Search for


TEXT SIZE

search for



CrossRef (0)
Development of an Automatic PCR System Combined with Magnetic Bead-based Viral RNA Concentration and Extraction
Biomed Sci Letters 2023;29:363-370
Published online December 31, 2023;  https://doi.org/10.15616/BSL.2023.29.4.363
© 2023 The Korean Society For Biomedical Laboratory Sciences.

MinJi Choi1,*, Won Chang Cho2,**, Seung Wook Chung2,**, Daehong Kim3,*** and Il-Hoon Cho1,4,†,***

1Department of Senior Healthcare, Eulji University, Seongnam 13135, Korea
2Department of Research Institute of Bitvalue, Guro Digital G Valley Complex, Seoul 08390, Korea
3Department of Radiological Science, College of Health Science, Eulji University, Seongnam 13135, Korea
4Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Korea
Correspondence to: Il-Hoon Cho. Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Korea.
Tel: +82-31-740-7397, Fax: +82-31-740-7284, e-mail: ihcho@eulji.ac.kr
*Graduate student, **Researcher, ***Professor.
Received December 1, 2023; Revised December 7, 2023; Accepted December 8, 2023.
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 respiratory viral infections such as COVID-19 are highly contagious, so continuous management of airborne viruses is essential. In particular, indoor air monitoring is necessary because the risk of infection increases in poorly ventilated indoors. However, the current method of detecting airborne viruses requires a lot of time from sample collection to confirmation of results. In this study, we proposed a system that can monitor airborne viruses in real time to solve the deficiency of the present method. Air samples were collected in liquid form through a bio sampler, in which case the virus is present in low concentrations. To detect viruses from low-concentration samples, viral RNA was concentrated and extracted using silica-magnetic beads. RNA binds to silica under certain conditions, and by repeating this binding reaction, bulk samples collected from the air can be concentrated. After concentration and extraction, viral RNA is specifically detected through real-time qPCR (quantitative polymerase chain reaction). In addition, based on liquid handling technology, we have developed an automatic machine that automatically performs the entire testing process and can be easily used even by non-experts. To evaluate the system, we performed air sample collection and automated testing using bacteriophage MS2 as a model virus. As a result, the air-collected samples concentrated by 45 times then initial volume, and the detection sensitivity of PCR also confirmed a corresponding improvement.
Keywords : Aerosols, Quantitative real-time PCR, Point-of-care systems, Respiratory viruses, Automation
꽌 濡

COVID-19 궗깭 씠썑, 理쒓렐 샇씉湲 諛붿씠윭뒪 愿由ъ뿉 븳 愿떖怨 洹 以묒슂꽦씠 쟾 꽭怨꾩쟻쑝濡 몢릺怨 엳떎. 샇씉湲 諛붿씠윭뒪쓽 쟾뙆 寃쎈줈뒗 洹 諛⑸쾿뿉 뵲씪 젒珥 媛먯뿼, 鍮꾨쭚 媛먯뿼, 怨듦린 媛먯뿼쑝濡 援щ텇맂떎. 닕二 諛 媛먯뿼 媛쒖껜쓽 吏곸젒쟻씤 젒珥됱뿉 쓽빐 蹂묒썝泥닿 쟾뙆릺뒗 젒珥 媛먯뿼怨 떖由, 鍮꾨쭚 媛먯뿼 諛 怨듦린 媛먯뿼 鍮꾨쭚 삉뒗 뿉뼱濡쒖「뿉 쓽빐 쟾뙆媛 씠猷⑥뼱吏꾨떎. 鍮꾨쭚 媛먯뿼옄쓽 湲곗묠씠굹 옱梨꾧린瑜 넻빐 띁吏뒗 5 μm 씠긽 겕湲곗쓽 遺꾨퉬臾쇱쓣 쓽誘명븯硫, 빟 1 m源뚯留 遺꾩궗릺뼱 諛젒븳 긽깭뿉꽌쓽 쟾뿼 媛뒫꽦씠 넂떎. 뿉뼱濡쒖「 5 μm 씠븯쓽 옉 엯옄뱾씠硫, 씠瑜 넻빐 諛붿씠윭뒪媛 怨듦린 以묒뿉 쟾뙆맆 寃쎌슦 洹 踰붿쐞媛 긽쟻쑝濡 꼻湲 븣臾몄뿉 鍮꾨쭚 媛먯뿼뿉 鍮꾪빐 쟾뙆젰씠 留ㅼ슦 넂떎(Drossionos et al., 2021). COVID-19쓽 寃쎌슦뿉룄 궗깭 珥덈컲뿉뒗 諛붿씠윭뒪쓽 쟾뙆媛 鍮꾨쭚濡쒕쭔 씠猷⑥뼱吏뒗 寃껋쑝濡 뙋떒릺뿀쑝굹, 怨듦린 媛먯뿼쓽 媛뒫꽦씠 닔 李⑤ 젣湲곕맂 諛 엳떎(Blocken et al., 2021; Buonanno et al., 2020; Guo et al., 2020; Kutter et al., 2021). 씠 媛숈씠 쟾뙆젰씠 媛뺥븳 怨듦린 以 諛붿씠윭뒪瑜 愿由ы븯湲 쐞빐꽌뒗 吏뿭궗쉶 닔以쓽 떊냽븯怨 吏냽쟻씤 紐⑤땲꽣留 떆뒪뀥씠 븘슂븯떎. 쁽옱 怨듦린 以 깮臾쇳븰쟻 쑀빐臾쇱쭏쓣 寃궗븯뒗 諛⑸쾿쑝濡쒕뒗 쁽옣쓽 怨듦린瑜 룷吏묓븳 썑 빐떦 떆猷뚮 遺꾩꽍븯湲 쐞빐 떎뿕떎濡 슫諛섑븯뒗 諛⑹떇씠 쟻슜릺怨 엳떎. 씠윭븳 寃궗踰뺤 쁽옣쓽 怨듦린 吏덉쓣 떎떆媛꾩쑝濡 紐⑤땲꽣留 븯湲곗뿉 쟻젅븯吏 븡 諛⑹떇씠硫, 씠뒗 떊냽븳 議곗튂쓽 遺옱濡 씠뼱吏寃 맂떎(Puthussery et al., 2023). 삁瑜 뱾뼱, 怨듦린 以 遺쑀븯뒗 誘몄깮臾 삉뒗 諛붿씠윭뒪瑜 遺꾩꽍븯湲 쐞빐꽌뒗 怨듦린 떆猷 梨꾩랬湲(air sampler)뿉 븘꽣 삉뒗 諛곗뼇 諛곗瑜 옣李⑺븯뿬 씠뿉 怨듦린 떆猷뚮 룷吏묓븳 썑 諛곗뼇 怨쇱젙쓣 嫄곗퀜 寃곌낵瑜 솗씤븯뒗 寃껋씠 씪諛섏쟻씤 諛⑹떇씠떎(Pasquarella et al., 2008). 빐떦 諛⑸쾿 寃곌낵瑜 솗씤븯湲 쐞빐 듅젙 議곌굔뿉꽌 씪젙 떆媛 룞븞 寃異 긽쓣 諛곗뼇빐빞 븳떎뒗 떒젏씠 議댁옱븳떎. 삉븳, 븸긽쑝濡 怨듦린瑜 룷吏묓븯뿬 떆猷뚮 뼸뒗 寃쎌슦뿉뒗 룷吏묐맂 긽 誘몄깮臾 諛 諛붿씠윭뒪쓽 냽룄媛 留ㅼ슦 궙븘 寃異쒖쓽 誘쇨컧룄媛 븯맆 슦젮媛 엳떎(Breshears et al., 2022). 蹂 뿰援ъ뿉꽌뒗 씠윭븳 臾몄젣젏쓣 蹂댁셿븯뿬 怨듦린 以 샇씉湲 諛붿씠윭뒪瑜 떎떆媛꾩쑝濡 愿由ы븷 닔 엳뒗 떆뒪뀥쓣 젣떆븯怨좎옄 븯떎. 빐떦 떆뒪뀥 怨듦린 룷吏 썑 쁽옣뿉꽌 諛붾줈 떆猷뚯쓽 쟾泥섎━遺꽣 諛붿씠윭뒪瑜 듅씠쟻쑝濡 寃異쒗븯湲 쐞븳 PCR 寃궗源뚯 媛뒫븯룄濡 븯뒗 寃껋쓣 紐⑺몴濡 媛쒕컻릺뿀떎. 怨듦린 룷吏 떆猷뚯쓽 궙 냽룄瑜 洹밸났븯湲 쐞븯뿬 떎由ъ뭅-옄꽦鍮꾨뱶瑜 씠슜븳 怨듦린 룷吏 떆猷뚯쓽 냽異뺢낵 諛붿씠윭뒪쓽 빑궛 異붿텧 諛⑹떇쓣 룄엯븯쑝硫, 씠瑜 넻빐 諛붿씠윭뒪쓽 怨좉컧룄 깘吏媛 媛뒫븯떎. 理쒖쥌쟻쑝濡, 怨듦린 떆猷 룷吏 씠썑쓽 쟾 寃궗 怨듭젙 옄룞솕 옣鍮꾨 넻빐 궗슜옄쓽 빖뱾留곸쓣 理쒖냼솕븿쑝濡쒖뜥 쑀빐臾쇱쭏 끂異쒖뿉 븳 쐞뿕쓣 以꾩씠怨 寃궗 寃곌낵쓽 옱쁽꽦쓣 뼢긽떆궎뒗 寃껋쓣 紐⑺몴濡 븯떎.

