Hidayet TUTUN, Nadir KOÇAK, Özlem ALTINTAŞ, Tutku ARSLANTAŞ, Alper KARAGÖZ, Levent ALTINTAŞ

SARS-CoV-2’nin beş primer seti kullanılarak belirlenmesi

Pnömoni ile ilişkili solunum bozukluğundan (COVID-19) sorumlu yeni bir koronavirüs (SARS-CoV-2) salgını Aralık 2019'un başında Çin'in Wuhan şehrinde başladı ve hızla dünyaya yayıldı. Hızlı ve doğru teşhis testleri COVID-19 salgını ile mücadelede çok önemli bir rol oynar. Bu çalışmada SARS-CoV-2’nin saptanması için farklı bölgelerinin amplifiye edilmesi amacıyla tasarlanmış 5 primer setin karşılaştırılması ve sekans analizinin yapılması amaçlanmıştır. Konvansiyonel RT-PCR, COVID-19 tanısı için ORF1ab, Zarf (E), RNA-bağlı RNA polimeraz (RdRp), Spike (S) ve Nükleokapsid (N) genleri içeren virüs genomunun farklı bölgelerini hedefleyen primerler kullanıldı ve ORF1ab geninin DNA dizisi, GenBank SARS-CoV-2 DNA dizisi verileriile karşılaştırılarak, ORF1ab filogenetik ağacı oluşturuldu. ORF1ab, S, E, N ve RdRp genlerinin amplikon boyutları, sırasıyla 588bp, 440 bp, 145 bp, 323 bp ve 196 bp idi. Toplam örneklerin %74’ünde RdRp geni, %87’sinde N geni, %74’ünde S geni, %61’inde Egeni ve %82’sinde ORF1ab geni tespit edildi. 82 hastadan SARS-CoV-2'nin ORF1ab dizileri, Wuhan izolat dizisi ve kendi aralarında %100 özdeşliğe sahipti. Filogenetik analiz, tüm izolatların bir küme oluşturduğunu ortaya çıkarmıştır. Bu çalışmanın sonuçları, N bölgesinin SARS-CoV-2 tespiti için en iyisi olduğunu göstermektedir.

Detection of SARS-CoV-2 using five primer sets

A novel coronavirus (SARS-CoV-2) outbreak, responsible for a pneumonia-associated respiratory disorder (COVID-19), has started in early December 2019 in Wuhan, China, and has rapidly spread around the world. Rapid and accurate diagnostic testing plays a crucial role in tackling the COVID-19 pandemic. In this study, it was aimed to compare 5 primer sets designed to amplify different regions for the detection of SARS-CoV-2 and to perform sequence analysis. Conventional RT-PCR was carried out using primers targeting different regions of the virus genome including ORF1ab, Envelope (E), RNA-dependent RNA polymerase (RdRp), Spike (S) and Nucleocapsid (N) genes for the diagnosis of COVID-19. DNA sequence of ORF1ab gene from each sample were compared with the DNA sequence data of SARS-CoV-2 stored in the GenBank and ORF1ab phylogenetic tree was constructed. The amplicon sizes of ORF1ab, S, E, N and RdRp genes were 588 bp, 440 bp, 145 bp, 323 bp and 196 bp, respectively. The SARS-CoV-2RNA was detectedfrom 74% of total samples from RdRp gene, 87% for N gene, 74% for S gene, 61% for E gene and 82% for ORF1ab region. The ORF1ab sequences of SARS-CoV-2 from 82 patients were had 100% identity to the sequence of Wuhan isolate and among themselves. The phylogenetic analysis revealed that all isolates formed a cluster. The results of this study suggest that the N region is the best for SARS-CoV-2 identification.

