Optimization and performance evaluation of PCR primers for human respiratory viruses detection

Abstract

Background: Respiratory viral infections are a main cause of acute respiratory disease and contribute to morbidity worldwide. The wide diversity of viruses presents challenges for comprehensive detection.

Objectives: To develop, optimize, and evaluate polymerase chain reaction (PCR) primers targeting genomic regions of human respiratory viruses, including Adenovirus, Bocavirus, Parvovirus B19, Influenza virus, Respiratory syncytial virus, Metapneumovirus, Rhinovirus, Enterovirus A71, Coxsackievirus A16, Parainfluenza virus, Measles virus, Mumps virus, and Coronaviruses.

Methods: Genome reference sequences (2019–2025) were obtained from NCBI and BV-BRC databases. Nucleotide sequences were aligned using MEGA X to identify conserved regions for each virus and designed as primers with degenerate bases to support sequence variation based on standard parameters, which consist of melting temperature (55℃–65℃), GC content (35%–60%), amplicon length (380–700 bp), and predicted secondary structure using the OligoAnalyzer™ and Oligos. Primer specificity was assessed using sequence alignment and similarity searches using the NCBI BLAST to minimize off-target sequences. Then, gradient PCR was performed to optimize the annealing temperature, and the limit of detection was evaluated using a 10-fold serial dilution of the standard plasmid containing the specific gene of each virus.

Results: This study demonstrated that 16 primer pairs with suitable thermodynamic properties exhibited minimal secondary structures. Preliminary results showed that all primers successfully produced PCR products of the expected amplicon size, without cross-amplification with human cDNA samples. The limit of detection ranged from 102 to 105 copies/µL.

Conclusion: These findings provide a preliminary result of suitable candidate primers for human respiratory viral detection.