옱猷 諛 諛⑸쾿

떎由ъ뭅-옄꽦鍮꾨뱶瑜 씠슜븳 RNA쓽 냽異 諛 異붿텧

긽 諛붿씠윭뒪쓽 RNA瑜 냽異 諛 異붿텧븯湲 쐞븯뿬 MagListoTM 5 M Viral DNA/RNA Extraction Kit (Bioneer, Korea)瑜 씠슜븯떎. 빐떦 extraction kit뒗 몴硫댁뿉 떎由ъ뭅媛 泥섎━맂 옄꽦鍮꾨뱶瑜 쟻슜븯뿬 빑궛쓣 젙젣븯뒗 諛⑹떇씠떎. 떎由ъ뭅-옄꽦鍮꾨뱶瑜 씠슜븳 빑궛쓽 異붿텧 諛⑹떇 씪諛섏쟻쑝濡 寃고빀, 꽭泥, 슜異쒖쓽 怨쇱젙쑝濡 닔뻾맂떎. 怨좊냽룄쓽 chaotropic salts 議댁옱 븯뿉 빑궛 떎由ъ뭅 몴硫댁뿉 寃고빀븯寃 릺硫, 씠썑 븣肄붿삱 湲곕컲쓽 꽭泥 怨쇱젙쓣 넻빐 빑궛 쇅쓽 떎떦瑜 諛 떒諛깆쭏怨 媛숈 鍮꾨컲쓳臾쇱쭏뱾씠 젣嫄곕맂떎(Sun et al., 2014). 理쒖쥌쟻쑝濡 궙 뿼(salts) 議곌굔뿉꽌 빑궛쓣 슜異쒖떆궗 닔 엳쑝硫, 씠 媛숈 諛⑹떇 由ы대뱶 빖뱾留곸쓣 넻빐 옄룞솕 옣鍮꾩뿉 蹂댄렪쟻쑝濡 쟻슜릺怨 엳떎(Adams et al., 2015). 蹂 뿰援ъ뿉꽌뒗 씠瑜 씠슜븯뿬 슜웾 떆猷 궡 議댁옱븯뒗 냽룄쓽 RNA瑜 냽異뺥븯怨좎옄 떎由ъ뭅 RNA쓽 諛섏쓳 떒怨꾨 嫄곕벊 諛섎났븯뒗 諛⑹떇쓣 梨꾪깮븯떎. 빐떦 怨듭젙뿉꽌 RNA 떎由ъ뭅쓽 寃고빀 슚쑉쓣 理쒕솕븷 닔 엳룄濡 씪遺 쟻슜 議곌굔쓽 理쒖쟻솕媛 씠猷⑥뼱議뚮떎.

One-step RT-qPCR

RNA쓽 냽異 諛 異붿텧 슚쑉 AccuPower® Dual-HotStartTM RT-qPCR Master Mix (Bioneer, Korea)瑜 씠슜븯뿬 one-step reverse transcription-quantitative PCR (RT-qPCR)쓣 넻빐 솗씤릺뿀떎. PCR 蹂 뿰援щ 넻빐 젣옉븳 옣鍮꾨 씠슜븯뿬 50꼦뿉꽌 15遺꾧컙 뿭쟾궗瑜 넻븳 cDNA 빀꽦 썑 씠瑜 95꼦뿉꽌 5遺 룞븞 pre-denaturation 떆궓 썑, 95꼦, 30珥 denaturation - 60꼦, 35珥 annealing쓽 PCR 떒怨꾨 40 cycle 吏꾪뻾븯뒗 議곌굔쑝濡 닔뻾릺뿀떎. 빐떦 PCR Taq DNA 以묓빀슚냼瑜 쟻슜븳 諛⑹떇쑝濡 5' 留먮떒뿉 FAM, 3' 留먮떒뿉 냼愿묒젣媛 媛곴컖 몴吏맂 TaqMan 봽濡쒕툕瑜 씠슜븯뿬 떎떆媛꾩쑝濡 삎愿 떊샇媛 痢≪젙릺룄濡 븯떎(Hoffmann et al., 2016).

