Comparative Analysis of In-House RT-qPCR Detection of SARS-CoV-2 for Resource-Constrained Settings

We developed and standardized an efficient and cost-effective in-house RT-PCR method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated sensitivity, specificity, and other statistical parameters by different RT-qPCR methods including triplex, duplex, and simplex ass...

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Bibliographic Details
Published in:Diagnostics (Basel) 2022-11, Vol.12 (11), p.2883
Main Authors: Bello-Lemus, Yesit, Anaya-Romero, Marco, Gómez-Montoya, Janni, Árquez, Moisés, González-Torres, Henry J, Navarro-Quiroz, Elkin, Pacheco-Londoño, Leonardo, Pacheco-Lugo, Lisandro, Acosta-Hoyos, Antonio J
Format: Article
Language:English
Subjects:
Accuracy
Coronaviruses
COVID-19
Diagnostic tests
Genes
Genetic engineering
in-house PCR
Infections
Laboratories
Pandemics
RNA polymerase
RT-PCR
SARS-CoV-2 diagnostics
Severe acute respiratory syndrome coronavirus 2
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Summary:We developed and standardized an efficient and cost-effective in-house RT-PCR method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated sensitivity, specificity, and other statistical parameters by different RT-qPCR methods including triplex, duplex, and simplex assays adapted from the initial World Health Organization- (WHO) recommended protocol. This protocol included the identification of the E envelope gene (E gene; specific to the Sarvecovirus genus), RdRp gene of the RNA-dependent RNA polymerase (specific for SARS-CoV-2), and RNase P gene as endogenous control. The detection limit of the E and the RdRp genes were 3.8 copies and 33.8 copies per 1 µL of RNA, respectively, in both triplex and duplex reactions. The sensitivity for the RdRp gene in the triplex and duplex RT-qPCR tests were 98.3% and 83.1%, respectively. We showed a decrease in sensitivity for the RdRp gene by 60% when the E gene acquired Ct values > 31 in the diagnostic tests. This is associated with the specific detection limit of each gene and possible interferences in the protocol. Hence, developing efficient and cost-effective methodologies that can be adapted to various health emergency scenarios is important, especially in developing countries or settings where resources are limited.
ISSN:2075-4418
2075-4418
DOI:10.3390/diagnostics12112883