Low-cost SYBR Green-based RT-qPCR assay for detecting SARS-CoV-2 in an Indonesian setting using WHO-recommended primers.
Heliyon
; 8(11): e11130, 2022 Nov.
Article
in English
| MEDLINE | ID: covidwho-2179006
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.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Type of study:
Diagnostic study
/
Experimental Studies
/
Prognostic study
Language:
English
Journal:
Heliyon
Year:
2022
Document Type:
Article
Affiliation country:
J.heliyon.2022.e11130
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