Characterizing a visual lateral flow device for rapid SARS-CoV-2 virus protein detection: pre-clinical and system assessment.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections have affected more than 769 million individuals worldwide over the last few years. Although the pandemic is transitioning into an endemic, the COVID-19 outbreak is still a global concern. A rapid screening platform is needed for effective preventive and control measures. Herein, a visual rapid lateral flow platform for SARS-CoV-2 nucleocapsid protein detection is developed. Under optimal conditions, the system demonstrated good detection sensitivity and selectivity against tested respiratory viruses. The system provides direct visual detection with a limit of 0.7 ng of the nucleocapsid protein per mL of a sample (0.7 ng mL-1) within 15 minutes. Further, a correlation between direct visual detection and semi-quantitative analysis using a reader showed a similar detection limit (R2 = 0.9571). The repeatability and reproducibility studies highlighted the potential of the system for the rapid screening of SARS-CoV-2 infection, with variations within 5% and 10% at high and low protein concentrations, respectively. Subsequent pre-clinical validation to correlate the performance with the standard molecular approach (RT-PCR) using 170 nasopharyngeal swabs demonstrated 98% estimated sensitivity (95% CI, 89.35-99.95%) and 100% specificity (95% CI, 96.38-100%). The positive and negative predictive values were reported to be 100% and 99%, respectively, with an accuracy of 99.3%. With high viral load samples (Ct value ≤25, n = 47), the system demonstrated 100% detection sensitivity and specificity. The proposed technique provides a valuable platform for potential use in rapid screening, particularly during pandemics, where diagnostic capacity and mass screening are crucial.

Characterization and CRISPR-based genotyping of clinical trh-positive Vibrio parahaemolyticus.

BACKGROUND: Vibrio parahaemolyticus is a causative agent of gastroenteritis. Most of the clinical isolates carry either tdh and/or trh genes which are considered as the major virulence genes of this pathogen. In this study, the clinical isolates of V. parahaemolyticus carrying trh gene (n = 73) obtained from 1886 to 2012 from various countries were investigated for the urease production, haemolytic activity, and biofilm formation. In addition, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping among these isolates was investigated. RESULTS: In this study, no significant differences were observed in the urease production between tdh + trh1+ and tdh + trh2+ isolates (p = 0.063) and between the tdh - trh1+ and tdh - trh2+ isolates (p = 0.788). The isolates carrying only the trh gene showed variation in their haemolytic activity. The ratio of urease production and haemolytic activity between the trh1+ and trh2+ isolates and biofilm formation of trh + V. parahaemolyticus isolates were not significantly different. Sixteen of thirty-four tested isolates (47.0%) of trh + V. parahaemolyticus were positive for CRISPR detection. The discriminatory power index (DI) of CRISPR-virulence typing was higher than the DI obtained by CRISPR typing alone. CONCLUSION: The tdh and trh genes were not involved in urease production in the trh + V. parahaemolyticus, and variation of haemolytic activity detected in V. parahaemolyticus carrying only the trh gene might be correlated to the sequence variation within trh1 and trh2 genes. Additionally, biofilm production of V. parahaemolyticus was not associated with harboring of virulence genes. For genotyping, CRISPR sequences combined with virulence genes can be used as genetic markers to differentiate trh + V. parahaemolyticus strains.

Isolation of Bdellovibrio and like organisms and potential to reduce acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease, a severe disease of shrimp, is caused by Vibrio parahaemolyticus (AHPND Vp), a halophilic bacterium harboring a plasmid that contains toxin genes homologous to Photorhabdus insect-related toxins. We obtained 9 isolates of Bdellovibrio and like organisms (BALOs) from water and sediment samples in Thailand. Using 16S rRNA sequencing, all of the organisms were identified as Bacteriovorax spp. and were able to attack all tested AHPND Vp isolates. In addition, their various susceptible hosts, including Gram-positive and Gram-negative bacteria, were observed. The optimal ratio for interaction between the Bacteriovorax isolate BV-A and AHPND Vp was determined to be 1:10. The suitable conditions applied for co-culture between BV-A and AHPND Vp were 30°C, 2% NaCl, and pH 7.6. The capability of BV-A to reduce numbers of AHPND Vp in vitro was observed in co-culture after incubation for 2 d and continued until the end of the incubation period. In vivo, BV-A was able to reduce mortality of shrimp post-larvae infected with AHPND Vp. In addition, BV-A significantly decreased the formation of biofilm by AHPND Vp. These findings provide evidence for using Bacteriovorax as a biocontrol of AHPND Vp in shrimp aquaculture.