Multiplex Lateral Flow Assay for Rapid Visual Blood Group Genotyping

Abstract

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.

Rapid and Ultrasensitive Quantification of Multiplex Respiratory Tract Infection Pathogen via Lateral Flow Microarray based on SERS Nanotags

“…In order to obtain a uniform microarray of suitable dimensions onto the LFM, we firstly used a pipette (0.01-2 μL) to immobilize a 0.01 μL aliquot of capture nucleic acids onto the NC membrane. However, it was practically impossible to precisely align the T dots manually, which was evident in other LFM strips that have been constructed previously 12. The alignment of T dots in a microarray is important in low sensitivity detection in which a color change on the NC membrane cannot be observed by eye and the position for signal detection is dependent on the coordinates of each T dot in the microarray.…”

“…There are a number of studies that describe the construction of a lateral flow microarray (LFM) that combines a microarray with an LFA. This greatly increases detection throughput, with as many as eight biomarkers able to be analyzed per strip 12, 13, although the colloidal gold labels used in these assays restrict the quantitative capability and sensitivity of an LFM. In addition, the size of the strip restricts the quantity of dots for each microarray and it is not possible to greatly increase the throughput of detection of the LFM to any great extent.…”

Genotyping of Multiple Clinical Samples with a Combined Direct PCR and Magnetic Lateral Flow Assay

“…Remarkably, Gomez-Martinez et al. have developed a multiplex linear-after-the-exponential (LATE)-PCR for visual detection of blood group genotype with turnaround time of approximately 1 hr by using a KAPA2G Fast HotStart DNA Polymerase ( Gomez-Martinez et al., 2018 ). However, it is not clear whether this LATE-PCR method can be applicable to different sample types.…”

Lateral flow assay ruler for quantitative and rapid point-of-care testing

“…More biomarkers and more "ruler" channels could be added to the device to achieve multiplexed quantitation. 31,32 To further improve the user interface and ease the operation in the POC settings, we generated a 3D-printed LFA ruler with a slider to transfer the pads conveniently without cutting, as shown in Figure S7. This prototype will be further validated in future studies to achieve the goal of multiplexed quantification and ease of use.…”