Low-cost SYBR Green-based RT-qPCR assay for detecting SARS-CoV-2 in an Indonesian setting using WHO-recommended primers

Ratika Rahmasari, Muhareva Raekiansyah, Syifa Naura Azallea, Marvella Nethania, Navany Bilqisthy, Anna Rozaliyani, Anom Bowolaksono, Rani Sauriasari

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

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. The compatibility of the two primers was tested in silico with Indonesian SARS-CoV-2 genome sequences retrieved from the GISAID database and using 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 the detection of SARS-CoV-2 RNA for diagnostic purposes.

Original languageEnglish
Article numbere11130
JournalHeliyon
Volume8
Issue number11
DOIs
Publication statusPublished - Nov 2022

Keywords

  • Intercalating dye SYBR Green
  • One-step RT-qPCR
  • SARS-CoV-2

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