Development of a triplex endpoint PCR assay for the detection of SARS-CoV-2: insights on cost-efficiency and method design.

Introduction: Lower respiratory tract infections, including COVID-19, have a substantial global impact, making the development of diagnostic tests crucial. The study aimed to develop a new, accurate, fast, and cost-effective PCR-based detection method for SARS-CoV-2, applicable in limited settings and capable of detecting all current variants and potential future pathogens.

Methods: The study was conducted between 2020 and 2022 at the molecular biology department of Mures County Clinical Hospital, Romania. Initially, pharyngeal and nasal secretions were collected and processed using the real-time qRT-PCR method for routine COVID-19 diagnosis. Ninety-two samples were randomly selected to develop the assay, including samples from different infection periods and negative controls. Complementary DNA (cDNA) was prepared from the selected samples, and the presence and integrity of the extracted RNA were evaluated by amplifying the GAPDH housekeeping gene. Primers for three specific viral genes (N, ORF1ab, and S) were designed, and their efficiency was evaluated using endpoint PCR and sequencing. Finally, the method was optimized and implemented as a one-step triplex PCR assay for routine diagnostic use.

Results: The molecular laboratory at the Mures County Clinical Hospital (MCCH) analyzed a total of 41,818 samples between June 2020 and December 2022. Among these samples, 26.15% tested positive for SARS-CoV-2, while 70.9% were negative and 2.95% were inconclusive or invalid. Three peaks of positive tests were observed in November 2020, April 2021, and February 2022. The study selected 92 preserved RNA samples for triplex-PCR assay development, validating the primers' specificity and confirming the quality of the nucleic acids. The comparative analysis showed the efficiency and accuracy of the endpoint reverse-transcription triplex-PCR method (RT-PCR), indicating its potential as a cost-effective alternative to real-time reverse-transcription PCR (qRT-PCR) in low-income countries with limited infrastructure for COVID-19 testing.

Conclusion: This method has the potential to facilitate large-scale diagnosis of SARS-CoV-2 infections, allowing for rapid and appropriate therapeutic management and ongoing monitoring of patients. Additionally, the method can be easily adapted for the detection of other pathogens.