PDF

___

1. Alsuliman T, Sulaiman R, Ismail S, et al (2020): COVID- 19 paraclinical diagnostic tools: Updates and future trends. Curr Res Transl Med. 68, 83-91.
2. Bustin SA, Nolan T (2020): RT-qPCR testing of SARSCoV- 2: a primer. Int J Mol Sci, 21, 3004.
3. Cai Q, Yang M, Liu D, et al (2020): Experimental treatment with favipiravir for COVID-19: an open-label control study. Engineering.
4. Chen B, Tian EK., He B, et al (2020): Overview of lethal human coronaviruses. Signal Transduct Tar, 5, 1-16.
5. Chen N, Zhou M, Dong X, et al (2020): Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. The Lancet, 395, 507-513.
6. Corman VM, Landt O, Kaiser M, et al (2020): Detection of 2019 novel coronavirus (2019-nCoV) by real-time RTPCR. Eurosurveillance, 25, 2000045.
7. Emery SL, Erdman DD, Bowen MD, et al (2004): Realtime reverse transcription–polymerase chain reaction assay for SARS-associated coronavirus. Emerg Infect Dis, 10, 311-316.
8. Holshue ML, DeBolt C, Lindquist S, et al (2020): First case of 2019 novel coronavirus in the United States. N Engl J Med, 36, 929-936.
9. Kilic AU, Kara F, Alp E, et al (2020): New threat: 2019 novel Coronavirus infection and infection control perspective in Turkey. North Clin Istanb, 7, 95-98.
10. Kodaz H (2020): Editorial: Successful Treatment Strategy of Turkey against Covid-19 Outbreak. EJMO, 4, 177-178.
11. Kumar S, Nyodu R, Maurya VK, et al (2020): Morphology, Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV- 2). 23-31. In: SK Saxena (Ed) Coronavirus Disease 2019 (COVID-19). Springer, Singapore.
12. Luan J, Lu Y, Jin X, et al (2020): Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection. Biochem Biophys Res Commun, 526, 165-169.
13. Mathuria JP, Yadav R (2020): Laboratory diagnosis of SARS-CoV-2- a review of current methods. J Infect Public Heal, 13, 901-905.
14. Mollaei HR, Afshar AA, Kalantar-Neyestanaki D, et al (2020): Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR. Iran J Microbiol, 12, 185-193.
15. Omolo CA, Soni N, Fasiku VO, et al (2020): Update on therapeutic approaches and emerging therapies for SARSCoV- 2 virus. Eur J Pharmacol, 883, 173348.
16. Rodríguez-Morales AJ, Balbin-Ramon GJ, Rabaan AA, et al (2020): Genomic Epidemiology and its importance in the study of the COVID-19 pandemic. InfezMed, 2, 139- 142.
17. Sağlık Bakanlığı (2020): Genel Koronavirus Tablosu. Available at https://covid19. saglik.gov.tr/TR-66122/genelkoronavirus- tablosu.html (Accessed September 13, 2020).
18. Singh AK, Singh A, Shaikh A, et al (2020): Chloroquine and hydroxychloroquine in the treatment of COVID-19 with or without diabetes: A systematic search and a narrative review with a special reference to India and other developing countries. Diabetes Metab Syndr, 14, 241-246.
19. Stebbing J, Phelan A, Griffin I, et al (2020): COVID-19: combining antiviral and anti-inflammatory treatments. Lancet Infect Dis, 20, 400-402.
20. Tang YW, Schmitz JE, Persing DH, et al (2020): Laboratory diagnosis of COVID-19: current issues and challenges. J Clin Microbiol, 58, e00512-20.
21. The Republic of Turkey Ministry of Health (2020): COVID-19 (SARS-CoV-2 Infection) Guide. July 17, 2020. Available at https://hsgm.saglik.gov.tr/en/covid-19-ingilizce- dokumanlar/rehberler.html. (Accessed July 20, 2020).
22. Udugama B, Kadhiresan P, Kozlowski HN, et al (2020): Diagnosing COVID-19: the disease and tools for detection. ACS Nano, 14, 3822-3835.
23. Wei Q, Wang Y, Ma J, et al (2020): Description of the First Strain of 2019-nCoV, C-Tan-nCoV Wuhan Strain— National Pathogen Resource Center, China, 2020. China CDC Weekly, 2, 81-82.
24. Weiss P, Murdoch DR (2020): Clinical course and mortality risk of severe COVID-19. The Lancet, 395, 1014- 1015.
25. Woo PC, Lau SK, Lam CS, et al (2012): Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol, 86, 3995-4008.
26. Woolhouse M, Scott F, Hudson Z, et al (2012): Human viruses: discovery and emergence. Philos T R Soc B, 367, 2864-2871.
27. Wordometers (2020): COVID-19 Coronavirus Pandemic. Available at https://www.worldometers.info/coronavirus/ (Accessed September 13, 2020).
28. Yang D, Leibowitz JL (2015): The structure and functions of coronavirus genomic 3′ and 5′ ends. Virus Res, 206, 120- 133.
29. Yoldar ZA, Koç BT, Oğuzoğlu TÇ (2020): Phylogenetic analysis of partial transmembrane protein gene of canine coronaviruses detected in Turkey. Ankara Univ Vet Fak Derg, 67, 265-271.
30. Yousefifard M, Zali A, Ali KM, et al (2020): Antiviral therapy in management of COVID-19: a systematic review on current evidence. Arch Acad Emerg Med, 8, e45.
31. Zhang J, Zhou L, Yang Y, et al (2020): Therapeutic and triage strategies for 2019 novel coronavirus disease in fever clinics. Lancet Respir Med, 8, e11-e12.
32. Zhu H, Wei L, Niu P (2020): The novel coronavirus outbreak in Wuhan, China. Global Health Research and Policy, 5, 1-3.