RESUMEN
Due to the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N, and DNA/RNA Shield™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis.
Asunto(s)
COVID-19 , COVID-19/diagnóstico , Prueba de COVID-19 , Chile , Medios de Cultivo , Humanos , Nasofaringe , Pandemias , ARN , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/genética , Manejo de Especímenes/métodosRESUMEN
Mitochondrial tRNAs are processed at their 5'ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2. The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p.
Asunto(s)
Regulación Fúngica de la Expresión Génica/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN de Hongos/biosíntesis , ARN Mitocondrial/biosíntesis , ARN de Transferencia/biosíntesis , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , ARN de Hongos/genética , ARN Mitocondrial/genética , ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.
Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Vacuna contra la Fiebre Amarilla/genética , Vacuna contra la Fiebre Amarilla/normas , Fiebre Amarilla/inmunología , Fiebre Amarilla/prevención & control , Virus de la Fiebre Amarilla/genética , Animales , Especificidad de Anticuerpos , Chlorocebus aethiops , Humanos , Plásmidos/genética , Control de Calidad , ARN Viral/inmunología , ARN Viral/aislamiento & purificación , Estándares de Referencia , Reproducibilidad de los Resultados , Células Vero , Carga Viral , Viremia/virología , Fiebre Amarilla/virología , Vacuna contra la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
RNase P is a ribozyme originally identified for its role in maturation of tRNAs by cleavage of precursor tRNAs (pre-tRNAs) at the 5'-end termini. RNase P is a ribonucleoprotein consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. The site of cleavage of a pre-tRNA is identified by its tertiary structure; and any RNA molecule can be cleaved by RNase P as long as the RNA forms a duplex that resembles the regional structure in the pre-tRNA. When the antisense sequence that forms the duplex with the strand that is subsequently cleaved by RNase P is in a separate molecule, it is called an external guide sequence (EGS). These fundamental observations are the basis for EGS technology, which consists of inhibiting gene expression by utilizing an EGS that elicits RNase P-mediated cleavage of a target mRNA molecule. EGS technology has been used to inhibit expression of a wide variety of genes, and may help development of novel treatments of diseases, including multidrug-resistant bacterial and viral infections.