븯뒗 紐뉖챺쓽 諛⑸쾿怨 븿猿 솢슜맆 닔룄 엳떎. 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 몴以뼇꽦議곌뎔 쑀쟾옄 겢濡쒕떇쓣 넻빐 븞젙쟻쑝濡 蹂닿씠 媛뒫븯怨, 듅젙 냽룄濡 젣怨 諛 怨듦툒씠 맆 닔 엳뼱 蹂 뿰援ъ뿉꽌 媛쒕컻븳 봽씪씠癒 議고빀뱾怨 븿猿 SARS-CoV2 吏꾨떒뿉 쑀슜븯寃 솢슜맆 닔 엳쓣 寃껋쑝濡 湲곕맂떎.

Fig. 3. Information and evaluation of standard positive control developed in this study. Panel A, Sequence information of standard positive control for the detection of SARS-CoV2. Panel B, Restriction enzyme EcoR Ⅴ (GATATC) digested using the three RT-PCR products, respectively. Lane M, 100 bp DNA Ladder maker (Enzynomics); amplicon, RT-PCR product from standard positive control as a template; digestion, EcoR Ⅴ digestion of RT-PCR product.

媛쒕컻 봽씪씠癒 諛 몴以뼇꽦議곌뎔쓣 씠슜븳 떆뿕 怨쇱젙 諛 怨좎같

SARS-CoV2쓽 듅젙 빑궛쓣 利앺룺븯뒗 寃궗 뵆옯뤌쓣 궗슜븯뒗 寃쎌슦뒗 꽭怨꾨낫嫄닿린援 諛 쑀읇뿰빀 벑쓽 湲곌怨 Corman et al. (2020), Mollaei et al. (2020) 벑 떎닔쓽 뿰援 蹂닿퀬뿉꽌 ORF1ab (RdRp), E 諛 N 쑀쟾옄瑜 긽쑝濡 븯怨 엳떎. 룞씪븳 떆猷뚯뿉꽌 異붿텧맂 珥 빑궛쑝濡쒕꽣 떎뼇븳 醫낅쪟쓽 蹂묒썝泥 삉뒗 쑀쟾옄瑜 吏꾨떒븷 寃쎌슦, 寃궗 긽 빆紐⑹씠 留롮븘 吏덉닔濡 PCR 議곗꽦 諛 議곌굔씠 긽씠븷 寃쎌슦 寃궗옄 쁽옣뿉꽌쓽 솢슜꽦뿉 젣븳맆 닔 엳떎(Lee, 2013; Lee et al., 2015). 洹몃윭굹 湲곗〈쓽 뿰援 蹂닿퀬뿉꽌뒗 떎닔쓽 寃궗옄뿉 쓽빐 媛쒕컻맂 諛⑸쾿뱾濡 蹂닿퀬맂 諛⑸쾿留덈떎 PCR 議곗꽦臾쇨낵 議곌굔씠 긽씠뻽쑝硫, 룞씪븳 蹂닿퀬 궡뿉꽌룄 봽씪씠癒 諛 뼱땺留 Tm 媛믪씠 떖씪꽌 議곌굔씠 떎瑜 寃쎌슦룄 議댁옱븯떎. 洹몃윭굹 蹂 뿰援ъ뿉꽌뒗 媛쒕컻븳 3媛쒖쓽 諛⑸쾿뿉 븳 議곗꽦臾 諛 議곌굔씠 紐⑤몢 룞씪븯寃 媛쒕컻릺硫댁꽌 씠윭븳 臾몄젣젏뱾쓣 洹밸났븯怨좎옄 븯떎. RT-PCR 議곗꽦臾쇱 AccuPower® RT/PCR PreMix (Bioneer, Daejeon, Korea) dry type, 젙諛⑺뼢 諛 뿭諛⑺뼢 봽씪씠癒 媛곴컖 25 pmol 냽룄濡 1 μL뵫(珥 2 μL), 二쇳삎 빑궛 1 μL 諛 nucleic acid free water 17 μL濡 珥 20 μL媛 궗슜릺硫, 3媛 쑀쟾옄뒗 媛곴컖 떎瑜 봽씪씠癒멸 궗슜릺硫 洹 諛뽰쓽 議곗꽦臾쇰뱾쓽 냽룄 遺뵾뒗 룞씪븯떎. RT-PCR 議곌굔 뿭쟾궗(42℃, 60遺), 珥덇린 蹂꽦(95℃, 5遺), 35쉶 諛섎났[蹂꽦(95℃, 45珥), 寃고빀(55℃, 60珥) 諛 떊옣(72℃, 60珥)], 理쒖쥌 떊옣(72℃, 5遺)쑝濡 룞씪븯뿬 媛곸옄 떎瑜 뒠釉뚮 궗슜빐빞 븯吏留 븳 쓽 thermocycler뿉꽌 룞떆 諛섏쓳씠 媛뒫븯寃 븯뿬 寃궗옄 移쒗솕쟻(user friendly) 諛 쁽옣 솢슜꽦쓣 怨좊젮븯떎. 븳렪, 쐞뼇꽦쓣 寃젙븷 닔 엳뒗 몴以뼇꽦議곌뎔쓽 媛쒕컻濡 SARS-CoV2 吏꾨떒뿉 넂 젙솗꽦쓣 吏썝븷 닔 엳쓣 寃껋쑝濡 蹂댁씤떎. 媛쒕컻븳 몴以뼇꽦議곌뎔쑝濡쒕꽣 利앺룺씠 씪뼱굹硫 젣븳슚냼 EcoRV 諛섏쓳쑝濡 諛대뱶뙣꽩뿉 쓽빐 쐞뼇꽦 뙋젙씠 媛뒫븯떎. 洹몃윭굹 삉븳 媛쒕컻븳 몴以뼇꽦議곌뎔 DNA 삎깭씠誘濡 떆猷뚯뿉꽌 total RNA瑜 異붿텧븯뿬 떆뿕쓣 吏꾪뻾븯뿬 異붿젙 뼇꽦 諛대뱶媛 솗씤릺뿀떎硫, AccuPower® HotStart PCR PreMix (Bioneer) 벑 뿭쟾궗 슚냼媛 룷븿릺吏 븡 PCR kit瑜 궗슜븯뒗 옱 떆뿕쓣 넻빐 삤뿼 뿬遺瑜 솗씤븷 닔룄 엳떎. 떆猷뚯뿉꽌 total RNA 異붿텧 썑 RT-PCR 떆뿕 꽕怨 떆 媛쒕컻븳 몴以뼇꽦議곌뎔쓣 뼇꽦, nucleic acid free water瑜 쓬꽦議곌뎔쑝濡 꽕젙븯뿬 3媛 쑀쟾옄 긽쑝濡 紐⑤몢 씪諛 RT-PCR 寃궗瑜 닔뻾븯뿬 2% agarose gel뿉꽌 쟾湲곗쁺룞 諛 媛곴컖쓽 異붿젙 뼇꽦쓣 뙋젙 븳 썑, 異붿젙 뼇꽦 諛대뱶媛 議댁옱븷 寃쎌슦 쐞뼇꽦 뙋젙쓣 닔뻾븳떎. 쐞뼇꽦씠 븘땶 寃껋쑝濡 遺꾩꽍릺硫 異붿젙 뼇꽦 諛대뱶뿉 빐 sequencing븯뿬 뿼湲곗꽌뿴쓣 湲곗큹濡 NCBI BLAST 벑쑝濡 理쒖쥌 룞젙 諛 怨꾪넻닔 援ъ텞 벑怨 媛숈 쑀쟾삎쓣 遺꾩꽍븷 닔 엳떎. 洹몃윭굹 뼢썑 떎닔쓽 떆猷뚯뿉 븳 젏寃씠 븘슂븷 寃껋쑝濡 蹂댁씠硫, 쁽옣 紐⑤땲꽣留 寃궗 벑쓽 湲곌컙쓣 嫄곗퀜 뼢썑 샇씉湲 諛붿씠윭뒪 쑀愿 遺꾩빞쓽 吏꾨떒 諛 뿰援ъ뿉 솢슜꽦씠 湲곕맂떎.