諛뺥뀒由ъ삤뙆吏 MS2쓽 lysis

蹂 뿰援ъ뿉꽌뒗 씤泥닿컧뿼꽦씠 뾾뒗 諛뺥뀒由ъ삤뙆吏 MS2 (ATCC15597-B1)瑜 샇씉湲 諛붿씠윭뒪쓽 紐⑤뜽濡 꽑젙븯뿬 뀒뒪듃瑜 吏꾪뻾븯떎. MS2濡쒕꽣 RNA瑜 슚怨쇱쟻쑝濡 異붿텧븯湲 쐞빐꽌뒗 異⑸텇븳 lysis 怨듭젙씠 씠猷⑥뼱졇빞 븳떎. 뵲씪꽌 MS2 lysis 떆 쟻슜릺뒗 媛 議곌굔뱾뿉 븳 議곌굔 뀒뒪듃瑜 吏꾪뻾븯쑝硫, 뀒뒪듃맂 蹂닔뒗 heating 삩룄 諛 떆媛, proteinase K쓽 쟻슜, lysis buffer쓽 쟻슜 議곌굔씠떎. 媛 議곌굔뿉 뵲瑜 lysis 슚쑉뿉 븳 룊媛뒗 American Type Culture Collection (ATCC)뿉꽌 젣떆븳 soft agar overlay plaque assay瑜 씠슜븯怨좎옄 븯떎. 씠瑜 쐞빐 MS2쓽 host bacteria씤 E.coli (Escherichia coli C-3000, ATCC15597)瑜 17떆媛 諛곗뼇븳 썑 씠瑜 45꼦 긽깭쓽 0.5% soft agar 50 mL뿉 300 μL 젒醫낇븳 吏곹썑, 37꼦濡 썙諛띾맂 1.5% agar plate (90 mm)뿉 2.5 mL뵫 遺꾩<븯떎. 빐떦 agar plate뒗 20遺꾧컙 援논엺 썑 plaque assay뿉 궗슜릺뿀떎. Plaque assay뒗 lysis 議곌굔씠 쟻슜릺吏 븡 MS2 諛곗뼇븸쓣 議 몴以쑝濡 븯뿬 媛 議곌굔뿉 뵲씪 lysis媛 떆룄맂 떆猷뚮뱾쓣 10諛곗닔濡 怨꾨떒 씗꽍븯뿬 2 μL뵫 젒醫낇븯뒗 諛⑹떇쑝濡 닔뻾릺뿀떎(Reddy, 2007). 씠瑜 37꼦, 17떆媛 諛곗뼇 썑 愿李곕릺뒗 plaque瑜 怨꾩닔븯뿬 솢꽦씠 쑀吏맂 MS2쓽 냽룄瑜 怨꾩궛븿쑝濡쒖뜥 媛 議곌굔쓽 lysis 슚쑉쓣 솗씤븯떎. 냽룄쓽 怨꾩궛 떎쓬쓽 떇쓣 넻븯뿬 씠猷⑥뼱議뚮떎(Boone et al., 2001-2012).

pfu/mL=average plaque count/dilution factor2×103mL

怨듦린 룷吏 떆猷뚯쓽 쟻슜

怨듦린 룷吏묒 LMO 2벑湲 떎뿕떎 궡뿉 븘겕由대줈 젣옉맂 1 m3쓽 cubic쓣 꽕移섑븯뿬 吏꾪뻾릺뿀떎. MS2 諛곗뼇븸쓣 diethyl pyrocarbonate (DEPC) treated water뿉 1/10,000 씗꽍븯뿬 젣옉븳 떆猷뚮 怨좎븬 뒪봽젅씠瑜 넻빐 cubic 궡뿉 12珥 룞븞 遺꾩궗븯뿬 뿉뼱濡쒖「쓣 삎꽦븯쑝硫, 珥 遺꾩궗웾 20 mL씠떎. Cubic 궡뿉뒗 bio sampler (SKC, USA)媛 쐞移섑븯뿬 遺꾩궗媛 떆옉맂 떆젏遺꽣 媛룞릺뼱 collection vessel 궡 DEPC treated water 20 mL 긽뿉 10遺 媛 룷吏묒씠 吏꾪뻾릺뿀떎(Puthussery et al., 2023). 媛 떆猷뚯뿉꽌 諛뺥뀒由ъ삤뙆吏 MS2쓽 RNA瑜 냽異 諛 異붿텧븯湲 쐞븯뿬 긽湲 RNA 냽異 諛 異붿텧 諛⑹떇 以 理쒖쟻솕媛 닔뻾맂 議곌굔쓣 쟻슜븯떎. 떆猷뚮 媛곴컖 4.5 mL뵫 痍⑦븯뿬 怨듭젙쓣 닔뻾븯쑝硫, 理쒖쥌쟻쑝濡 異붿텧맂 슜웾 媛 100 μL濡 遺뵾 湲곗 45諛 냽異뺣맂 떆猷뚮 뼸쓣 닔 엳뿀떎. 怨듦린 룷吏 떆猷 궡쓽 MS2瑜 寃異쒗븯湲 쐞빐 븵꽌 RNA쓽 냽異 슚쑉쓣 룊媛븳 諛⑹떇怨 룞씪븯寃 one-step RT-qPCR쓣 닔뻾븯떎. PCR MS2쓽 mat gene쓣 寃잛쑝濡 븯뿬 닔뻾릺뿀쑝硫, 媛 떆猷 궡 MS2쓽 copy 닔瑜 젙웾븯怨좎옄 MS2 RNA (Roche, Germany)瑜 10諛 닔濡 怨꾨떒 씗꽍븯뿬 몴以 떆猷뚮줈뜥 쟻슜븯떎. 媛 怨듦린 룷吏 떆猷뚮뒗 PCR뿉 template濡 쟻슜 떆 5 μL뵫 궗슜릺뿀쑝硫, 냽異 諛 異붿텧쓽 쟾泥섎━ 뾾씠 諛붾줈 쟻슜븯뒗 寃쎌슦, MagListoTM 5 M Viral DNA/RNA Extraction Kit쓽 봽濡쒗넗肄쒖쓣 뵲씪 젙젣맂 RNA瑜 쟻슜븯뒗 寃쎌슦, RNA쓽 냽異 怨듭젙쓣 嫄곗튇 썑 異붿텧맂 RNA瑜 쟻슜븳 寃쎌슦濡 鍮꾧탳 룊媛븯떎. 媛 떆猷뚯쓽 RNA copy 닔뒗 몴以 떆猷뚯쓽 利앺룺 寃곌낵瑜 넻빐 몴以 怨≪꽑쓣 옉꽦븳 썑 Ct (cycle threshold) 媛믪쓣 엯븯뿬 젙웾릺뿀떎(Bustin et al., 2009).

