Design and evaluation of degenerate primers targeting the NS3 gene for detection of dengue virus by RT-PCR
Abstract
Background: Dengue fever, caused by the dengue virus, is hyper-endemic in Indonesia. Since no protective vaccine or specific treatments are available, accurate diagnosis is crucial for the early implementation of preventive measures to limit dengue transmission and reduce economic losses. Various diagnostic techniques have been developed, including reverse transcriptase-polymerase chain reaction (RT-PCR) for detecting viral nucleic acids using specific primers.
Objective: This study aimed to design and evaluate the effectiveness of a new primer targeting the NS3 gene of the dengue virus for molecular detection in clinical samples.
Methods: A cross-sectional molecular study was conducted in Banjarnegara, Indonesia. Serum samples were collected from dengue-suspected patients attending hospitals in the city. The diagnosis was initially performed using dengue NS1 antigen and IgG/IgM antibody detection. Dengue virus (DENV) serotyping was conducted using Simplexa Dengue real-time RT-PCR with a newly designed NS3 gene primer. The effectiveness of the new primer was assessed by comparing its performance with a commercial primer.
Results: The primers, DenVNS3F (5’-CGAGTAGGAATGGGWGARGCAGC-3’) and DenVNS3R (5’-CTGTCCAGTGAGCRYGGTCTT-3’) were able to detect the NS3 gene of the dengue virus. However, the level of agreement in detecting the dengue virus compared to the commercial primer showed a moderate agreement (k = 0.60) with a low confidence level.
Conclusion: The newly designed primers DenVNS3F and DenVNS3R are capable of detecting the NS3 gene. However, the primers may require further optimization to achieve a higher level of accuracy and confidence in detecting the dengue virus, and additional validation through sequencing is necessary to confirm the specificity of the amplified product.
References
Nagar PK, Savargaonkar D, Anvikar AR. Detection of Dengue Virus-Specific IgM and IgG Antibodies through Peptide Sequences of Envelope and NS1 Proteins for Serological Identification. J Immunol Res. 2020;2020: 1-8. https://doi.org/10.1155/2020/1820325
Candra K, Asnia Z, Asriati A. Evaluasi Pelaksanaan Program Pencegahan dan Penanggulangan Penyakit Demam Berdarah Dengue di Kota Kendari. Jurnal Ilmiah Obsgin. 2022;14: 226-241. doi:10.36089/job.v14i3.835
Kementerian Kesehatan RI. Profil Kesehatan Indonesia Tahun 2021. Jakarta: Kementrian Kesehatan Republik Indonesia; 2021.
Rahman NAA, Djamil FM, Balasubramaniam VR, Hassan SS, Yap WB. Dengue Virus Non-structural (NS) 1 Gene as A Molecular Marker for Early Detection of in vitro Dengue Virus Infection. Sains Malays. 2021;50: 3035-3043. https://doi.org/10.17576/jsm-2021-5010-16
Sasmono RT, Aryati A, Wardhani P, Yohan B, Trimarsanto H, Fahri S, et al. Performance of Simplexa dengue molecular assay compared to conventional and SYBR Green RT-PCR for detection of dengue infection in Indonesia. PLoS One. 2014;9. https://doi.org/10.1371/journal.pone.0103815
Prayitno PA, Kusmintarsih ES, Wahyono DJ. Deteksi molekuler virus Dengue serotipe 3 pada nyamuk aedes aegypti di wilayah Purwokerto Timur. BioEksakta Jurnal Ilmiah Biologi Unsoed. 2020;2: 215. https://doi.org/10.20884/1.bioe.2020.2.2.1826
Messe Y, Budiarsa IM, Laenggeng AH. Desain Primer Polymerase Chain Reaction (PCR) secara In Silico untuk Amplifikasi Gen gyrA Extensively Drug Resistant Tuberculosis (XDR-TB). jurnalfkipuntad.com. 2020. doi:10.22487/jbse.v8i2.1169
Ayub A, Ashfaq UA, Idrees S, Haque A. Global Consensus Sequence Development and Analysis of Dengue NS3 conserved domains. Biores Open Access. 2013;2: 392-396. https://doi.org/10.1089/biores.2013.0022
Costa SM, Yorio AP, Ajs G, Vidale MM, Ecb C, Mohana-Borges R, et al. Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines. PLoS One. 2011;6: e25685. https://doi.org/10.1371/journal.pone.0025685
Gurukumar K, Priyadarshini D, Patil J, Bhagat A, Singh A, Shah P, et al. Development of real time PCR for detection and quantitation of Dengue Viruses. Virol J. 2009;6. https://doi.org/10.1186/1743-422X-6-10
Johnson BW, Russell BJ, Lanciotti RS. Serotype-Specific detection of dengue viruses in a fourplex Real-Time Reverse Transcriptase PCR assay. J Clin Microbiol. 2005;43: 4977-4983. https://doi.org/10.1128/JCM.43.10.4977-4983.2005
McHugh ML. Interrater reliability: the kappa statistic. Biochem Med (Zagreb). 2012;22: 276-282. https://doi.org/10.11613/BM.2012.031
Sariyanti M, Dewi BE, Sudiro TM. Pengklonaan Gen Nonstruktural 3 Virus Dengue Serotipe 2 (NS3 DENV-2) Strain Indonesia ke dalam Plasmid UMVC4A. Medical Journal of the Christian University of Indonesia. 2014;30: 2-7.
