Abstract

OPTIMIZED PRIMER & PROBE DESIGN FOR TARGETING THE RAPIDLY EVOLVING SARS-COV-2 GENOME USING NCBI AND INTEGRATED DNA TECHNOLOGY (IDT): ENSURING SPECIFICITY AND SENSITIVITY IN REAL-TIME DETECTION

S. K. Erfanul Haque*, Dr. Sukanta Bhadra and Dr. Nishith Kumar Pal

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Abstract

The fast genetic alterations in SARS-CoV-2 genomic sequences affect PCR diagnostic tests by decreasing their sensitivity which produces incorrect negative results. We created an integrated computational workflow relying on NCBI's BLAST, Primer-BLAST along with GenBank databases and IDT OligoAnalyzer and PrimerQuest tools for designing primers and probes which specifically target SARS-CoV-2 genomic sequences prone to conservation. The diagnostic approach achieves both high specificity and sensitivity together with automatic genetic change detection related to the virus. Assay Set-3 proved most efficient from the designed primer sets because it showed a detection threshold at 10 copies/μL and no false-positive results towards HCoV- 229E or HCoV-OC43 human coronaviruses along with human DNA. The experimental tests of clinical samples demonstrated an efficiency level ranging from 98 to 102 percent while maintaining minimal variability at less than 4.35% CV% and precise 95% confidence interval ranges. The statistical analysis validated the testing accuracy through p-values less than 0.001. The flowchart offers a flexible approach to primer creation which enables sustainable yet precise diagnostic testing for viral changes.

Keywords: SARS-CoV-2, NCBI tools, IDT platforms, primer design, PCR-based diagnosis, specificity, sensitivity.