RESUMO
The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.
Assuntos
COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , SARS-CoV-2/fisiologia , Replicação Viral/fisiologia , Proteínas do Nucleocapsídeo de Coronavírus/análise , Células Gigantes/efeitos dos fármacos , Células Gigantes/virologia , Células HEK293 , Humanos , Limite de Detecção , Nasofaringe/virologia , Fosfoproteínas/análise , Fosfoproteínas/genética , RNA Antissenso/farmacologia , RNA Viral , Ribonuclease P/genética , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Sensibilidade e Especificidade , Isolamento Social , Carga Viral , Proteínas Viroporinas/genética , Replicação Viral/efeitos dos fármacosRESUMO
Porcine rotavirus type A (PoRVA) is the main cause of dehydration and diarrhea in piglets, which has a great impact on the development of the pig industry worldwide. A rapid, accurate and sensitive detection method is conducive to the monitoring, control, and removal of PoRVA. In this study, a PoRVA real-time fluorescent reverse transcription recombinase-aided amplification (RT-RAA) assay was developed. Based on the PoRVA VP6 gene, specific primers and probes were designed and synthesized. The sensitivity of RT-RAA and TaqMan probe-based RT-qPCR was 7 copies per reaction and 5 copies per reaction, respectively. The sensitivity of the RT-RAA method was close to TaqMan probe-based RT-qPCR. The detection results of RT-RAA and TaqMan probe-based quantitative real-time RT-PCR methods were completely consistent in 241 clinical samples. Therefore, we successfully established a rapid and specific RT-RAA diagnostic method for PoRVA.