An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid...

Background: The COVID-19 pandemic relies on real-time polymerase chain reaction (qRT-PCR) for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to facilitate roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers' recommendations to sustain the testing capability in a resource-limited setting. Methods: We used a SARS-CoV-2 positive control RNA sample to generate several 10-fold dilution series that were used for optimization and comparison of the performance of the four qRT-PCR assays: i) Charité Berlin primer-probe set, ii) European Virus Archive - GLOBAL (EVAg) primer-probe set, iii) DAAN premixed commercial kit and iv) Beijing Genomics Institute (BGI) premixed commercial kit. We adjusted the manufacturer- and protocol-recommended reaction component volumes for these assays and assessed the impact on cycle threshold (Ct) values. Results: The Berlin and EVAg E gene and RdRp assays reported mean Ct values within range of each other across the different titrations and with less than 5% difference. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit improved in performance following a reduction of the reaction components. Conclusion: We achieved a 2.6-fold and 4-fold increase in the number of tests per kit for the commercial kits and the primer-probe sets, respectively. All the assays had optimal performance when the primers and probes were used at 0.375X, except for the Berlin N gene assay. The DAAN kit was a reliable assay for primary screening of SARS-CoV-2 whereas the BGI kit's performance was dependent on the volumes and concentrations of both the reaction buffer and enzyme mix. Our recommendation for SARS-CoV-2 diagnostic testing in resource-limited settings is to optimize the assays available to establish the lowest volume and suitable concentration of reagents required to produce valid results.