Positive control synthesis method for COVID-19 diagnosis by one-step real-time RT-PCR

Cho, Hyejin; Hwan Jung, Young; Bum Cho, Hong; Kim, Hee-tae; Kim, Kwang-sun

Backgrounds The coronavirus disease 2019 (COVID-19) pandemic is still ongoing. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is regarded as a gold-standard method. However, COVID-19 test kits, especially the positive control samples that are synthesized by laboratories or companies, have increased the inconclusiveness of disease interpretation. Therefore, development of new methods for the preparation of reliable positive controls that are not impaired by contamination for diagnosing COVID-19 remains a challenge. Methods A new approach for producing synthetic positive controls using synthetic positive template (SPT) oligonucleotides was designed. SPT oligonucleotides contain probe binding and virus-irrelevant regions as templates for detecting SARS-CoV-2 genes (RdRP, E, and N SARS-CoV-2) by real-time RT-PCR was performed in a concentration-dependent manner. The limit of detection (LOD) for individual SARS-CoV-2 genes by Ct values with concentration ranges was determined with SPT templates and genomic RNAs prepared from SARS-CoV-2. Results LODs with SPT templates were >10-15 (atto) M for RdRP, 10-12 (femto) to 10-13 (100 atto) M for E gene, and 10-12 to 10-14 (10 atto) M for N gene, respectively. Real-time RT-PCR assay using serially diluted genomic RNAs prepared from SARS-CoV-2 showed that picogram quantities of RNAs prepared from COVID-19 virus cultures resulted in the LOD. The sensitivity based on Ct values for RdRP and E genes were less sensitive to this platform than N gene. Conclusion This method significantly decreased the risk of contamination and false-positive reactions that result from contamination of the synthesis procedures that are used to produce positive control materials. Therefore, this approach may be integrated into the molecular diagnosis of COVID-19 and provides a general method for preparing positive controls for diagnosing emerging RNA virus infections.