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The ABO blood group plays an important role in blood transfusion, linkage analysis, individual identification, etc. Serologic methods of blood typing are gold standards for the time being, which require stable typing antisera and fresh blood samples and are labor intensive. At present, reliable determination of ABO blood group genotypes based on single-nucleotide polymorphisms (SNPs) among A, B, and O alleles remains necessary. Thus, in this work, CRISPR/Cas13a-mediated genotyping for the ABO blood group by detecting SNPs between different alleles was proposed. The ABO*O.01.01(c.261delG) allele (G for the A/B allele and del for the O allele) and ABO*B.01(c.796C > A) allele (C for the A/O allele and A for the B allele) were selected to determine the six genotypes (AA, AO, BB, BO, OO, and AB) of the ABO blood group. Multiplex PCR was adapted to simultaneously amplify the two loci. CRISPR/Cas13a was then used to specifically differentiate ABO*O.01.01(c.261delG) and ABO*B.01(c.796C > A) of A, B, and O alleles. Highly accurate determination of different genotypes was achieved with a limit of detection of 50 pg per reaction within 60 min. The reliability of this method was further validated based on its applicability in detecting buccal swab samples with six genotypes. The results were compared with those of serological and sequencing methods, with 100% accuracy. Thus, the CRISPR/Cas13a-mediated assay shows great application potential in the reliable identification of ABO blood group genotypes in a wide range of samples, eliminating the need to collect fresh blood samples in the traditional method. [ABSTRACT FROM AUTHOR]

MiRNAs play important regulatory roles in numerous biological processes and serve as significant biomarkers for the development and prognosis of several diseases. Their unique characteristics, such as short size, high sequence homology among family members, low abundance, and easy degradability, have hindered their specific and highly sensitive detection. Herein, a duplex-specific nuclease (DSN)-assisted target recycling signal amplification-based fluorescent lateral flow assay was demonstrated for the point-of-care detection of cancer-related miRNA-21. In this assay, digoxin/biotin-labeled DNA probes were selectively cleaved by the DSN enzyme in the rounds of hybridization with the miRNA-21 target and cleavage cycle. Subsequently, the resulting mixture, containing the miRNA-21 target and intact and cleaved DNA probes, was loaded onto the lateral flow strip with digoxin antibody-conjugated quantum dot nanobeads and the streptavidin-coated test line. The increase in the proportion of cleaved DNA probes can induce a weakened response signal, which is directly associated with the amount of the miRNA target. Thus, highly sensitive quantification of miRNA-21 was achieved at a low limit of detection of 0.16 pM within 2 h of assay time. Assay specificity toward miRNA-21 was validated by testing several other miRNAs, including let-7b, let-7d, miRNA-141, and miRNA-200a. Moreover, the assay can quantify miRNA-21 spiked in human serum samples with acceptable recovery values, thus indicating its considerable clinical feasibility. [ABSTRACT FROM AUTHOR]

Several virulent variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged along with the spread of this virus throughout the population. Some variants can exhibit increased transmissibility and reduced immune neutralization reactivity. These changes are deeply concerning issues that may hinder the ongoing effort of epidemic control measures, especially mass vaccination campaigns. The accurate discrimination of SARS-CoV-2 and its emerging variants is essential to contain the coronavirus disease 2019 pandemic. Herein, we report a low-cost, facile, and highly sensitive diagnostic platform that can simultaneously distinguish wild-type (WT) SARS-CoV-2 and its two mutations, namely, D614G and N501Y, within 2 h. WT or mutant (M) nucleic acid fragments at each allelic locus were selectively amplified by the tetra-primer amplification refractory mutation system (ARMS)-PCR assay. Allele-specific amplicons were simultaneously detected by two test lines on a quantum dot nanobead (QB)-based dual-color fluorescent test strip, which could be interpreted by the naked eye or by a home-made fluorescent strip readout device that was wirelessly connected to a smartphone for quantitative data analysis and result presentation. The WT and M viruses were indicated and were strictly discriminated by the presence of a green or red band on test line 1 for the D614G site and test line 2 for the N501Y site. The limits of detection (LODs) for the WT and M D614G were estimated as 78.91 and 33.53 copies per μL, respectively. This assay was also modified for the simultaneous detection of the N and ORF1ab genes of SARS-CoV-2 with LODs of 1.90 and 6.07 copies per μL, respectively. The proposed platform can provide a simple, accurate, and affordable diagnostic approach for the screening of SARS-CoV-2 and its variants of concern even in resource-limited settings.