Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, laboratory testing to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription PCR (RT-qPCR) has played a central role in mitigating the spread of the virus (1). Soon after the viral genome sequences were available, several RT-qPCR assays were developed and made available by World Health Organization (WHO) for public use (https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf). The primer and probe sequences for these assays were chosen from multiple target genes within the viral genome such as the E gene, RdRp gene, ORF1ab and N gene. Many commercial and laboratory-developed assays were developed for SARS-CoV-2 detection based on these primer and probe sequences. The large-scale sustained person-to-person transmission of SARS-CoV-2 has led to many mutational events, some of which may affect the sensitivity and specificity of available PCR assays (2). Recently, mutations in the E gene (C26340T) and N gene (C29200T) were reported affecting the detection of target genes by two commercial assays in 8 and 1 patients, respectively. Interestingly, both mutations are of C>T type, a common single nucleotide polymorphism (SNP) that may be associated with strong host cell mRNA editing mechanisms known as APOBEC cytidine deaminase (3, 4). Another study found a G to U substitution in position 29140 that affected the sensitivity of detection of N gene-based assays (5). Here we report a novel N gene mutation (C29200A) seen in 3 patients, which affected the detection of SARS-CoV-2 N gene by a commercial assay.

Background: Marine algae used as a food source for ocean life and range in color from red to green to brown grow along rocky shorelines around the world. The synthesis of silver nanoparticles by marine alga Padina sp. and its characterization were fulfilled by using UV-visible spectrophotometer, Fourier transform infrared spectroscopy, scanning electron microscopy and field emission scanning electron microscopy, and energy-dispersive X-ray spectroscopy. Results: UV-visible absorption spectrum revealed that the formation of Ag nanoparticles was increased by the addition of marine algae and the spectral peak observed between a wavelength of~420 nm and 445 nm. In addition, SEM and FESEM images examined the surface morphology and the size of the synthesized NPs was relatively uniform in size~25-60 nm. Energy-dispersive X-ray spectroscopy analysis confirmed the purity of Ag NPs with atomic percentage of 48.34% Ag. The synthesized Ag NPs showed highly potent antibacterial activity. The Staphylococcus aureus and Pseudomonas aeruginosa were found to be more susceptible to silver nanoparticles by forming 15.17 ± 0.58 mm and 13.33 ± 0.76 mm of diameter of the inhibition zone, respectively. Conclusions: The study suggested that marine alga Padina sp. could be an alternative source for the production of Ag nanoparticles and are efficient antimicrobial compounds against both gram-negative and gram-positive bacteria which can be a promising material against infectious bacteria.

To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65˚C for 10 minutes along with the use of TaqPath™ 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x10 3 copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR C T values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, p = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

No abstract