옄룞솕 옣鍮

蹂 뿰援щ 넻빐 媛쒕컻맂 옄룞솕 옣鍮꾨뒗 鍮꾩븘씠떚諛몃쪟쓽 蹂댁쑀 湲곗닠쓣 넻빐 젣옉릺뿀쑝硫, 겕寃 냽異뺣 利앺룺遺濡 援щ텇맂떎. 由ы대뱶 빖뱾留 諛⑹떇쓣 湲곕컲쑝濡 븳 옄룞솕 옣鍮 援ъ텞쓣 쐞빐 x異, y異, z異뺤쓽 꽭 媛쒖쓽 異뺤쑝濡 援ъ꽦릺뼱 엳뒗 濡쒕큸 紐⑤뱢쓣 쟻슜븯쑝硫, z異뺤쓽 釉뚮씪耳볦뿉 뙆씠렖 紐⑤뱢쓣 議곕┰븯떎. 씠瑜 넻빐 뙆씠렖 紐⑤뱢 媛 諛⑺뼢쑝濡쒖쓽 씠룞씠 媛뒫븯硫, 떎由곗 뵾뒪넠씠 쟻슜릺뼱 떆猷뚯쓽 씉엯 諛 遺꾩<瑜 닔뻾븯寃 맂떎. 냽異뺣쓽 痢〓㈃뿉뒗 옄꽍쓣 꽕移섑븯쑝硫, 諛섏쓳 뒠釉뚯쓽 젒珥됯낵 遺꾨━瑜 諛섎났븯뿬 옄꽦 遺꾨━ 怨듭젙쓣 닔뻾븷 닔 엳룄濡 븯떎. 由ы대뱶 빖뱾留곸쓣 넻빐 냽異 諛 異붿텧맂 빑궛 떆猷뚮뒗 씠썑 利앺룺遺濡 씠룞븯寃 릺硫, 봽씪씠癒 諛 봽濡쒕툕瑜 鍮꾨’빐 PCR 닔뻾뿉 븘슂븳 援ъ꽦슂냼뱾怨쇱쓽 샎빀씠 씠猷⑥뼱吏꾨떎. 利앺룺遺뒗 븯遺뿉 엳듃떛겕 깋媛곹뙩씠 꽕移섎맂 렆떚뼱냼옄瑜 씠슜븯뿬 삩룄쓽 긽듅 諛 븯媛뺤씠 씠猷⑥뼱吏硫, 뒠釉뚯뿉 삩룄瑜 쟾떖븯뒗 엳똿 뵆젅씠듃뿉뒗 삩룄꽱꽌媛 옣李⑸릺뼱 삩룄 젣뼱媛 媛뒫븯룄濡 븯떎. 삉븳, PCR 怨쇱젙 以 뒠釉 궡뿉꽌 諛쒖깮븷 닔 엳뒗 떆猷뚯쓽 利앸컻 쁽긽쓣 理쒖냼솕븯湲 쐞빐 뒠釉뚯쓽 긽遺뿉 엳꽣瑜 쟻슜븯떎. 떎떆媛꾩쑝濡 PCR쓣 紐⑤땲꽣留 븯湲 쐞빐 諛쒓킅 떎씠삤뱶(Light emitting diode, LED)瑜 愿묒썝쑝濡 븯怨, 遺遺 諛⑹쟾 諛⑹떇쓽 뵒뀓꽣(Partial discharge detector)瑜 쟻슜븳 삎愿 愿묓븰 紐⑤뱢쓣 궗슜븯떎. 빐떦 愿묓븰 紐⑤뱢 2媛쒖쓽 삎愿 뙆옣 痢≪젙씠 媛뒫븯룄濡 젣옉븯뿬 룞떆뿉 몢 媛吏 쑀쟾옄瑜 긽쑝濡 PCR 닔뻾씠 媛뒫븯떎. 삎愿 痢≪젙 愿묓븰怨꾧 뒠釉뚯쓽 痢〓㈃뿉꽌 씠룞븯硫 뒪罹뷀븯뒗 諛⑹떇쓣 쟻슜븯쑝硫, 씠뒗 뒪뀦紐⑦꽣 由 諛 踰⑦듃瑜 씠슜븳 援щ룞遺뿉 쓽빐 닔뻾맂떎.

寃 怨

怨듦린 以 諛붿씠윭뒪쓽 빑궛쓣 寃異쒗븯湲 쐞븳 옄룞솕 遺꾩꽍 떆뒪뀥

怨듦린 以 諛붿씠윭뒪瑜 寃異쒗븯湲 쐞빐 援ъ텞븳 寃궗 怨쇱젙 떎쓬怨 媛숇떎. 슦꽑 寃궗븯怨좎옄 븯뒗 쁽옣뿉꽌 씪젙떆媛 룞븞 bio sampler 궡뿉 븸긽쑝濡 怨듦린瑜 룷吏묓븳떎. 씠瑜 쐞빐 쓬븬 럩봽媛 궗슜릺硫, 븸긽 궡 룷吏묐맂 떆猷뚮뒗 씠썑 옄룞솕 옣鍮꾩뿉 쟻슜릺뼱 빑궛 냽異 諛 異붿텧怨 PCR 寃궗媛 씠猷⑥뼱吏꾨떎. 옄룞솕 옣鍮꾩뿉뒗 슜웾쓽 떆猷뚮 痍④툒븷 닔 엳뒗 reservoir媛 議댁옱븯뿬 怨듦린 룷吏 떆猷뚮 쐞移섏떆궗 닔 엳떎. 옄룞솕 옣鍮꾩쓽 옉룞씠 떆옉릺硫 reservoir뿉 쐞移섑븳 떆猷뚮뒗 뙆씠렖쓣 넻빐 냽異뺣濡 씪젙웾뵫 遺꾩<릺뼱 냽異 怨듭젙씠 닔뻾맂떎. 떎由ъ뭅-옄꽦鍮꾨뱶瑜 씠슜븯뿬 냽異 썑 異붿텧맂 빑궛 씠썑 利앺룺遺濡 삷寃⑥졇 one-step RT-qPCR씠 씠猷⑥뼱吏꾨떎. 寃異 寃곌낵뒗 옣鍮 쇅遺뿉 꽕移섎맂 LCD 솕硫댁쓣 넻빐 솗씤씠 媛뒫븯룄濡 븯떎(Fig. 1).

