The design of Indonesian SARS‐CoV‐2 primers based on phylogenomic analysis of the SARS‐CoV‐2 clades

Tsania Taskia Nabila(1), Ata Rofita Wasiati(2), Afif Pranaya Jati(3), Annisa Khumaira(4*)

(1) Biotechnology Study Program, Faculty of Science and Technology, Universitas ‘Aisyiyah Yogyakarta, Jl. Ringroad Barat No.63, Nogotirto, Gamping, Sleman, Daerah Istimewa Yogyakarta 55592, Indonesia
(2) Biotechnology Study Program, Faculty of Science and Technology, Universitas ‘Aisyiyah Yogyakarta, Jl. Ringroad Barat No.63, Nogotirto, Gamping, Sleman, Daerah Istimewa Yogyakarta 55592, Indonesia
(3) Masyarakat Bioinformatika dan Biodiversitas Indonesia (MABBI), Ruang 613, Lantai 6, Program Studi Bioteknologi, Universitas Esa Unggul, Jl. Arjuna Utara No. 9, Jakarta Barat 11510, Indonesia
(4) Biotechnology Study Program, Faculty of Science and Technology, Universitas ‘Aisyiyah Yogyakarta, Jl. Ringroad Barat No.63, Nogotirto, Gamping, Sleman, Daerah Istimewa Yogyakarta 55592, Indonesia
(*) Corresponding Author


Molecular detection needs to be augmented for COVID‐19 detection in Indonesia using the PCR method with primer‐based gene analysis. This is necessary because the RNA of the SARS‐CoV‐2 virus, the causative infectious agent of the pandemic, has been mutated. Therefore, this study aimed to develop a primer design for determining SARS‐CoV‐2 clades in Indonesia using phylogenomic analysis. Data were obtained from 38 GISAID (Global Initiative on Sharing All Influenza Data) viruses and the relationships were analyzed using maximum likelihood (ML) phylogenomic analysis with a substitution model of generalized time‐reversible (GTR) to construct the tree topology. The results showed that the five types of SARS‐CoVs‐2 clades in Indonesia were L, G, GH, GR, and O. It also indicated that the GH region had the highest rate of clade at 50%, with the S clade affecting its formation. Furthermore, the genome sequences of the GH type used to design its primer were based on three genes, namely RdRp, S, and N. The RdRp and N genes were found to be conserved and hardy mutants, while the S gene occurred repeatedly. Several previous studies have stated that the designed primers produced missense mutations compared to another in silico. Therefore, three sets of primers were achieved from the GC contents and clamps, Tm range, and structural secondary indicator standards.


maximum likelihood method; phylogenomic analysis; primer design; SARS‐CoV‐2

Full Text:



Ansori ANM, Kharisma VD, Antonius Y, Tacharina MR, Rantam FA. 2020. Immunobioinformatics analysis and phylogenetic tree construction of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) in Indonesia: spike glycoprotein gene. J. Teknol. Lab. 9(1). doi:10.29238/teknolabjournal.v9i1.221.

Arenas M. 2015. Trends in substitution models of molecular evolution. Front. Genet. 6(OCT). doi:10.3389/fgene.2015.00319.

Boopathi S, Poma AB, Kolandaivel P. 2020. Novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment. doi:10.1080/07391102.2020.1758788.

Bustin SA, Nolan T. 2020. RT­QPCR testing of SARS­COV­2: A primer. Int. J. Mol. Sci. 21(8). doi:10.3390/ijms21083004.

Davi MJP, Jeronimo SM, Lima JP, Lanza DC. 2021. Design and in silico validation of polymerase chain reaction primers to detect severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2). Sci. Rep. 11(1). doi:10.1038/s41598­021­91817­9.

Dhar A, Minin V. 2016. Maximum Likelihood Phylogenetic Inference. Oxford: Academic Press. doi:­0­12­800049­ 6.00207­9. URL nce/article/pii/B9780128000496002079.

