Development and Performance Evaluation of a Low-Cost In-House rRTPCR Assay in Ecuador for the Detection of SARS-CoV-2

Author(s): Marco Salinas-Pinta, Diana Aguirre, David De la Torre, Jorge Pérez-Galarza, Ronny Pibaque, Paul Beltran, Tatiana Veloz, Lucy Baldeón-Rojas

Background: Ecuador has had the greatest fatality rate from Coronavirus (COVID-19) in South America during the SARS-CoV-2 pandemic. To control the pandemic, it is necessary to test as much population as possible to prevent the spread of the SARS-CoV-2 infection. For the Ecuadorian population, accessing a PCR test is challenging, since commercial screening kits tend to be expensive.

Aim: The objective of this study was to develop an in-house duplex rRTPCR protocol for the detection of SARS-CoV-2 that contributes to the screening while keeping quality and low testing costs.

Results: An in-house duplex rRT-PCR protocol based on the viral envelope (E) gene target of SARS-CoV-2 and human ribonuclease P gene (RP) as an internal control is reported. The protocol was optimized to obtain with the primers E an efficiency of up to 94.45% and detection of 100% of SARSCoV- 2 up to 15 copies per uL. The clinical performance was determined by a sensibility of 93.8% and specificity of 98.3%.

Conclusion: We developed, standardized, and validated a low-cost, sensitive in-house duplex rRT-PCR assay that may be utilized in lowincome countries.