[논문]Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR

Park, Myungsun; Won, Joungha; Choi, Byung Yoon; Lee, C. Justin

doi:10.1038/s12276-020-0452-7

[국내논문] Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR 원문보기

Experimental & molecular medicine : EMM, v.52 no.6, 2020년, pp.963 - 977

Park, Myungsun (Center for Cognition and Sociality, Cognitive Glioscience Group, Institute for Basic Science, Daejeon, 34126 Republic of Korea) , Won, Joungha (Center for Cognition and Sociality, Cognitive Glioscience Group, Institute for Basic Science, Daejeon, 34126 Republic of Korea) , Choi, Byung Yoon (Department of Otorhinolaryngology, Seoul National University Bundang Hospital, Seongnam, 13620 Korea) , Lee, C. Justin (Center for Cognition and Sociality, Cognitive Glioscience Group, Institute for Basic Science, Daejeon, 34126 Republic of Korea)

Abstract ▼ AI-Helper

SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.Diagnostic microbiology: Boosting confidence in COVID-19 detectionA design strategy for virus detection tests could lead to improved detection of SARS-CoV-2 coronavirus. Laboratories currently perform SARS-CoV-2 diagnostic assays based on a technique called the polymerase chain reaction (PCR), which enables targeted amplification of viral nucleic acids. PCR assays are sensitive but can be plagued by false positives or false negatives. C. Justin Lee of the Institute for Basic Science, Daejon, Republic of Korea, and coworkers have devised a protocol for optimizing assay performance. The success of a PCR assay is determined by the selection of ‘primers’, short DNA strands that determine which genomic sequence gets amplified. The researchers identify a strategy for designing effective primer sets, and selecting reaction conditions that make best use of those primers. Although the focus here is on SARS-CoV-2, this approach should be applicable to other viruses.

참고문헌 (27)

1. Cascella, M., Rajnik, M., Cuomo, A., Dulebohn, S. C. & Di Napoli, R. Features, Evaluation and Treatment Coronavirus (COVID-19) (StatPearls Publishing, Treasure Island, FL, 2020).
2. Chan JF A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster Lancet 2020 395 514 523 10.1016/S0140-6736(20)30154-9 31986261

상세보기
3. Xu Z Pathological findings of COVID-19 associated with acute respiratory distress syndrome Lancet Respir. Med. 2020 8 420 422 10.1016/S2213-2600(20)30076-X 32085846

상세보기
4. Worldometer. COVID-19 CORONAVIRUS PANDEMIC. https://www.worldometers.info/coronavirus/? (2020).
5. (CDC), U.S.C.f.D.C.a.P. How COVID-19 Spreads. https://www.cdc.gov/coronavirus/2019-ncov/prevent-getting-sick/how-covid-spreads.html?CDC_AA_refVal=https%3A%2F%2Fwww.cdc.gov%2Fcoronavirus%2F2019-ncov%2Fprepare%2Ftransmission.html (2020).
6. News, N. How does coronavirus spread? https://www.nbcnews.com/health/health-news/how-does-new-coronavirus-spread-n1121856 (2020).
7. Wolfel, R. et al. Virological assessment of hospitalized patients with COVID-2019. Nature 581 , 465？469 (2020).
8. Kupferschmidt, K. Study claiming new coronavirus can be transmitted by people without symptoms was flawed. Science 10.1126/science.abb1524 (2020).
9. Won, J. et al. Development of a laboratory-safe and low-cost detection protocol for SARS-CoV-2 of the coronavirus disease 2019 (COVID-19). Exp. Neurobiol . 29 , 107？119 (2020).
10. Liu, Y., Gayle, A. A., Wilder-Smith, A. & Rocklov, J. The reproductive number of COVID-19 is higher compared to SARS coronavirus. J. Travel Med. 10.1093/jtm/taaa021 (2020).
11. Sanche, S. et al. High contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2. Emerg. Infect. Dis. 10.3201/eid2607.200282 (2020)
12. Wallinga J Teunis P Different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures Am. J. Epidemiol. 2004 160 509 516 10.1093/aje/kwh255 15353409

