Low Cost SYBR Green-Based RT-qPCR for Detecting SARS-CoV-2 in Indonesia Setting Using WHO-Recommended Primers

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the ongoing coronavirus disease 2019 (COVID-19) pandemic. For laboratory diagnosis, low-cost detection of SARS-CoV-2 is urgently needed, particularly in developing countries with limited resources. Probe- or TaqMan-based real-time reverse transcription polymerase chain reaction (RT-qPCR) is currently the gold standard for diagnosing infected individuals, as recommended by the World Health Organization (WHO). However, this assay is expensive, making it difficult to use for diagnosis on a large scale. Therefore, in this study we develop and validate an alternative approach for RT-qPCR diagnosis by employing the DNA intercalating dye SYBR Green. We evaluate and use two WHO-recommended primers, namely CCDC-N and HKU-ORF1b-nsp14. Compatibility of the two primers was tested in silico with Indonesian SARS-CoV-2 genome sequences retrieved from the GISAID database and using a bioinformatic tools. Using in vitro-transcribed RNA, optimization, sensitivity, and linearity of the two assays targeting the N and Nsp-14 genes were carried out. For further evaluation, we used clinical samples from patients and performed the SYBR Green-based RT-qPCR assay protocol in parallel with TaqMan-based commercial assay. Our results show that our methodology performs similarly to the broadly used TaqMan-based detection method in terms of specificity and sensitivity and thus offers an alternative assay for detection of SARS-CoV-2 RNA for diagnostic purposes.Funding: This research was supported financially by a University of Indonesia Grant.Declaration of Interests: The authors declare no conflicts of interest.Ethics Approval Statement: The study protocol was approved by the Research Ethics Committee of the Faculty of Medicine, University of Indonesia, with Approval number 21-05-0523.