Dados do Trabalho

Título

Development of an isothermal Reverse Transcriptase – Recombinase Polymerase Amplification (RT-RPA) to detect SARS-CoV-2

Introdução

The gold standard diagnostics for COVID-19 is the RT-qPCR. However, the RT-qPCR is expensive and requires specialized human resources to execute and analyze the results. Since COVID-19 is a fast-spreading disease, a large number of tests has been necessary for disease control. Therefore, easy and fast-executable tests are needed for COVID-19.

Objetivo (s)

To develop a prototype of a molecular diagnostic test based on the isothermal PCR Recombinase Polymerase Amplification (RPA) for COVID-19.

Material e Métodos

RPA primers and FAM-containing probes were designed to target the nucleoprotein (N) of SARS-CoV-2 (Wuhan strain), based on the literature data and sequence alignments. Protocol optimizations were performed with the aim to reduce reagent use and improve the detection. In vitro transcription (IVT) was performed to produce mRNA encoding the N protein using a EcoRV-linearized pET21a vector. Thirty-three clinical samples from the Molecular Diagnostic Laboratory of SENAI CIMATEC were used for test validation. All reactions were performed at 42° C for 30 minutes in QuantiStudio1.

Resultados e Conclusão

First, the manufacturing protocol for RPA was optimized. The reaction final volume was reduced from 50 to 10 µL, and the sample volume was doubled, which reduced sample dilution by 10-fold, improving the detection. mRNA encoding N protein produced by IVT was detected as low as 1 pg/reaction. The reverse transcriptase (RT)-RPA standardized presented 60% of sensitivity and 100% of specificity in human swab samples previously characterized by RT-qPCR. The RT-RPA false negative results were associated with samples presenting a RT-qPCR(Ct) median of 32, while the RT-RPA true positive results were associated with a lower RT-qPCR(Ct) median equal to 24.
The RT-RPA shown in this work presented low sensibility, despite the detection at picogram levels of IVT-produced mRNA, but high specificity to clinical samples. Thus, RT-RPA can be a promising alternative for high-cost molecular tests due to its faster, easier, and cheaper performance than the conventional methods.

Palavras-chave

Diagnostics. Reverse transcriptase-recombinase polymerase amplyfication. COVID-19. SARS-CoV-2