Utilization of in silico-designed primers for SARS-CoV-2 molecular surveillance using Direct PCR Product Sequencing Surveillance (DPPSS) method.

Sarah Jane DATAY-LIM; Flyndon Mark DAGALEA; Michael Reigh GUEVARRA; Kristine AVILA; Kim Claudette FERNANDEZ; Francisco HERALDE III

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Utilization of in silico-designed primers for SARS-CoV-2 molecular surveillance using Direct PCR Product Sequencing Surveillance (DPPSS) method.

Author: Sarah Jane DATAY-LIM ¹ ; Flyndon Mark DAGALEA ² ; Michael Reigh GUEVARRA ³ ; Kristine AVILA ³ ; Kim Claudette FERNANDEZ ³ ; Francisco HERALDE III ³
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1. Department of Laboratory Medicine and Pathology, The Medical City, Pasig, Metro Manila, NCR, Philippines;Department of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines Manila, Philippine General Hospital, Manila, Metro Manila, NCR, Philippines
2. Department of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines Manila, Philippine General Hospital, Manila, Metro Manila, NCR, Philippines;Department of Chemistry, College of Science, University of Eastern Philippines, Catarman, Northern Samar, Region 08, Philippines
3. Department of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines Manila, Philippine General Hospital, Manila, Metro Manila, NCR, Philippines
Publication Type:Journal Article, Original
Keywords: Virus Variants; Sequencing
MeSH: Covid-19; Polymerase Chain Reaction; Molecular Diagnostic Techniques
From: Philippine Journal of Pathology 2026;11(1):12-19
CountryPhilippines
Abstract: BACKGROUND
The COVID-19 pandemic caused by SARS-CoV-2 significantly strained healthcare systems in the Philippines, highlighting the critical importance of reliable molecular diagnostics and genomic surveillance. Although vaccination efforts and public health measures mitigated disease impact, the continued emergence of viral variants underscores the need for sustainable local surveillance strategies. Strengthening in-country capacity through the development of in silico–designed primers and cost-effective sequencing approaches can enhance rapid variant detection and improve preparedness for future emerging infectious diseases.
OBJECTIVE
This paper offers a method in detecting the SARS-CoV-2 virus and its variants. A direct PCR product sequencing surveillance or DPPSS offers a new possibility of detecting emerging disease by using PCR products and using it as templates in determining the base sequence.
METHODOLOGY
A total of 20 random positive samples for SARS-COV-2 from March 2022 sample pool in Metro Manila, Philippines was used in this study. The RNA was extracted using Purelink™ RNA Mini Kit, quantified with NanoDrop, and subjected to one-step RT-PCR. An in-house designed in silico primers were used in this study by using thermodynamic parameters to optimize specificity and amplification efficiency, considering GC contents, balanced Tm, minimal secondary structures and cross-dimers and in silico validation via Basic Local Alignment Search Tool (BLAST) against reference databases.
Amplicons were analyzed through gel electrophoresis, sequenced, and analyzed using BioEdit software. A nucleotide BLAST search identified COVID-19 variants, confirmed using Cov-Lineages website.
RESULTS
In silico designed primers (S1, S2, E/M, Orf1ab) collectively exhibited 100% sensitivity in detecting SARS-CoV2 in nasopharyngeal swab samples. Individual primer sensitivities varied, with Orf1ab at 58.82% and E/M at 90.91%. Our analysis revealed the prevalence of Omicron sublineage BA.2 in the Philippines, aligning with local data showing more BA.2 cases than the global predominance of BA.1.
CONCLUSION
Combined in silico primers (S1, S2, E/M, ORF1ab) accurately detect SARS-COV-2 and its variants. This method provides a valuable diagnostic and surveillance tool for public health management.