Djalante R, Lassa J, Setiamarga D, Sudjatma A, Indrawan M, Haryanto B, Mahfud C, Sinapoy MS, Djalante S, Rafliana I, Gunawan LA, Surtiari GAK, Warsilah H. 2020. Review and analysis of current responses to COVID­19 in Indonesia: Period of January to March 2020. Prog. Disaster Sci. 6. doi:10.1016/j.pdisas.2020.100091.

Eaaswarkhanth M, Al Madhoun A, Al­Mulla F. 2020. Could the D614G substitution in the SARS­CoV­ 2 spike (S) protein be associated with higher COVID­19 mortality? Int. J. Infect. Dis. 96. doi:10.1016/j.ijid.2020.05.071.

Githinji G, de Laurent ZR, Mohammed KS, Omuoyo DO, Macharia PM, Morobe JM, Otieno E, Kinyanjui SM, Agweyu A, Maitha E, Kitole B, Suleiman T, Mwakinangu M, Nyambu J, Otieno J, Salim B, Kasera K, Kiiru J, Aman R, Barasa E, Warimwe G, Bejon P, Tsofa B, Ochola­Oyier LI, Nokes DJ, Agoti CN. 2021. Tracking the introduction and spread of SARS­CoV­2 in coastal Kenya. Nat. Commun. 12(1). doi:10.1038/s41467­021­25137­x.

Guindon S, Lethiec F, Duroux P, Gascuel O. 2005. PHYML Online ­ A web server for fast maximum likelihood­based phylogenetic inference. Nucleic Acids Res. 33(SUPPL. 2). doi:10.1093/nar/gki352.

Hamed SM, Elkhatib WF, Khairalla AS, Noreddin AM. 2021. Global dynamics of SARS­CoV­2 clades and their relation to COVID­19 epidemiology. Sci. Rep. 11(1). doi:10.1038/s41598­021­87713­x.

Islam MT, Alam ARU, Sakib N, Hasan MS, Chakrovarty T, Tawyabur M, Islam OK, Al­Emran HM, Jahid MIK, Anwar Hossain M. 2021. A rapid and costeffective multiplex ARMS­PCR method for the simultaneous genotyping of the circulating SARSCoV­2 phylogenetic clades. J. Med. Virol. 93(5). doi:10.1002/jmv.26818.

Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, Hengartner N, Giorgi EE, Bhattacharya T, Foley B, Hastie KM, Parker MD, Partridge DG, Evans CM, Freeman TM, de Silva TI, Angyal A, Brown RL, Carrilero L, Green LR, Groves DC, Johnson KJ, Keeley AJ, Lindsey BB, Parsons PJ, Raza M, Rowland­Jones S, Smith N, Tucker RM, Wang D, Wyles MD, McDanal C, Perez LG, Tang H, Moon­Walker A, Whelan SP, LaBranche CC, Saphire EO, Montefiori DC. 2020. Tracking Changes in SARS­CoV­2 Spike: Evidence that D614G Increases Infectivity of the COVID­19 Virus. Cell 182(4). doi:10.1016/j.cell.2020.06.043.

Kuo L, Masters PS. 2003. The Small Envelope Protein E Is Not Essential for Murine Coronavirus Replication. J. Virol. 77(8). doi:10.1128/jvi.77.8.4597­4608.2003.

Li D, Zhang J, Li J. 2020. Primer design for quantitative real­time PCR for the emerging Coronavirus SARS­CoV­2. Theranostics 10(16):7150– 7162. doi:10.7150/thno.47649.

Makarenkov V, Kevorkov D, Legendre P. 2006. Phylogenetic Network Construction Approaches, volume 6. Elsevier. doi:­ 5334(06)80006­7.

Mercatelli D, Giorgi FM. 2020. Geographic and Genomic Distribution of SARS­CoV­2 Mutations. Front. Microbiol. 11. doi:10.3389/fmicb.2020.01800.

