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All Journal Jurnal Riset Kimia Chimica et Natura Acta
Shabrinna, Hanif
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemistry Environmental Science Materials Science & Nanotechnology

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New Custom Primers for the Detection of SARS-CoV-2 using the Singleplex rRT‒PCR SYBR Green-Based Method with the NSP10 and N genes as Targets Gaffar, Shabarni; Shabrinna, Hanif; Putri, Rafika; Wiraswati, Hesti Lina; Hartati, Yeni Wahyuni; Ishmayana, Safri; Faridah, Lia; Ekawardhani, Savira
Chimica et Natura Acta Vol 13, No 1 (2025)
Publisher : Departemen Kimia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24198/cna.v13.n1.53493

Abstract

Although COVID-19 is no longer a global health emergency, rapid, sensitive, and specific detection tests are still needed. In this study, we developed a cost-effective test, the SYBR Green-based rRT‒PCR kit, using new custom primers targeting the N and NSP10 genes of the SARS-CoV-2 virus. The specificity of the designed primers was determined through agarose gel electrophoresis. A standard curve generated from a ten-fold dilution of SARS-CoV-2 RNA was used to determine the efficiency and sensitivity of the kit. Validation of this protocol was carried out on ten clinical specimens. As expected, the results showed that the N and NSP10 gene primers produced 134 and 161 bp products, respectively. The limits of detection and limit of quantification with N gene primers were 7.74 and 23.46 copies/μL, respectively, and those with the NSP10 gene primers were 4.69 and 14.21 copies/μL, with a PCR efficiency of 102.5% and 110.6%, respectively. The validation results with clinical specimens revealed that seven samples were true-positive for COVID-19 (Ct range 15.09–21.33), and three were confirmed to be true-negative. Costs associated with COVID-19 patient testing can be anticipated to decrease with the use of custom primers for the detection of SARS-CoV-2 via the use of the singleplex rRT‒PCR mix SYBR Green.
In-house Multiplex rRT-PCR Assay for Sars-Cov2 Detection in Indonesia using a new primer design Gaffar, Shabarni; Putri, Rafika Nanda; Shabrinna, Hanif; Supriyadi, Isma Yustifania; Wiraswati, Hesti Lina; Ekawardani, Savira; Ishmayana, Safri; Hartati, Yeni Wahyuni; Faridah, Lia
Jurnal Riset Kimia Vol. 16 No. 1 (2025): March
Publisher : Universitas Andalas

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.25077/jrk.v16i1.781

Abstract

During the COVID-19 pandemic, we attempted to develop an in-house rRT-PCR kit, utilizing custom primers targeting NSP14 and RdRp, with the RPP30 gene as an internal control. This kit will support Indonesian independence in enhancing COVID-19 diagnostics. The primer and probe were designed by a bioinformatics tool, determining primer specificity and sensitivity, optimizing probe concentrations, establishing LoD (Limit of Detection), LoQ (Limit of Quantification), and rRT-PCR efficiency, multiplex testing of the rRT-PCR kit on clinical samples, and testing the kit's stability. The in-house rRT-PCR kit can detect NSP14, RdRp, and RPP30 genes. The optimal concentrations for the NSP14, RdRp, and RPP30 probes are 1 μM, 1.5 μM, and 1.5 μM, respectively. The LoD and LoQ for the NSP14 are 0.22 ng/μL and 0.67 ng/μL, and for the RdRp are 1.08 ng/μL and 3.28 ng/μL. The rRT-PCR efficiencies for the NSP14, RdRp, and RPP30 are 80.3%, 100.6%, and 106%, respectively. Detection of ten clinical samples, comprising seven true positive and three true negative samples, showed Ct (Cycle threshold) values of 28–31 for the RPP30 gene, Ct 21–27 for the RdRp gene, and Ct 30–34 for the NSP14 gene. Stability testing of the rRT-PCR kit demonstrated promising results, where the kit stored at -20°C for seven days showed almost no difference in Ct values. This in-house multiplex rRT-PCR will support Indonesian independence in enhancing COVID-19 diagnostics, providing a dependable method for detecting SARS-CoV-2 in clinical samples.
Co-Authors Ekawardani, Savira Hesti Lina Wiraswati Lia Faridah PUTRI, RAFIKA Putri, Rafika Nanda Safri Ishmayana Savira Ekawardhani Shabarni Gaffar -, Shabarni Gaffar Supriyadi, Isma Yustifania Yeni Wahyuni Hartati