Conventional blood group phenotyping by hemagglutination assays, carried out pretransfusion, is unsuitable in certain clinical situations. Molecular typing offers an alternative method, allowing the deduction of blood group phenotype from genotype. However, current methods require a long turnaround time and are not performed on-site, limiting their application in emergency situations. Here, we report the development of a novel, rapid multiplex molecular method to identify seven alleles in three clinically relevant blood group systems (Kidd, Duffy, and MNS). Our test, using a dry-reagent allele-specific lateral flow biosensor, does not require DNA extraction and allows easy visual determination of blood group genotype. Multiplex linear-after-the-exponential (LATE)-PCR and lateral flow parameters were optimized with a total processing time of 1 h from receiving the blood sample. Our assay had a 100% concordance rate between the deduced and the standard serological phenotype in a sample from 108 blood donors, showing the accuracy of the test. Owing to its simple handling, the assay can be operated by nonskilled health-care professionals. The proposed assay offers the potential for the development of other relevant single nucleotide polymorphism (SNP) panels for immunohematology and new applications, such as for infectious diseases, in the near future.

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Identification of the main SARS-CoV-2 variants in real time is of interest to control the virus and to rapidly devise appropriate public health responses. The RT-qPCR is currently considered to be the reference method to screen SARS-CoV-2 mutations, but it has some limitations. The multiplexing capability is limited when the number of markers to detect increases. Moreover, the performance of this allele-specific method may be impacted in the presence of new mutations. Herein, we present a proof-of-concept study of a simple molecular assay to detect key SARS-CoV-2 mutations. The innovative features of the assay are the multiplex asymmetric one-step RT-PCR amplification covering different regions of SARS-CoV-2 S gene and the visual detection of mutations on a lateral flow DNA microarray. Three kits (Kit 1: N501Y, E484K; Kit 2: L452R, E484K/Q; Kit 3: K417N, L452R, E484K/Q/A) were developed to match recommendations for surveillance of SARS-CoV-2 variants between January and December 2021. The clinical performance was assessed using RNA extracts from 113 SARS-CoV-2-positive samples with cycle thresholds <30, and results demonstrated that our assay allows specific and sensitive detection of mutations, with a performance comparable to that of RT-qPCR. The VAR-CoV assay detected four SARS-CoV-2 targets and achieved specific and sensitive screening of spike mutations associated with the main variants of concern, with a performance comparable to that of RT-qPCR. With well-defined virus sequences, this assay can be rapidly adapted to other emerging mutations; it is a promising tool for variant surveillance.