Fig. 1. The automatic system for detecting airborne viruses.
The air-collected samples through a bio sampler are transferred to an automatic machine and then undergo sequential process from RNA concentration and extraction to PCR analysis. Finally, the results are displayed on the LCD monitor.

떎由ъ뭅-옄꽦鍮꾨뱶瑜 씠슜븳 RNA 냽異

떎由ъ뭅-옄꽦鍮꾨뱶瑜 씠슜븳 RNA쓽 냽異 슚쑉쓣 솗씤븯湲 쐞븯뿬 Influenza A H1N1 (pdm09) RNA (ATCC#VR-1894DQ)瑜 긽쑝濡 뀒뒪듃瑜 吏꾪뻾븯떎. MagListoTM 5 M Viral DNA/RNA Extraction Kit쓽 湲곕낯 봽濡쒗넗肄쒖뿉꽌 떆猷뚯 떎由ъ뭅-옄꽦鍮꾨뱶瑜 寃고빀떆궎뒗 떒怨꾨 5쉶 닔뻾븯쓣 븣, 湲곗〈 議곌굔(1쉶 닔뻾)鍮 理쒖쥌쟻쑝濡 異붿텧맂 RNA쓽 냽룄瑜 鍮꾧탳븯떎. 媛 寃고빀 떒怨꾩뿉꽌 쟻슜맂 RNA쓽 냽룄뒗 1,000 copies/μL씠硫, 理쒖쥌쟻쑝濡 異붿텧맂 媛 RNA瑜 one-step RT-qPCR뿉 template濡 궗슜븯뿬 Ct 媛믪쓣 鍮꾧탳븯떎. 룞씪 議곌굔쓽 뀒뒪듃瑜 3쉶 諛섎났 닔뻾븳 寃곌낵, 湲곗〈 諛⑹떇 鍮 떎由ъ뭅-옄꽦鍮꾨뱶쓽 寃고빀 떒怨꾨 5쉶 쟻슜븳 議곌굔뿉꽌 Ct 媛믪씠 2.5~3 cycle쓽 踰붿쐞濡 떒異뺣릺뒗 슚怨쇰 솗씤븯떎(Fig. 2). 씠뒗 PCR efficiency (E)媛 100%씪 寃쎌슦 RNA copy 닔媛 5諛 씠긽 넂 寃껋쑝濡 異붿젙븷 닔 엳떎.

Fig. 2. RNA concentration using silica-magnetic bead.
In case of performing silica-RNA binding 5 times, the Ct values decreased by more than 2.5 cycles compared to the control protocol.

諛뺥뀒由ъ삤뙆吏 MS2쓽 lysis 슚쑉 鍮꾧탳

諛뺥뀒由ъ삤뙆吏 MS2 lysis 슚쑉쓣 솗씤븯湲 쐞븳 蹂닔뒗 MagListoTM 5 M Viral DNA/RNA Extraction Kit쓽 湲곕낯 봽濡쒗넗肄쒖쓣 넗濡 뀒뒪듃릺뿀떎. 빐떦 kit뿉꽌뒗 諛붿씠윭뒪 lysis 諛 떎由ъ뭅쓽 寃고빀 議곌굔 議곗꽦쓣 異⑹”븯湲 쐞빐 VB buffer瑜 궗슜븯怨 엳쑝硫, lysis 떒怨꾩뿉꽌 proteinase K媛 븿猿 쟻슜맂떎. 씠븣 proteinase K쓽 솢꽦솕瑜 쐞빐 60꼦뿉꽌 10遺꾧컙 heating씠 씠猷⑥뼱吏꾨떎. Proteinase K 쟻슜 뿬遺 VB buffer 궗슜웾뿉 뵲瑜 lysis 슚쑉쓣 룊媛븯湲 쐞븯뿬 VB buffer瑜 MS2 諛곗뼇븸怨 1:1쓽 鍮꾩쑉濡 궗슜븳 寃쎌슦 씠뿉 젅諛섏뿉 빐떦븯뒗 뼇留 쟻슜븳 寃쎌슦(0.5:1)濡 굹늻뼱 뀒뒪듃븯쑝硫, 뿬湲곗뿉 proteinase K瑜 궗슜븳 寃쎌슦 洹몃젃吏 븡 寃쎌슦룄 븿猿 鍮꾧탳븯떎. Heating 옄泥닿 MS2쓽 lysis뿉 쁺뼢쓣 誘몄튌 닔 엳쑝誘濡, proteinase K쓽 궗슜 뿬遺 愿怨꾩뾾씠 紐⑤뱺 議곌굔쓽 떆猷뚯뿉 湲곗〈 諛⑹떇怨 룞씪븳 heating 議곌굔쓣 쟻슜븯떎. 洹 寃곌낵, lysis 議곌굔씠 쟻슜릺吏 븡 議 떆猷 쇅뿉 紐⑤뱺 議곌굔뿉꽌 MS2쓽 利앹떇쓣 솗씤븷 닔 뾾뿀떎. 씠 媛숈 寃곌낵瑜 넻빐 heating쓽 쟻슜씠 MS2쓽 lysis뿉 吏븳 쁺뼢쓣 誘몄튇 寃껋쑝濡 궗猷뚮릺뿀쑝硫, 씠瑜 솗씤븯湲 쐞빐 proteinase K뒗 紐⑤몢 쟻슜븯吏 븡怨 긽湲 VB buffer 蹂닔뿉 heating 쟻슜 뿬遺瑜 異붽 蹂닔濡 븯뿬 뀒뒪듃瑜 吏꾪뻾븯떎. Plaque assay瑜 넻빐 媛 議곌굔뿉 븳 MS2쓽 솢꽦쓣 솗씤븳 寃곌낵, 議 떆猷뚮뒗 5 × 1012 pfu/mL쓽 냽룄濡 솗씤맂 寃껋뿉 鍮꾪빐 VB buffer:MS2 諛곗뼇븸 = 1:1 議곌굔뿉 heating쓣 쟻슜븳 떆猷뚯뿉꽌뒗 利앹떇씠 솗씤릺吏 븡븯떎. 븳렪, VB buffer쓽 궗슜웾씠 洹몃濡쒖씠硫 heating씠 쟻슜릺吏 븡 議곌굔뿉꽌뒗 5 × 109 pfu/mL, VB buffer쓽 궗슜웾씠 젅諛섏씠굹 heating씠 쟻슜맂 議곌굔쓽 떆猷뚮뒗 1.5 × 106 pfu/mL쓽 닔以쑝濡 怨꾩닔릺뿀떎(Table 1 & Fig. 3). 빐떦 떎뿕쓣 넻빐 諛뺥뀒由ъ삤뙆吏 MS2뿉 媛옣 겙 쁺뼢쓣 誘몄튂뒗 寃껋 heating씠硫, 洹 떎쓬쑝濡 以묒슂븳 슂냼뒗 VB buffer쓽 쟻슜 議곌굔씠씪뒗 寃껋쓣 솗씤븯떎. VB buffer쓽 궗슜웾쓣 蹂몃옒 봽濡쒗넗肄쒕濡 떆猷뚯 룞씪븯寃 쟻슜 떆 怨듦린 룷吏 떆猷뚯쓽 슜웾씠 留ㅼ슦 而ㅼ硫 냽룄 삉븳 씗꽍맆 슦젮媛 엳떎. 뵲씪꽌 蹂 뿰援ъ뿉꽌뒗 VB buffer瑜 bio sampler쓽 collection vessel뿉 쟻슜 썑 씠뿉 諛붾줈 怨듦린瑜 룷吏묓븯뒗 諛⑹떇쓣 理쒖쥌쟻쑝濡 쟻슜븯떎. 삉븳, VB buffer뿉 諛붾줈 怨듦린瑜 룷吏묓븷 寃쎌슦 諛쒖깮븷 닔 엳뒗 buffer쓽 꽍異 쁽긽쓣 諛⑹븯怨좎옄 ½쓽 냽룄濡 씗꽍븯뿬 궗슜븯떎.

