Khanna, T. | bioRxiv Science

bioRxiv2020

Development of RNA-based assay for rapid detection of SARS-CoV-2 in clinical samples

The ongoing spread of pandemic coronavirus disease (COVID-19) is caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). In the lack of specific drugs or vaccines for SARS-CoV-2, demands rapid diagnosis and management are crucial for controlling the outbreak in the community. Here we report the development of the first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 minutes. We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and changes in gold-colloid color from pink to blue in visible range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus (HPV) infected women. The results were validated using nasopharangeal RNA samples from suspected COVID-19 subjects (n=136). Using RT-PCR as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. Overall, the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.

microbiology↗

bioRxiv2020

Structure, function and variants analysis of the androgen-regulated TMPRSS2, a drug target candidate for COVID-19 infection

SARS-CoV-2 is a novel virus causing mainly respiratory, but also gastrointestinal symptoms. Elucidating the molecular processes underlying SARS-CoV-2 infection, and how the genetic background of an individual is responsible for the variability in clinical presentation and severity of COVID-19 is essential in understanding this disease. Cell infection by the SARS-CoV-2 virus requires binding of its Spike (S) protein to the ACE2 cell surface protein and priming of the S by the serine protease TMPRSS2. One may expect that genetic variants leading to a defective TMPRSS2 protein can affect SARS-CoV-2 ability to infect cells. We used a range of bioinformatics methods to estimate the prevalence and pathogenicity of TMPRSS2 genetic variants in the human population, and assess whether TMPRSS2 and ACE2 are co-expressed in the intestine, similarly to what is observed in lungs. We generated a 3D structural model of the TMPRSS2 extracellular domain using the prediction server Phyre and studied 378 naturally-occurring TMPRSS2 variants reported in the GnomAD database. One common variant, p.V160M (rs12329760), is predicted damaging by both SIFT and PolyPhen2 and has a MAF of 0.25. Valine 160 is a highly conserved residue within the SRCS domain. The SRCS is found in proteins involved in host defence, such as CD5 and CD6, but its role in TMPRSS2 remains unknown. 84 rare variants (53 missense and 31 leading to a prematurely truncated protein, cumulative minor allele frequency (MAF) 7.34x10-4) cause structural destabilization and possibly protein misfolding, and are also predicted damaging by SIFT and PolyPhen2 prediction tools. Moreover, we extracted gene expression data from the human protein atlas and showed that both ACE2 and TMPRSS2 are expressed in the small intestine, duodenum and colon, as well as the kidneys and gallbladder. The implications of our study are that: i. TMPRSS2 variants, in particular p.V160M with a MAF of 0.25, should be investigated as a possible marker of disease severity and prognosis in COVID-19 and ii. in vitro validation of the co-expression of TMPRSS2 and ACE2 in gastro-intestinal is warranted.

bioinformatics↗