Various reports of decreased analytical sensitivities of real-time PCR-based detection of Coronavirus Disease 2019 (COVID-19) have been associated with occurrence of mutations in the target area of primer/probe binding. Knowledge about propensities of different genes to undergo mutation can inform researchers to select optimal genes to target for the qPCR design. We analyzed supplementary data from over 45 thousand SARS-CoV-2 genomes provided by Mercatelli et al to calculate the unique and prevalent mutations in different genes of SARS-CoV-2. We found that non-structural proteins in the ORF1ab region were more conserved compared to structural genes. Further factors which need to be relied upon for proper selection of genes for qPCR design are discussed.
This research paper presents the design, construction, and performance testing of an automatic electrically powered egg incubator utilizing the horizontal placement of eggs. This experimental research was driven by the specific design and construction of a well-insulated rectangular egg-incubating box of dimension 400 mm × 600 mm × 500 mm and thickness 30 mm, where sample eggs were kept for experimental test analysis under the favorable temperature and humidity inside the box, which was automatically controlled. The device was made to operate under the temperature range of 37°C – 38°C, which was found to be adequate for developing embryos and also maintaining relative humidity at the range of 45–60% for the first 18 days and 60–90% for the last three days. Finally, in this machine, the egg tray was adjusted horizontally, and a motor clamped mechanism was used 5 times a day which was controlled automatically by a micro-controller for the motion of eggs up to 18 days of incubation. In conclusion, this egg-incubating machine has an efficiency and hatchability of 72.22%.
The genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been evolving via genomic drifts resulting in “emerging/drifting variants” circulating worldwide. The construction of polymerase chain reaction (PCR) assays for the reliable, efficient, and specific diagnosis of the drifting variants of SARS-CoV-2 is specifically governed by the selection and construction of primers and probes. The efficiency of molecular diagnosis is impacted by the identity/homology of the genome sequence of SARS-CoV-2 with other coronaviruses, drifting variants or variants of concern (VOCs) circulating in communities, inherent capacity of mutation(s) of various target genes of SARS-CoV-2, and concentration of genes of interest in host cells. The precise amplicon selection and construction of primers and probes for PCR-based assays can efficiently discriminate specific SARS-CoV-2 drifting variants. The construction of single nucleotide polymorphism (SNP)-specific primers and probes for PCR assays is pivotal to specifically distinguish SARS-CoV-2 variants present in the communities and contributes to better diagnosis and prevention of the ongoing COVID-19 pandemic. In this study, we have utilized in silico-based bioinformatic tools where the alignment for genes, the positions and types of SNPs/mutations of VOCs, and the relative number of SNPs per nucleotide in different genomic regions were investigated. Optimal and specific genome region (amplicon) selection with comparatively lower mutability in the SARS-CoV-2 genome should be prioritized to design/construct PCR assays for reliable and consistent diagnosis in various regions of the world for a longer duration of time. Further, the rational selection of target genes that is at an optimal detectable concentration in biological samples can bolster PCR assays of high analytical sensitivity. Hence, the construction of primers and probes with the rational selection of targeting specific E gene, genomic regions with highly conserved sequences, multiple target genes with relatively lower mutability and detectable level of concentration, SNP-specific binding regions of spike (S gene) protein, and shorter amplicon size (100–150 bp) are vital for the PCR assays to achieve optimal efficiency in the point-of-care laboratory diagnosis of circulating drifting variants of SARS-CoV-2 with optimal accuracy.
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