Fig. 3. Lysis of bacteriophage MS2 according to application conditions of VB buffer and heating temperature.
The lytic efficiency of bacteriophage MS2 was confirmed by plaque assay. When applying heating to the sample(몼, 몿), lytic efficiency is increased. Table 1 shows the concentration of live bacteriophage MS2 for each condition.

Lysis of bacteriophage MS2 according to application conditions of VB buffer and heating temperature

No. Sample : VB buffer (關L) Heating (60꼦) pfu/mL
C - - 5 횞 1012
200:100 X 5 횞 1012
O 1.5 횞 106
200:200 X 5 횞 109
몿 O -


슜웾 떆猷뚯쓽 냽異

怨듦린 룷吏 吏곹썑쓽 떆猷뚮뒗 슜웾쓽 븸긽 긽깭씠硫, 씠瑜 怨좊냽룄 슜웾 긽깭濡 냽異뺤떆궎뒗 諛⑹떇뿉 븳 뿰援щ 吏꾪뻾븯떎. 紐⑤뜽諛붿씠윭뒪濡쒖뜥 쟻슜븳 諛뺥뀒由ъ삤뙆吏 MS2 諛곗뼇븸쓣 룞씪븳 냽룄濡 븯뿬 200 μL, 2 mL, 4 mL쓽 슜웾 긽깭濡 媛곴컖 냽異뺤쓣 떆룄븯쑝硫, 씠븣 떎由ъ뭅-옄꽦鍮꾨뱶 떆猷뚯쓽 諛섏쓳 떒怨 諛 옄꽦 遺꾨━ 怨듭젙쓣 紐⑤몢 븳 踰덉뿉 닔뻾븯뒗 諛⑹떇(method 1)怨 媛 떒怨꾩뿉꽌 쟻슜릺뒗 떆猷뚮 씪젙 슜웾쑝濡 굹늻뼱 吏꾪뻾븯뒗 諛⑹떇(method 2)쓣 鍮꾧탳븯떎. 냽異 슚쑉뿉 븳 寃곌낵뒗 媛 諛⑹떇쓣 넻빐 異붿텧맂 RNA瑜 one-step RT-qPCR뿉 template濡 쟻슜븯뿬 Ct 媛믪쓣 鍮꾧탳븿쑝濡쒖뜥 솗씤븯떎. 쟾옄쓽 寃쎌슦 떆猷 200 μL濡쒕꽣 異붿텧맂 RNA瑜 template濡 쟻슜븳 Ct 媛믪쓣 湲곗쑝濡 븯쓣 븣, 2 mL 떆猷뚯쓽 寃쎌슦 1.862 cycle, 4 mL 떆猷뚯뿉꽌뒗 1.692 cycle쓽 Ct 媛믪씠 떒異뺣릺뒗 寃껋쓣 솗씤븷 닔 엳뿀떎. 諛섎㈃, 썑옄쓽 諛⑹떇뿉꽌뒗 200 μL 떆猷 鍮 2 mL 떆猷뚯 4 mL 떆猷뚯뿉꽌 媛곴컖 2.734 cycle, 3.432 cycle쓽 Ct 媛 떒異뺤쓽 寃곌낵瑜 蹂댁떎(Fig. 4). 씠뒗 썑옄쓽 諛⑹떇뿉꽌 떆猷뚯 諛섏쓳븯뒗 떎由ъ뭅-옄꽦鍮꾨뱶쓽 냽룄媛 긽쟻쑝濡 怨좊냽룄濡 쟻슜릺뼱 寃고빀 슚쑉씠 넂怨, 옄꽦遺꾨━ 怨쇱젙뿉꽌 옄꽦鍮꾨뱶쓽 넀떎 슦젮媛 쟾옄뿉 鍮꾪빐 궙븘 굹굹뒗 쁽긽쑝濡 깮媛곷맂떎. 뵲씪꽌, RNA쓽 쉶닔 슚쑉씠 긽쟻쑝濡 슦닔븳 썑옄쓽 諛⑹떇쓣 냽異 諛⑸쾿쑝濡 理쒖쥌 梨꾪깮븯뿬 옄룞솕 떆뒪뀥뿉 쟻슜븯떎.

Fig. 4. Concentration efficiency depending on the methods.
Method 1: RNA-silica binding and magnetic separation in one-step, Method 2: Perform RNA-silica binding and magnetic separation with multiple split volumes. Method 2 is more effective for RNA concentration due to less magnetic bead loss.

