Novel Lateral Flow-Based Assay for Simple and Visual Detection of SARS-CoV-2 Mutations.

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.

Near-point-of-care assay with a visual readout for detection of HIV-1 drug resistance mutations: A proof-of-concept study.

Human immunodeficiency virus (HIV) infection is a chronic disease that can be treated with antiretroviral (ARV) therapy. However, the success of this treatment has been jeopardized by the emergence of HIV infections resistant to ARV drugs. In low-to middle-income countries (LMICs), where transmission of resistant viruses has increased over the past decade, there is an urgent need to improve access to HIV drug resistance testing. Here, we present a proof-of-concept study of a rapid and simple molecular method to detect two major mutations (K103 N, Y181C) conferring resistance to first-line nonnucleoside reverse transcriptase inhibitor regimens. Our near-point-of-care (near-POC) diagnostic test, combining a sequence-specific primer extension and a lateral flow DNA microarray strip, allows visual detection of HIV drug resistance mutations (DRM) in a short turnaround time (4 h 30). The assay has a limit of detection of 100 copies of plasmid DNA and has a higher sensitivity than standard Sanger sequencing. The analytical performance was assessed by use of 16 plasma samples from individuals living with HIV-1 and results demonstrated the specificity and the sensitivity of this approach for multiplex detection of the two DRMs in a single test. Furthermore, this near-POC assay could be easily taylored to detect either new DRMs or DRM of from various HIV clades and might be useful for pre-therapy screening in LMICs with high levels of transmitted drug resistance.

An Innovative Multiplexed and Flexible Molecular Approach for the Differential Detection of Arboviruses.

Nucleic acid testing during the preseroconversion viremic phase is required to differentially diagnose arboviral infections. The continuing emergence of arboviruses, such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV), necessitates the development of a flexible diagnostic approach. Similar clinical signs and the priority to protect pregnant women from ZIKV infection indicate that the differential diagnosis of arboviruses is essential for effective patient management, clinical care, and epidemiologic surveillance. We describe an innovative diagnostic approach that combines generic RT-PCR amplification and identification by hybridization to specific probes. Original tetrathiolated probes were designed for the robust, sensitive, and specific detection of amplified arboviral genomes. The limit of detection using cultured and quantified stocks of whole viruses was 1 TCID50/mL for DENV-1, DENV-3, and CHIKV and 10 TCID50/mL for DENV-2, DENV-4, and ZIKV. The assay had 100% specificity with no false-positive results. The approach was evaluated using 179 human samples that previously tested as positive for the presence of ZIKV, DENV, or CHIKV genomes. Accordingly, the diagnostic sensitivity for ZIKV, DENV, and CHIKV was 87.88% (n = 58/66), 96.67% (n = 58/60), and 94.34% (n = 50/53), respectively. This method could be easily adapted to include additional molecular targets. Moreover, this approach may also be adapted to develop highly specific, sensitive, and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses.

Multiplex Lateral Flow Assay for Rapid Visual Blood Group Genotyping.

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.

Flexible automated platform for blood group genotyping on DNA microarrays.

The poor suitability of standard hemagglutination-based assay techniques for large-scale automated screening of red blood cell antigens severely limits the ability of blood banks to supply extensively phenotype-matched blood. With better understanding of the molecular basis of blood antigens, it is now possible to predict blood group phenotype by identifying single-nucleotide polymorphisms in genomic DNA. Development of DNA-typing assays for antigen screening in blood donation qualification laboratories promises to enable blood banks to provide optimally matched donations. We have designed an automated genotyping system using 96-well DNA microarrays for blood donation screening and a first panel of eight single-nucleotide polymorphisms to identify 16 alleles in four blood group systems (KEL, KIDD, DUFFY, and MNS). Our aim was to evaluate this system on 960 blood donor samples with known phenotype. Study data revealed a high concordance rate (99.92%; 95% CI, 99.77%-99.97%) between predicted and serologic phenotypes. These findings demonstrate that our assay using a simple protocol allows accurate, relatively low-cost phenotype prediction at the DNA level. This system could easily be configured with other blood group markers for identification of donors with rare blood types or blood units for IH panels or antigens from other systems.

Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays.

We are reporting here a low cost colorimetric device for high-throughput multiplexed blood group genotyping and allergy diagnosis, displayed as an automated 96-well microtiter plate format. A porous polymeric membrane sealed at the bottom of each well accounts for the sensor support. For each sensing unit, a 6×6 matrix of specific probes is spotted on the external surface of the membrane resulting in 5 mm(2) microarrays. Thanks to the membrane porosity, reagents dispensed into the well can be eliminated through vacuum soaking. This unusual design drastically reduces the assay background signal. The system was first validated on robust models composed of either two complementary oligonucleotide sequences or one allergen/specific rabbit IgG pair. The quality of both oligonucleotide and protein immobilisation on the membrane substrate was then demonstrated together with the capacity to use the arrayed biomolecules as probes for the quantitative detection of specific targets (respectively complementary oligonucleotide and specific antibody). On the basis of these good results, two multiplex assays were developed for crude biological samples testing, focussing on two human in vitro diagnosis applications: a hybridisation assay for multiplex blood group genotyping and a multiparametric immunoassay for allergy diagnosis. In both cases, the transfer to crude biological samples testing was successful i.e. high signal to noise ratio of the stained membranes, reproducibility and good correlation with results obtained using routine testing procedures.

Robust, high-throughput solution for blood group genotyping.

With the concomitant increase of blood transfusions and safety rules, there is a growing need to integrate high-throughput and multiparametric assays within blood qualification centers. Using a robust and automated solution, we describe a new method for extended blood group genotyping (HiFi-Blood 96) bringing together the throughput possibilities of complete automation and the microarray multiplexed analysis potential. Our approach provides a useful resource for upgrading blood qualification center facilities. A set of six single-nucleotide polymorphisms (SNPs) associated with clinically important blood group antigens (Kell, Kidd, Duffy, and MNS systems) were selected and the corresponding genotyping assays developed. A panel of 293 blood samples was used to validate the approach. The resulting genotypes were compared to phenotypes previously determined by standard serologic techniques, and excellent correlations were found for five SNPs out of six. For the Kell, Kidd, Duffy, and MNS3/MNS4 systems, high matching percentages of 100%, 98.9%, 97.7%, and 97.4% were obtained, respectively, whereas a concordance percentage of 83.3% only was attained for the MNS1/MNS2 polymorphism.