Sasongko ND, Aziz S, Sulisyono Y. Degenerated primers for the KCS enzyme gene encoding erucic acid content in winged bean seeds. IOP Conference Series Earth and Environmental Science 2020;550. 2020. https://doi.org/10.1088/1755-1315/550/1/012032
Purwakasih DB, Achyar A. Desain Primer dan PCR In Silico untuk Deteksi Shigella sp. pada Sampel Air Minum Isi Ulang. Serambi Biologi. 2021;6: 1-6.
Nugraha R, Dewi PS, Nurilmala M. Evaluasi Primer Gen COI sebagai Biomarker Ketertelusuran Ikan menggunakan Bioinformatika. J Pengolah Has Perikan Indones. 2022;25: 67-79. https://doi.org/10.17844/jphpi.v25i1.36501
Fakih TM, Wijaya S, Priani SE. Desain Primer Gen 12S sRNA dari DNA Mitrokondria Babi (Sus scrofa) secara In Silico sebagai Kandidat Primer dalam Analisis Molekuler Kehalalan Produk. Jurnal Sains Farmasi & Klinis. 2021;8: 316. https://doi.org/10.25077/jsfk.8.3.316-322.2021
Yunita R, Rosadi FN, Jamsari N, Azizah A. Optimization of annealing temperature for amplification of the exon one region of the HPPD gene in HA1 accession sunflowers. IOP Conference Series Earth and Environmental Science 2023;1160. 2023. https://doi.org/10.1088/1755-1315/1160/1/012002
Trianom B, Arwiyanto T, Joko T. Perancangan Primer Spesifik Subspesies Berbasis Gen Endoglukanase untuk Deteksi Ralstonia syzygii subsp. syzygii. Jurnal Perlindungan Tanaman Indonesia. 2018;22: 124. https://doi.org/10.22146/jpti.32217
Kesuma S. Uji Diagnosis NS 1. IgG dan IgM Dengue Metode Immunokromatografi dan Elisa. Jurnal Analis Laboratorium Medik. 2022;7: 72-85. https://doi.org/10.51544/jalm.v7i2.3374
Tonglolangi OS, Pratiningrum M, Yadi H. Gejala Klinis dengan Nilai CT pada Pemeriksaan Realtime PCR SARS-CoV-2. Jurnal Kedokteran. 2021;8: 89-99. https://doi.org/10.30872/j.ked.mulawarman.v8i3.6559
Yuenleni. Langkah-Langkah Optimasi PCR. Indonesian Journal of Laboratory. 2019;1: 51-56. https://doi.org/10.22146/ijl.v1i3.48723
Jannah M. Optimalisasi Kondisi PCR untuk Amplifikasi Sekuen Gen HBB. Jurnal Pendidikan Biologi. 2023;12: 36-42. https://doi.org/10.33627/oz.v12i1.1057
Setyawati R, Zubaidah S. Optimasi Konsentrasi Primer dan Suhu Annealing dalam Mendeteksi Gen Leptin pada Sapi Peranakan Ongole (PO) Menggunakan Polymerase Chain Reaction (PCR). Indonesian Journal of Laboratory. 2021;4: 36-40. https://doi.org/10.22146/ijl.v4i1.65550
Mollaei HR, Afshar AA, Neyestanaki DK, 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. https://doi.org/10.18502/ijm.v12i3.3234
Ardiyanto D, Wuryastuty H, Wasito R. Deteksi Brucella abortus dari Sampel Darah-Utuh dengan Uji Polymerase Chain Reaction Tanpa Ekstraksi DNA. Jurnal Sain Veteriner. 2020;38: 222. doi:10.22146/jsv.53506 https://doi.org/10.22146/jsv.53506
Vilahur N, Baccarelli AA, Bustamante M, Agramunt S, Byun H-M, Fernandez MF, et al. Storage conditions and stability of global DNA methylation in placental tissue. Epigenomics. 2013;5: 341-348. https://doi.org/10.2217/epi.13.29
Damo NY, Porotu?o JP, Rambert GI, Disease RFDC. (COVID-19) dengan Pemeriksaan Laboratorium Mikrobiologi Klinik. Jurnal e-Biomedik. 2019;2021: 9. https://doi.org/10.35790/ebm.v9i1.31899
Sulistyowatiningsih WCDW, Wijaya H. Evaluasi Penyebab Hasil Invalid pada Pemeriksaan RT-PCR Pasien COVID-19. Jurnal SainHealth. 2022;6: 1-7. https://doi.org/10.51804/jsh.v6i1.1727.1-7
Martel F, Grundemann D, Schomig E. A simple method for elimination of false positive results in RT-PCR. BMB Rep. 2002;35: 248-250. https://doi.org/10.5483/BMBRep.2002.35.2.248
Merdekawati F, Nurhayati B. Desain primer gen pengkode RNA Dependent RNA Polimerase (RdRp) untuk deteksi SARSCOV2 dengan menggunakan real time polymerase chain reaction. Jurnal Riset Kesehatan Poltekkes Depkes Bandung. 2023;15: 30-36. https://doi.org/10.34011/juriskesbdg.v15i1.2179
Mawarnursavira K, Sari R, Apridamayanti P. Optimasi Melting Temperature Primer Degenerate pada Suhu 60?C Gen ERM(T) (Erythromycin Ribosome Methylase) yang Resistensi terhadap Bakteri Streptococcus pyogenes. Jurnal Mahasiswa Farmasi Fakultas Kedokteran dan Ilmu Kesehatan UNTAN. 2019;4: 1-12.
Copyright (c) 2025 Authors
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
