Dados do Trabalho

Título

Evaluation of the gene encoding the SARS-CoV-2 E protein for qualitative diagnosis by RT-PCR

Introdução

SARS-CoV-2 is a positive-sense RNA virus of the Coronavidae family, discovered in China in December 2019 and globally disseminated, causing thousands of infections and deaths. Vaccines are essential for reducing COVID-19 morbidity; however, the circulation of the virus in the population persists, with the risk of the emergence of new variants with unpredictable clinical characteristics. Epidemiological surveillance of circulating viruses and the search for molecular markers that facilitate diagnosis and study of genetic variability are of particular importance to prevent SARS-CoV-2 indiscriminate dissemination. Envelope (E) protein of SARS-CoV-2 is one of the structural proteins of the virus involved in various stages of the virus replication cycle. 

Objetivo (s)

To investigate the genetic variability of the SARS-CoV-2 E protein, for the use of the gene encoding it in diagnostic tests and epidemiological surveillance.

Material e Métodos

Standardize a qualitative detection method for SARS-CoV-2, using the E protein gene, and investigate genetic polymorphisms in samples collected in 2021 and 2022 in the Metropolitan Region of São Paulo. The research, approved by the Ethics Committee on Human Research of UFABC (CAAE 39420920.1.0000.0082), involved 1729 saliva RNA samples from individuals diagnosed by RT-PCR. Synthesis of the complementary DNA (cDNA) was performed by using the Reverse Transcriptase from Cellco according to manufacturer instructions. cDNA quality was verified by amplification of a 430 bp DNA fragment corresponding to the human mitochondrial hypervariable region (HV1). To amplify the SARS-CoV-2 E protein-encoding gene, a pair of oligonucleotides was designed to obtain the complete sequence. 

Resultados e Conclusão

All cDNA samples produced amplified the HV1 430 bp DNA fragment. The SARS-CoV-2 E encoding gene fragment is bein amplified from samples with RTqPCR CT value lower than 35. PCR products obtained will be sent for sequencing to study the genetic variability of the virus E encoding gene. The methodology using self-collected saliva samples showed efficiency in detecting SARS-CoV-2, allowing direct genetic investigations in clinical samples.

Palavras Chave

SARS-CoV-2; Molecular diagnosis; sequencing.