怨듦린 룷吏 떆猷뚯뿉꽌쓽 諛뺥뀒由ъ삤뙆吏 MS2 寃異 諛 젙웾

怨듦린 룷吏 떆猷뚯쓽 MS2瑜 젙웾븯湲 쐞빐 MS2 RNA 10~105 copies/reaction 냽룄 援ш컙뿉 븳 PCR 몴以 怨≪꽑쓣 옉꽦븯쑝硫, 씠뿉 媛 寃궗 떆猷뚯쓽 Ct 媛믪쓣 엯븯뿬 copy 닔瑜 鍮꾧탳븯떎. 洹 寃곌낵 蹂꾨룄쓽 쟾泥섎━瑜 븯吏 븡 怨듦린 룷吏 떆猷뚯 씪諛 異붿텧 諛⑹떇쓣 넻빐 뼸 떆猷뚯쓽 Ct 媛믪 媛곴컖 36.384, 33.144濡 痢≪젙릺뿀쑝硫, 냽異 怨듭젙 씠썑 젙젣맂 떆猷뚯쓽 寃쎌슦 Ct 媛 30.721쓽 寃곌낵瑜 蹂댁떎. 빐떦 寃곌낵瑜 몴以 怨≪꽑쓣 넻빐 젙웾 떆 媛곴컖 19, 171, 885 copies濡 異붿젙븷 닔 엳떎(Fig 5(A)). 냽異 怨듭젙씠 쟻슜맆 寃쎌슦 쟾泥섎━媛 릺吏 븡 떆猷 鍮 빟 46諛, 湲곗〈 RNA 異붿텧 諛⑹떇 鍮 빟 5諛곗쓽 RNA 냽異 슚怨쇰 뼸쓣 닔 엳뿀떎. 異붽濡 one-step RT-qPCR쓽 利앺룺궛臾쇱 TaqMan 봽濡쒕툕뿉 쓽빐 諛쒖깮릺뒗 삎愿묒떊샇 諛 븘媛濡쒖쫰寃 쟾湲곗쁺룞쓣 넻빐 꽌뿴 듅씠쟻쑝濡 利앺룺릺뿀쓬쓣 솗씤븯떎(Fig. 5(B)).

Fig. 5. Detection of bacteriophage MS2 in aerosols.
Non-purification: Sample without any treatment after air collection, Extraction only: Sample with RNA extraction only, Concentration + Extraction: Sample extracted after concentrating RNA 45 times. (A) As a result of one-step RT-qPCR for each sample, the RNA copy of the concentration + extraction sample was approximately 45 times more than that of non-purification sample. (B) Each amplification product was confirmed to be specific for mat gene of bacteriophage MS2 through agarose gel electrophoresis.
怨 李

蹂 뿰援ъ뿉꽌뒗 쁽議댄븯怨 엳뒗 怨듦린 以 깮臾쇳븰쟻 쑀빐臾쇱쭏쓽 愿由 諛⑹떇쓣 蹂댁셿븷 닔 엳뒗 븞쓣 젣떆븯怨좎옄 븯떎. 怨듦린 룷吏 떆猷뚮뒗 洹 怨쇱젙뿉 뵲씪 긽쓽 遺덊솢꽦솕媛 諛쒖깮븷 닔 엳떎. 빐떦 떆猷뚮 諛곗뼇쓣 넻빐 寃곌낵瑜 솗씤븯뒗 寃쎌슦, 誘몄깮臾쇱씠굹 諛붿씠윭뒪쓽 깮議대쪧씠 궙븘 떎젣 怨듦린 삤뿼룄瑜 怨쇱냼 룊媛븯寃 맆 슦젮媛 엳떎(Hinds, 1999). 뵲씪꽌 諛붿씠윭뒪쓽 깮議닿낵 愿怨꾩뾾씠 듅씠쟻쑝濡 寃異쒖씠 媛뒫븳 RT-qPCR 諛⑹떇쓣 蹂 뿰援ъ뿉꽌쓽 寃異쒕쾿쑝濡 궗슜븯떎. 쁽옣吏꾨떒寃궗(Point-of-care, POCT)뿉꽌뒗 鍮꾧탳쟻 鍮좊Ⅸ 遺꾩꽍 諛⑹떇씤 빆썝-빆泥 諛섏쓳쓽 硫댁뿭寃궗踰뺤씠 쑀由ы븷 닔 엳쑝굹 냽룄뿉 떆猷뚯뿉꽌뒗 쐞쓬꽦 寃곌낵瑜 珥덈옒븷 닔 엳쑝硫, 빆泥댁쓽 듅씠룄瑜 솗蹂댄븯吏 紐삵븷 寃쎌슦뿉뒗 쐞뼇꽦쓽 寃곌낵媛 諛쒖깮븷 닔 엳떎(Cassedy et al., 2021). 怨듦린 以묒뿉꽌 COVID-19쓽 썝씤 諛붿씠윭뒪씤 SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2)瑜 寃異쒗븯湲 쐞빐 닔뻾맂 理쒓렐 뿰援щ뱾뿉꽌룄 遺遺 PCR 諛⑹떇쓣 쟻슜븳 寃껋쑝濡 솗씤릺뿀떎(Liu et al., 2020; Moreno et al., 2021; Lednicky et al., 2020; Maestre et al., 2021). 씠 媛숈 궗빆쓣 怨좊젮빐蹂댁븯쓣 븣, 쁽옣뿉꽌 怨듦린 以 諛붿씠윭뒪瑜 寃異쒗븯湲 쐞븳 諛⑹떇쑝濡 one-step RT-qPCR쓣 梨꾪깮븯뒗 寃껋씠 쟻젅븯떎怨 궗猷뚮릺뿀떎. Bio sampler瑜 넻빐 룷吏묐맂 怨듦린 떆猷 궡쓽 諛붿씠윭뒪쓽 珥덇린 냽룄뒗 留ㅼ슦 냽룄 긽깭씪 媛뒫꽦씠 넂떎. 由ы대뱶 빖뱾留곸쓣 넻븳 떎由ъ뭅-옄꽦鍮꾨뱶 湲곕컲쓽 빑궛 냽異 諛⑹떇 怨듦린 以 遺쑀븯怨 엳뒗 諛붿씠윭뒪瑜 빑궛 닔以뿉꽌 怨좊냽룄濡 냽異뺥븯뿬 怨좉컧룄濡 깘吏븷 닔 엳떎. 떎젣 뿉뼱濡쒖「 긽깭쓽 諛뺥뀒由ъ삤뙆吏 MS2瑜 긽쑝濡 븯뿬 빐떦 諛⑸쾿쓣 쟻슜븯쓣 븣, 빟 45諛 닔以쓽 빑궛 냽異 슚怨쇰 솗씤븷 닔 엳뿀떎. 씠瑜 넻빐 怨듦린 以묒뿉 뿉뼱濡쒖「 긽깭濡 愿묐쾾쐞븯寃 遺꾪룷릺뼱 엳뒗 諛붿씠윭뒪瑜 븸긽쑝濡 1李 룷吏묓븳 썑, 씠瑜 빑궛 닔以뿉꽌 2李 냽異뺥븯뿬 怨좉컧룄濡 깘吏븷 닔 엳뒗 媛뒫꽦쓣 뼸쓣 닔 엳뿀떎. 삉븳, 빐떦 떆뒪뀥 옄룞솕瑜 넻븳 넂 솢슜꽦쓣 媛졇 鍮꾩쟾臾멸룄 떎뼇븳 遺꾩빞뿉꽌 넀돺寃 궗슜 媛뒫븯硫, 愿由 쁽옣뿉 뵲씪 寃異 긽쓣 쑀룞쟻쑝濡 蹂寃쏀븯뿬 쟻슜븷 닔 엳떎뒗 옣젏쓣 媛吏꾨떎. 긽뿉 븳 理쒖쥌 寃異쒖 PCR쓣 넻빐 씠猷⑥뼱吏湲 븣臾몄뿉, 寃異쒗븯怨좎옄 븯뒗 諛붿씠윭뒪 삉뒗 誘몄깮臾쇱쓽 쑀쟾옄뿉 듅씠쟻씤 봽씪씠癒 諛 봽濡쒕툕瑜 궗슜븿쑝濡쒖뜥 떎뼇븯寃 솢슜씠 媛뒫븷 寃껋쑝濡 湲곕맂떎. 異붽濡, 씠 媛숈 諛⑹떇 蹂꾨룄쓽 諛곗뼇 怨듭젙씠굹 깮솕븰룞젙쓣 븘슂濡 븯吏 븡湲 븣臾몄뿉 湲곗〈쓽 寃궗踰뺤뿉꽌 슂援щ릺뿀뜕 寃異 떆媛꾩쓣 슚怨쇱쟻쑝濡 떒異뺥븯뒗 寃껋씠 媛뒫븷 寃껋쑝濡 궗猷뚮맂떎.

