Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.

Alu siRNA to increase Alu element methylation and prevent DNA damage.

Global DNA hypomethylation promoting genomic instability leads to cancer and deterioration of human health with age. AIM: To invent a biotechnology that can reprogram this process. METHODS: We used Alu siRNA to direct Alu interspersed repetitive sequences methylation in human cells. We evaluated the correlation between DNA damage and Alu methylation levels. RESULTS: We observed an inverse correlation between Alu element methylation and endogenous DNA damage in white blood cells. Cells transfected with Alu siRNA exhibited high Alu methylation levels, increased proliferation, reduced endogenous DNA damage and improved resistance to DNA damaging agents. CONCLUSION: Alu methylation stabilizes the genome by preventing accumulation of DNA damage. Alu siRNA could be useful for evaluating reprograming of the global hypomethylation phenotype in cancer and aging cells.

Long interspersed nuclear element-1 hypomethylation and oxidative stress: correlation and bladder cancer diagnostic potential.

Although, increased oxidative stress and hypomethylation of long interspersed nuclear element-1 (LINE-1) associate with bladder cancer (BCa) development, the relationship between these alterations is unknown. We evaluated the oxidative stress and hypomethylation of the LINE-1 in 61 BCa patients and 45 normal individuals. To measure the methylation levels and to differentiate the LINE-1 loci into hypermethylated, partially methylated and hypomethylated, peripheral blood cells, urinary exfoliated cells and cancerous tissues were evaluated by combined bisulfite restriction analysis PCR. The urinary total antioxidant status (TAS) and plasma protein carbonyl content were determined. The LINE-1 methylation levels and patterns, especially hypomethylated loci, in the blood and urine cells of the BCa patients were different from the levels and patterns in the healthy controls. The urinary TAS was decreased, whereas the plasma protein carbonyl content was increased in the BCa patients relative to the controls. A positive correlation between the methylation of LINE-1 in the blood-derived DNA and urinary TAS was found in both the BCa and control groups. The urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content provided the best diagnostic potential for BCa prediction. Based on post-diagnostic samples, the combination test improved the diagnostic power to a sensitivity of 96% and a specificity of 96%. In conclusion, decreased LINE-1 methylation is associated with increased oxidative stress both in healthy and BCa subjects across the various tissue types, implying a dose-response association. Increases in the LINE-1 hypomethylation levels and the number of hypomethylated loci in both the blood- and urine-derived cells and increase in the oxidative stress were found in the BCa patients. The combination test of the urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content may be useful for BCa screening and monitoring of treatment.