Point of need testing device and methods of use thereof
Inventors
Kim, Chang Hee • Henderson, Wendy A. • Abey, Sarah K. • Fourie, Nicolaas H. • Ferguson, Eric G.
Assignees
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Publication Number
US-12247250-B2
Publication Date
2025-03-11
Expiration Date
2036-05-16
Abstract
The present invention relates to the rapid and electricity-free, point-of-care, multiplexed detection and quantification of at least one or more nucleic acid sequences from nucleic acids corresponding to a plurality of pathogens or biomarkers using a micropatterned lateral flow device. Rapid and molecular-level sensitive differential diagnosis of a disease condition may be enabled without the need for a delayed laboratory test so that timely treatment can be administered.
Core Innovation
The invention describes a rapid and electricity-free, point-of-care, multiplexed testing device and associated methods for detecting and quantifying one or more nucleic acid sequences from a plurality of pathogens or biomarkers using a micropatterned lateral flow device. This device enables molecular-level sensitive differential diagnosis of disease conditions directly at the point of need, eliminating the delays associated with laboratory testing and allowing timely administration of treatment.
The device comprises at least one lateral flow device including a sample loading area with debris trapping material, a detectably labelled probe specific for target nucleic acid sequences that is not bound to the device and capable of wicking across it, a capture probe immobilized on the device for the target sequence, and an absorbent material that facilitates sample migration across the device. Multiplex detection is achieved by including multiple lateral flow devices each specific for different target sequences, arranged radially or laterally around a central sample loading area.
The problem addressed is the need for rapid, sensitive, and specific point-of-care or point-of-need diagnostics for infectious and other diseases without requiring complex instrumentation, electricity, enzyme-based amplification, or laboratory purification steps. Existing rapid immunological tests lack sensitivity and specificity, and molecular tests requiring isothermal amplification enzymes are challenging in resource-limited settings due to refrigeration and power requirements. This invention solves these problems by providing a stable, durable, enzyme-free, antibody-free lateral flow nucleic acid test that operates directly on minimally processed or lysed biological samples.
Claims Coverage
The claims include two independent claims, each describing a method for detecting nucleic acid sequences in biological samples using a point of need testing device with lateral flow devices. The main inventive features cover device components, method steps, and multiplex capability.
Point of need testing device for detecting nucleic acids in stool samples
A lateral flow device comprising (a) a sample loading area with debris trapping material that receives a diluted stool sample, (b) an area with a detectably labelled probe specific for a microorganism or virus nucleic acid sequence that is not bound and can wick across the device, (c) an immobilized capture probe for the target sequence, and (d) absorbent material that wicks the aqueous sample. The detection involves trimolecular hybridization of the target nucleic acid, detectably labelled probe, and capture probe, without isolating or purifying nucleic acids from the lysed sample.
Target nucleic acid sequence is rDNA or rRNA from specific pathogens or viruses
The method detects rDNA or rRNA target sequences from microorganisms or viruses selected from a broad list including Escherichia, Campylobacter, Clostridium difficile, multiple E. coli strains, Salmonella, Shigella, viruses such as Zika, dengue, HIV, and others.
Debris trapping material comprising glass fiber
The sample loading area contains debris trapping material which includes glass fiber to capture debris from the sample during testing.
Inclusion of control probe area
The lateral flow device further includes an area comprising a capture probe for a control nucleic acid sequence complementary to the detectably labelled probe for the target, serving as an internal control on the device.
Detectably labelled probe labeling moieties
The detectably labelled probe is labeled with moieties selected from gold nanoparticles, protein binding ligands, haptens, antigens, fluorescent compounds, dyes, radioactive isotopes, and enzymes, with gold nanoparticles being a preferred label.
Multiplex testing capability with multiple lateral flow devices
The point of need testing device may comprise at least two lateral flow devices, each with probes specific for different microorganisms or viruses, allowing multiplex detection in a single assay.
Method steps for detecting target nucleic acid in diluted stool samples using the device
The method includes lysing cells within the diluted stool sample, adding the lysed sample to the sample loading area of the testing device, and detecting the presence of target nucleic acid sequences via trimolecular hybridization on the device without requiring nucleic acid purification or isolation.
Detection of gastrointestinal pathogens with control probe indicating absence of target
The method detects target nucleic acid sequence from gastrointestinal pathogens by trimolecular hybridization of target, detectably labelled, and capture probes to indicate presence and by bimolecular hybridization of detectably labelled probe and a second capture probe (control) for the target sequence portion to indicate absence of target. The stool sample is an aqueous mixture with lysis agent and unprocessed stool.
The independent claims provide methods for nucleic acid detection from minimally processed stool samples using a lateral flow device comprising debris trapping, detectably labelled probes, immobilized capture probes and absorbent material, with multiplexing capability and controls, enabling rapid, enzyme and instrument-free point of need diagnostics for a variety of pathogens.
Stated Advantages
Enables rapid, electricity-free, point-of-care nucleic acid detection without needing laboratory amplification or purification.
Provides molecular-level sensitivity and specificity superior to immunological tests in a stable, durable, and low-cost device.
Allows multiplexed detection of multiple pathogens or biomarkers simultaneously from minimally processed samples.
Suitable for use in remote or resource-limited settings without access to refrigeration, power, or complex laboratory infrastructure.
Eliminates the need for enzymatic or antibody-based components, reducing assay complexity and improving shelf-life and robustness.
Documented Applications
Rapid detection and quantification of nucleic acid sequences from pathogens causing infectious diseases at the point of care or need.
Differential diagnosis of diseases including infectious diseases and monitoring antibiotic susceptibility and resistance markers.
Multiplex detection of gastrointestinal pathogens from stool samples applied directly or diluted with a lysis agent.
Detection of nucleic acid biomarkers relevant to non-communicable chronic diseases such as cancers, autoimmune, cardiovascular, digestive, metabolic, and neurological diseases.
Use in remote or low-resource settings for health status monitoring and disease detection without laboratory infrastructure.
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