Pereira­Gómez M, Fajardo Á, Echeverría N, LópezTort F, Perbolianachis P, Costábile A, Aldunate F, Moreno P, Moratorio G. 2021. Evaluation of SYBR Green real time PCR for detecting SARSCoV­2 from clinical samples. J. Virol. Methods 289. doi:10.1016/j.jviromet.2020.114035.

Sanjuán R, Domingo­Calap P. 2016. Mechanisms of viral mutation. doi:10.1007/s00018­016­2299­6. S

cohy A, Anantharajah A, Bodéus M, Kabamba­Mukadi B, Verroken A, Rodriguez­Villalobos H. 2020. Low performance of rapid antigen detection test as frontline testing for COVID­19 diagnosis. J. Clin. Virol. 129. doi:10.1016/j.jcv.2020.104455.

Selberg AG, Gaucher EA, Liberles DA. 2021. Ancestral Sequence Reconstruction: From Chemical Paleogenetics to Maximum Likelihood Algorithms and Beyond. doi:10.1007/s00239­021­09993­1.

Shereen MA, Khan S, Kazmi A, Bashir N, Siddique R. 2020. Covid­19 infection: origin, transmission, and characteristics of human coronaviruses. Https:// 65.

World Health Organization (WHO).2020. Advice on the use of masks in the c. J. Adv. Res. 24. Tang YW, Schmitz JE, Persing DH, Stratton CW. 2020. Laboratory diagnosis of COVID­19: Current issues and challenges. doi:10.1128/JCM.00512­20.

Tavaré S. 1986. Some probabilistic and statistical problems in the analysis of DNA sequences. Turista DDR, Islamy A, Kharisma VD, Ansori ANM. 2020. Distribution of COVID­19 and phylogenetic tree construction of sars­CoV­ 2 in Indonesia. J. Pure Appl. Microbiol. 14. doi:10.22207/JPAM.14.SPL1.42.

Umair M, Ikram A, Salman M, Khurshid A, Alam M, Badar N, Suleman R, Tahir F, Sharif S, Montgomery J, Whitmer S, Klena J. 2021. Whole­genome sequencing of SARS­CoV­2 reveals the detection of G614 variant in Pakistan. PLoS One 16(3 March). doi:10.1371/journal.pone.0248371.

Van de Peer Y, Salemi M. 2012. Phylogenetic inference based on distance methods. Cambridge University Press. doi:10.1017/cbo9780511819049.007.

Vega­Magaña N, Sánchez­Sánchez R, Hernández­Bello J, Venancio­Landeros AA, Peña­Rodríguez M, VegaZepeda RA, Galindo­Ornelas B, Díaz­Sánchez M, García­Chagollán M, Macedo­Ojeda G, GarcíaGonzález OP, Muñoz­Valle JF. 2021. RT­qPCR Assays for Rapid Detection of the N501Y, 69­70del, K417N, and E484K SARS­CoV­2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact. Front. Cell. Infect. Microbiol. 11. doi:10.3389/fcimb.2021.672562.

Venkataraman S, Prasad BV, Selvarajan R. 2018. RNA dependent RNA polymerases: Insights from structure, function and evolution. doi:10.3390/v10020076.

Wang Y, Kang H, Liu X, Tong Z. 2020. Combination of RT­qPCR testing and clinical features for diagnosis of COVID­19 facilitates management of SARS­CoV­2 outbreak. doi:10.1002/jmv.25721.

Wurm T, Chen H, Hodgson T, Britton P, Brooks G, Hiscox JA. 2001. Localization to the Nucleolus Is a Common Feature of Coronavirus Nucleoproteins, and the Protein May Disrupt Host Cell Division. J. Virol. 75(19). doi:10.1128/jvi.75.19.9345­9356.2001.


Article Metrics

Abstract views : 1059 | views : 719


Copyright (c) 2022 The Author(s)

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.