A Rebuilding-Free Nucleic Acid Detection Strategy Enables Ultrasensitive Genotyping, N-in-1 Logic Screening and Accurate Multiplex Assay of Dangerous Pathogens.

Sensitive, rapid and low-cost nucleic acid detection is critical for controlling infectious pathogens. Here, we develop a ready-to-use and multimodal detection based on a rebuilding-free, ultrasensitive and selective strategy named dual hairpin ligation-induced isothermal amplification pro (DHLApro). Taking influenza A, influenza B, MERS-CoV, SARS-CoV-2 as model targets, we demonstrate DHLApro provides ≈zM level ultra-sensitivity, being equaling to 0.45 copy/µL in original sample. By simply changing the recognition module, a set of DHLApro components can be applied to a new target without performance loss. Moreover, DHLApro innovatively allows flexible logic/multiplex assay using one set of primer, for example, the "N pathogens-in-1" OR gate screening and accurate multi-channel multiplex assay. Compared with traditional methods, the cost of this logic/multiplex assay has been largely reduced and the cross-interference between the multiple primer sets is also avoided.

A novel single nucleotide polymorphism assay for the detection of N501Y SARS-CoV-2 variants.

The N501Y mutation in SARS-CoV-2 variants found in several strains from the UK, South Africa and Brazil has been linked to increased transmission. In order to discriminate N501Y variants quickly, a single nucleotide polymorphism (SNP) discrimination assay was designed and validated. It was then deployed prospectively in 757 nasopharyngeal swabs. Validation of the novel variant discrimination assay corroborated the results in all validation panel samples (n = 63) through sequencing. This novel variant discrimination assay was then deployed prospectively in 757 clinical nasopharyngeal swabs during the last week of January 2021. N501Y was found in 206 (27.4 %) of the samples: 94 (28.2 %) men and 112 (26.85 %) women (p = 0.73). The patients in whom it was identified had a mean age of 47.8 ± 25.8 (0-96) years, similar to that of patients without this variant: 51.7 ± 25.9 (0-104) years (p = 0.06). 501Y variant was confirmed in 34 samples by sequence method and 501 N wild type was confirmed in 67. This method is sensitive, specific, and simple to apply in any microbiology lab.