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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]

The pandemic outbreak of the 2019 coronavirus disease (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still spreading rapidly and poses a great threat to human health. As such, developing rapid and accurate immunodiagnostic methods for the identification of infected persons is needed. Here, we proposed a simple but sensitive on-site testing method based on spike protein-conjugated quantum dot (QD) nanotag-integrated lateral flow immunoassay (LFA) to simultaneously detect SARS-CoV-2-specific IgM and IgG in human serum. Advanced silica-core@dual QD-shell nanocomposites (SiO2@DQD) with superior luminescence and stability were prepared to serve as fluorescent nanotags in the LFA strip and guarantee high sensitivity and reliability of the assay. The performance of the SiO2@DQD-strip was fully optimized and confirmed by using 10 positive serum samples from COVID-19 patients and 10 negative samples from patients with other respiratory diseases. The practical clinical value of the assay was further evaluated by testing 316 serum samples (114 positive and 202 negative samples). The overall detection sensitivity and specificity reached 97.37% (111/114) and 95.54% (193/202), respectively, indicating the huge potential of our proposed method for the rapid and accurate detection of SARS-CoV-2-infected persons and asymptomatic carriers. [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]

In accidental irradiation situations, rapid in-field evaluation of acute radiation syndrome is critical for effective triage and timely medical treatment of irradiated individuals. A surface-enhanced Raman scattering (SERS)-based lateral flow assay was developed for the quantitative detection of C-reactive protein (CRP) as an early bio-indicator of a radiation-induced inflammatory response in nonhuman primates. Raman reporter-embedded gold-core silver-shell nanoparticles with built-in hot spots were synthesized and conjugated with a CRP detection antibody to serve as SERS tags in the lateral flow assay. The proposed SERS-based lateral flow assay can rapidly detect CRP with a limit of detection of 0.01 ng mL−1 and quantitative analysis ability. Furthermore, the assay was applied to evaluate the CRP levels in plasma samples of irradiated nonhuman primates at 0 to 80 h after exposure to sublethal (4 Gy) and lethal (8 Gy) doses of total body irradiation (n = 3 animals per group). The plasma CRP levels increase rapidly within few hours after irradiation. The CRP level peaks are observed at 12 or 24 h after irradiation, with a concentration of 201.30, 386.06 and 475.18 μg mL−1 for the 4 Gy irradiated animals and 197.14, 69.52 and 358.03 μg mL−1 for the 8 Gy irradiated animals. The results indicate the potential application of the proposed SERS-based lateral flow assay to serve as a rapid and accurate point-of-care biodosimetry assay for the quantitative detection of bio-indicators to triage irradiated individuals in the field of a radiation accident. [ABSTRACT FROM AUTHOR]

A rapid, sensitive, and label-free SERS detection method for bacteria pathogens is reported for the first time. The method, which is based on the combination of polyethylenimine (PEI)-modified Au-coated magnetic microspheres (Fe3O4@Au@PEI) and concentrated Au@Ag nanoparticles (NPs), was named the capture–enrichment–enhancement (CEE) three-step method. A novel Fe3O4@Au microsphere with monodispersity and strong magnetic responsiveness was synthesized as a magnetic SERS substrate and amino functionalized by PEI self-assembly. The negatively charged bacteria were quickly captured and enriched by the positively charged Fe3O4@Au@PEI microspheres, and the bacteria SERS signal was synergistically enhanced by using Fe3O4@Au@PEI microspheres and Au@Ag NPs in conjunction. The CEE three-step method proved useful in tap water and milk samples, and the total assay time required was only 10 min. Results further demonstrated that the CEE three-step method could be a common approach for detecting a wide range of bacteria, as verified by its detection of the Gram-positive bacterium E. coli and Gram-positive bacterium S. aureus at a detection limit of as low as 103 cells per mL. Therefore, our CEE three-step method offered the significant advantages of short assay time, simple operating procedure, and higher sensitivity than previously reported methods of SERS-based bacteria detection. [ABSTRACT FROM AUTHOR]