ACKNOWLEDGEMENT

This work was supported by the Technology development Program (S3194404) funded by the Ministry of SMEs and Startups (MSS, Korea).

List of abbreviations

PCR: Polymerase chain reaction

qPCR: Quantitative polymerase chain reaction

RT: Reverse transcription

DEPC: Diethyl pyrocarbonate

Ct: Cycle threshold

CONFLICT OF INTEREST

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

References
  1. Adams NM, Bordelon H, Wang KKA, et al. Comparison of three magnetic bead surface functionalities for RNA extraction and detection. ACS Appl Mater Interfaces. 2015. 7: 6062-6069.
    Pubmed CrossRef
  2. Blocken B, van Druenen T, Ricci A, et al. Ventilation and air cleaning to limit aerosol particle concentrations in a gym during the COVID-19 pandemic. Build Environ. 2021. 193: 107659.
    Pubmed KoreaMed CrossRef
  3. Boone DR, Castenholz RW, Garrity GM, et al. Bergey's Manual of Systematic Bacteriology, 2nd Edition. 2001-2012. Springer. NY, USA.
    CrossRef
  4. Breshears LE, Nguyen BT, Mata-Robles S, et al. Biosensor detection of airborne respiratory viruses such as SARS-CoV-2. SLAS Technology. 2022. 27: 4-17.
    Pubmed KoreaMed CrossRef
  5. Buonanno G, Stabile L, Morawska L, et al. Estimation of airborne viral emission: Quanta emission rate of SARS-CoV-2 for infection risk assessment. Environ Int. 2020. 141: 105794.
    Pubmed KoreaMed CrossRef
  6. Bustin SA, Benes V, Garson JA, et al. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin Chem. 2009. 55: 611-622.
    Pubmed CrossRef
  7. Cassedy A, Parle-McDermott A, O'Kennedy R. Virus Detection: A Review of the Current and Emerging Molecular and Immunological Methods. Front Mol Biosci. 2021. 20: 637559.
    Pubmed KoreaMed CrossRef
  8. Drossinos Y, Weber TP, Stilianakis NI. Droplets and aerosols: An artificial dichotomy in respiratory virus transmission. Health Sci Rep. 2021. 4: e275.
    Pubmed KoreaMed CrossRef
  9. Guo ZD, Wang ZY, Zhang SF, et al. Aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards, Wuhan, China, 2020. Emerg Infect Dis. 2020. 26: 1583-1591.
    Pubmed KoreaMed CrossRef
  10. Hinds WC. Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles, 2nd Edition. 1999. Wiley. NY, USA.
    CrossRef
  11. Hoffmann B, Hoffmann D, Henritzi D, et al. Riems influenza a typing array (RITA): An RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses. Sci Rep. 2016. 6: 27211.
    Pubmed KoreaMed CrossRef
  12. Kutter JS, de Meulder D, Bestebroer TM, et al. SARS-CoV and SARS-CoV-2 are transmitted through the air between ferrets over more than one meter distance. Nat Commun. 2021. 12: 1-8.
    Pubmed KoreaMed CrossRef
  13. Lednicky JA, Shankar SN, Elbadry MA, et al. Collection of SARS-CoV-2 Virus from the Air of a Clinic within a University Student Health Care Center and Analyses of the Viral Genomic Sequence. Aerosol Air Qual Res. 2020. 20: 1167-1171.
    Pubmed KoreaMed CrossRef
  14. Liu Y, Ning Z, Chen Y, et al. Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals. Nature. 2020. 582: 557-560.
    Pubmed CrossRef
  15. Maestre JP, Jarma D, Jia-Rong FY, et al. Distribution of SARS-CoV-2 RNA signal in a home with COVID-19 positive occupants. Sci Total Environ. 2021. 778: 146201.
    Pubmed KoreaMed CrossRef
  16. Moreno T, Pint처 RM, Bosch A, et al. Tracing surface and airborne SARS-CoV-2 RNA inside public buses and subway trains. Environ Int. 2021. 147: 106326.
    Pubmed KoreaMed CrossRef
  17. Pasquarella C, Albertini R, Dall'aglio P, et al. Air microbial sampling: the state of the art. Igiene e Sanita Pubblica. 2008. 64: 79-120.
  18. Puthussery JV, Ghumra DP, McBrearty KR, et al. Real-time environmental surveillance of SARS-CoV-2 aerosols. Nat Commun. 2023. 14: 3692.
    Pubmed KoreaMed CrossRef
  19. Reddy CA. Methods for general and molecular microbiology, 3rd Edition. 2007. pp 869-978. ASM Press. Washington D.C., USA.
    CrossRef
  20. Sun N, Deng C, Liu Y, et al. Optimization of influencing factors of nucleic acid adsorption onto silica-coated magnetic particles: Application to viral nucleic acid extraction from serum. J Chromatogr A. 2014. 1325: 31-39.
    Pubmed CrossRef