The Production of Standardized Samples with Known Concentrations for Severe Acute Respiratory Syndrome Coronavirus 2 RT-qPCR Testing Validation for Developing Countries in the Period of the Pandemic Era.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic of pneumonia spreading around the world, leading to serious threats to public health and attracting enormous attention. There is an urgent need for sensitive diagnostic testing implementation to control and manage SARS-CoV-2 in public health laboratories. The quantitative reverse transcription PCR (RT-qPCR) assay is the gold standard method, but the sensitivity and specificity of SARS-CoV-2 testing are dependent on a number of factors. METHODS: We synthesized RNA based on the genes published to estimate the concentration of inactivated virus samples in a biosafety level 3 laboratory. The limit of detection (LOD), linearity, accuracy, and precision were evaluated according to the bioanalytical method validation guidelines. RESULTS: We found that the LOD reached around 3 copies/reaction. Furthermore, intra-assay precision, accuracy, and linearity met the accepted criterion with an RSD for copies of less than 25%, and linear regression met the accepted R 2 of 0.98. CONCLUSIONS: We suggest that synthesized RNA based on the database of the NCBI gene bank for estimating the concentration of inactivated virus samples provides a potential opportunity for reliable testing to diagnose coronavirus disease 2019 (COVID-19) as well as limit the spread of the disease. This method may be relatively quick and inexpensive, and it may be useful for developing countries during the pandemic era. In the long term, it is also applicable for evaluation, verification, validation, and external quality assessment.

DNA-binding studies on the Bacillus subtilis transcriptional regulator and AbrB homologue, SpoVT.

The process of spore formation in Bacillus subtilis is dependent upon a sophisticated program of gene expression that is regulated both temporally and spatially by a series of alternate sigma factors, in conjunction with a number of transcriptional regulators. One of these, SpoVT, regulates forespore-specific sigmaG-dependent transcription and is related at the amino acid level to the major stationary phase sentinel, AbrB, whose mode of DNA recognition appears to be non-classical. Here, we report that the C-terminal domain of SpoVT is crucial to its correct folding and function, and how the DNA-binding domain from AbrB cannot complement the closely homologous domain of SpoVT in vivo. We also establish the oligomeric state of SpoVT and its component domains. Finally, we demonstrate that the regulation of transcriptional control by SpoVT is unexpectedly more complicated than its counterpart, AbrB, and that the latent non-specific DNA-binding activity of the N-terminal domain of SpoVT is modulated by the C-terminal domain, which perhaps in combination with another unknown factor, confers specificity.