A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.
Background: Oral submucous fibrosis (OSMF) is a premalignant condition mainly caused by areca nut chewing and is characterized by progressive fibrosis of submucosal tissues and epithelial atrophy. Activation of transforming growth factor beta (TGF-β) signaling is considered main causative event for increased collagen production and fibrosis. In this study, molecular pathogenesis of OSMF was investigated based on the expression of the TGF-β genes in OSMF tissues compared to normal controls. Methods: A total of 33 OSMF and 10 normal tissues were collected from patients and their clinic-epidemiological data was recorded. The expression of TGF-β isoform genes-TGF β1, TGF β2, TGF β3 and its receptor TGF βR1, TGF βR2 was studied by real time polymerase chain reaction (PCR). Comparison of the expression of these genes among normal controls and OSMF patients was done. The PCR results were confirmed by histopathological and immunohistochemical staining. Results: The histological changes included atrophic epithelium, loss of rete ridges, presence of inflammatory cells and dense collagen bundles in connective tissue. PCR showed statistically significant upregulation of TGF-β isoforms in OSMF as compared to normal tissues. Of the three isoforms, maximum fold change was observed in TGF-β1. Similarly, both TGF-βR1 and TGF-βR2 were found to be elevated in OSMF tissues compared to normal. The semi-quantitative analysis by immunohistochemical staining revealed statistically significant difference between normal and OSMF tissues. Conclusion: TGF-β signaling plays a major role in the molecular pathogenesis of OSMF as shown by increased mRNA expression of all the three TGF-β isotypes and their receptors.
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