상세보기
13. Chen, L. Y. Heartbreak in the Streets of Wuhan (Bloomberg Businessweek, 2020).
14. Andrew Ryan, J. H.a. T. A. State Figures on Testing Raise Questions About Efforts to Contain Outbreak (The Boston Globe, 2020).
15. Morning, P. 80% of Rapid COVID-19 Tests the Czech Republic Bought From China are Wrong Prague Morning (Prague Morning, 2020).
16. Chantal, B. F. et al. Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR assays. 10.1101/2020.03.30.20048108 (2020).
17. DuBridge RB Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system Mol. Cell Biol. 1987 7 379 387 10.1128/MCB.7.1.379 3031469

상세보기
18. Kim, D. et al. The architecture of SARS-CoV-2 transcriptome. Cell 181 , 914？921 (2020).
19. Govorkova EA Murti G Meignier B de Taisne C Webster RG African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses J. Virol. 1996 70 5519 5524 10.1128/JVI.70.8.5519-5524.1996 8764064

상세보기
20. Park WB Virus isolation from the first patient with SARS-CoV-2 in Korea J. Korean Med. Sci. 2020 35 e84 10.3346/jkms.2020.35.e84 32080990

상세보기
21. Untergasser A Primer3-new capabilities and interfaces Nucleic Acids Res 2012 40 e115 10.1093/nar/gks596 22730293

상세보기
22. Grantzdorffer, A. & Nemetz, C. in Cell-Free Protein Expression (ed. Swartz, J. R.) Ch. 5 (Springer, Berlin, Heidelberg, 2003).
23. Pfaffl, M. in Rapid Cycle Real-Time PCR (eds Meuer, S. et al.) 281？291 (Springer, Berlin, Heidelberg, 2001).
24. Ririe KM Rasmussen RP Wittwer CT Product differentiation by analysis of DNA melting curves during the polymerase chain reaction Anal. Biochem. 1997 245 154 160 10.1006/abio.1996.9916 9056205

상세보기
25. Ruiz-Villalba A van Pelt-Verkuil E Gunst QD Ruijter JM van den Hoff MJ Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR) Biomol. Detect Quantif. 2017 14 7 18 10.1016/j.bdq.2017.10.001 29255685

상세보기
26. Tsubouchi K The CD44 standard isoform contributes to radioresistance of pancreatic cancer cells J. Radiat. Res. 2017 58 816 826 10.1093/jrr/rrx033 29106581

상세보기
27. Song W Validation of housekeeping genes for the normalization of RT-qPCR expression studies in oral squamous cell carcinoma cell line treated by 5 kinds of chemotherapy drugs Cell Mol. Biol. (Noisy-le.-Gd.) 2016 62 29 34

LOADING...

활용도 분석정보

상세보기

다운로드

내보내기

활용도 Top5 논문

해당 논문의 주제분야에서 활용도가 높은 상위 5개 콘텐츠를 보여줍니다.
더보기 버튼을 클릭하시면 더 많은 관련자료를 살펴볼 수 있습니다.

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증

[국내논문] Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR 원문보기

Abstract ▼ AI-Helper

참고문헌 (27)

이 논문을 인용한 문헌

연구과제 타임라인

활용도 분석정보

활용도 Top5 논문

관련 콘텐츠

원문 보기

원문 URL 링크

오픈액세스(OA) 유형

이 논문과 함께 이용한 콘텐츠

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트

연합인증

[국내논문] Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR 원문보기

Abstract ▼ AI-Helper

참고문헌 (27)

이 논문을 인용한 문헌

연구과제 타임라인

전체(0) 논문(0) 특허(0) 보고서(0)

전체(0) 논문(0) 특허(0) 보고서(0)

활용도 분석정보

활용도 Top5 논문 더보기

관련 콘텐츠

원문 보기

원문 URL 링크

오픈액세스(OA) 유형

이 논문과 함께 이용한 콘텐츠

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트

활용도 Top5 논문