ACKNOWLEDGEMENT

This work was supported by the Shihan University Research Fund, 2021.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

References
  1. Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. The proximal origin of SARS-CoV-2. Nature Med. 2020. 26: 450-452.
    Pubmed KoreaMed CrossRef
  2. Carroll A, McNamara E. Comparison and correlation of commercial SARS-CoV-2 real-time-PCR assays. Euro Surveill. 2021. 26: 2002079.
    Pubmed KoreaMed CrossRef
  3. 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
  4. Corman VM, Landt O, Kaiser MKaiser M et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020. 25: 2000045.
    Pubmed KoreaMed CrossRef
  5. Domenico MD, Rosa AD, Boccellino M. Detection of SARS-COV-2 proteins using an ELISA test. Diagnostics (Basel). 2021. 11: 698.
    Pubmed KoreaMed CrossRef
  6. 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.
  7. Jung S, Lee DY, Choi W, Kang C. Introduction of reference materials for water- and food-borne disease viruses. Public Health Weekly Rep. 2018. 9: 254-259.
  8. Gorbalenya AE, Baker SC, Baric RSBaric RS et al. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nature Microbiol. 2020. 5: 536-544.
    Pubmed KoreaMed CrossRef
  9. 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.
  10. 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 Lett. 2019. 25: 331-338.
    CrossRef
  11. Lee S, Bae KS, Lee JYLee JY et al. Development of molecular diagnostic system with high sensitivity for the detection of human Sapovirus from water environments. Biomed Sci Lett. 2021. 27: 35-43.
    CrossRef
  12. Lee S, Lee JY, Moon BYMoon BY et al. Development of a diagnostic system for the detection of the Cowpea mild mottle virus specific gene in quarantine. Microbiol Biotechnol Lett. 2015. 43: 296-299.
    CrossRef
  13. 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
  14. Mollaei HR, Afshar AA, Kalantar-Neyestanaki D, Fazlalipour M, Aflatoonian B. Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR. Iran J Microbiol. 2020. 12: 185-193.
    Pubmed KoreaMed CrossRef
  15. Reuter G, Boldizsar A, Papp G, Pankovics P. Detection of Aichivirus shedding in a child with enteric and extraintestinal symptoms in Hungary. Arch Virol. 2009. 154: 1529-1532.
    Pubmed CrossRef
  16. 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.e1-799.e3.
    Pubmed CrossRef
  17. van Doremalen N, Bushmaker T, Morris DHMorris DH et al. Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1. New England J Med. 2020. 382: 1564-1567.
    Pubmed KoreaMed CrossRef
  18. 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
  19. Zhou P, Yang XL, Wang XGWang XG et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020. 579: 270-273.
    Pubmed KoreaMed CrossRef