JP2011528229A - Lateral flow nucleic acid detector - Google Patents

Abstract

Point-of-care binding assays multiplex with at least one sequence in a partner nucleic acid bound to a label by a complementary base pair between at least one sequence in the target nucleic acid and at least one sequence in the partner nucleic acid. It includes at least one target nucleic acid that binds by structure. The assay of the present invention overcomes the inherent deficiencies of antibody protein antigen assays. In a preferred embodiment, a nucleic acid sequence with a colored tag is used to bind a complementary target nucleic acid. The tagged nucleic acid sequence is preferably made from deoxyribonucleic acid, ribonucleotides or peptide nucleotides.
[Selection] Figure 2

Description

  This application claims one or more inventions, which were filed on July 15, 2008 and are entitled US Provisional Patent Application No. 61 / 080,879 entitled “Side Flow Nucleic Acid Detector”; Filed on September 22, 2009, and filed on May 18, 2009, entitled US Provisional Patent Application No. 61 / 098,935 entitled “In-situ Cell Lysis in Lateral Flow Immunoassay”. And US Provisional Patent Application No. 61 / 179,059, entitled “Method and Apparatus for Combined Detection of Bacterial Infections”. The benefit of US provisional patent application under 35 USC 119 (e) is hereby claimed and the aforementioned application is incorporated herein by reference.

  This application also claims one or more inventions, which were filed on July 14, 2009, and have been filed with US patent application Ser. No. 12 / 502,626, entitled “lateral flow nucleic acid detector”. No. 12 / 502,662, filed Jul. 14, 2009 and entitled “In situ Lysis of Cells in Lateral Flow Immunoassay”. The foregoing application is incorporated herein by reference.

  The present invention relates to the field of nucleic acid detection. More particularly, the invention relates to a lateral flow assay for the detection of nucleic acid targets.

  Lateral flow immunoassays are a subset of antibody-based immunoassays that combine various reagents and processing steps in one assay strip, and are therefore sensitive and sensitive for the detection of target molecules. Provides a quick means. Lateral flow immunoassays are available for large areas of target analytes and can be designed for sandwich or competitive testing. In general, high molecular weight analytes with several epitopes are analyzed in a sandwich format, whereas small molecules representing only one epitope are detected by a competitive assay. The first test was done for human chorionic gonadotropin (hCG). Today, there are tests available on the market to monitor ovulation, detect infectious diseased organs, analyze drug abuse, and measure other analytes of human physiology. Products for veterinary testing, environmental testing, and product monitoring are also introduced.

  Rapid point-of-care analysis is becoming increasingly important in the diagnosis and treatment of various viral or other pathogenic microbial agents. Prior art point-of-care tests, such as lateral flow immunochromatography tests, are immunoassays involving antibodies and their antigens. Such format binding assays operate on the ligand and its receptor and their associated binding constants. The essential lack of association between an antibody and its antigen is well known in the art. That is, ligand-receptor binding assays tend to degrade due to temperature cycling and thermal stress during storage, often causing interference from components in the sample matrix, causing non-specific binding during the assay, It will lead to wrong results. These inherent limitations do not provide sufficient specificity in the ligand-receptor interaction for reliable assay results. Ligand-receptor binding provides the specificity and limitations described above for protein antigen targets, but not for nucleic acid sequences that encode proteins.

  The binding assay of the invention comprises at least one sequence in another nucleic acid that is bound to a label by a complementary base pair between at least one sequence in the target nucleic acid having at least one sequence in the labeled nucleic acid. Includes target nucleic acid that binds to the sequence in multiple structures. The assay of the present invention overcomes the inherent deficiencies of antibody protein antigen assays. In a preferred embodiment, a nucleic acid sequence with a colored tag is used to bind a complementary target nucleic acid. The tagged nucleic acid sequence is preferably made from deoxyribonucleic acid, ribonucleotides or peptide nucleotides.

FIG. 3 shows a schematic top view of a test strip for a lateral flow binding assay of an embodiment of the present invention. 1 schematically shows a complex capable of positive detection of a single-stranded target nucleic acid according to the first embodiment of the present invention. 2 schematically shows a double-stranded target nucleic acid according to a second embodiment of the present invention. FIG. 4 schematically shows a first labeled complex comprising a nucleic acid sequence complementary to the X binding site of the target nucleic acid shown in FIG. 3 of the second embodiment of the present invention. Fig. 4 schematically shows a second labeled complex comprising a nucleic acid sequence complementary to the Y binding site of the target nucleic acid shown in Fig. 3 of the second embodiment of the invention. 3 schematically shows a third complex of a second embodiment of the invention. The 1st complex couple | bonded with the said X binding site of the target nucleic acid of 2nd embodiment of this invention is shown schematically. In 2nd embodiment of this invention, the 1st complex couple | bonded with the X binding site of a target nucleic acid and the 2nd complex couple | bonded with the Y binding site of a target nucleic acid are shown roughly. The complex and target nucleic acid which are combined and read by the test | inspection line of 2nd embodiment of this invention are shown.

  The lateral flow nucleic acid detector of the present invention preferably detects nucleic acid in a sample without utilizing the polymerase chain reaction as an amplification step. The amplification step is designed to increase the sensitivity of the target nucleic acid. In some embodiments, the detected nucleic acid is further quantified. Some examples of how nucleic acids are quantified include, but are not limited to, visual line intensity visual quantification or quantification via an electronic optical reader.

  The assays described herein are preferably point-of-care assays, including both lateral flow assays and flow-through assays as defined herein. These point-of-care assays are processed and read within minutes to hours after the sample is obtained, but alternatively are slow, such as 24-26 hours after the sample is obtained. Can be processed in time.

  Detector to detect target nucleic acids bound to any target virus, bacteria, fungi, protozoa, other pathogens, allergens, any genetic defect, or any other target nucleic acid in the sample Can be used. Other target nucleic acids include tumor markers (chemical carcinogens), cardiac markers (eg, myoglobin, troponin, creatine kinase, MMP-9, C-creative protein), inflammatory markers (cytokins, lymphokines, chemokines, cell signaling factors) , Chemoattractants, metalloproteins, interferons, MxA and growth factors), hormones, and nucleic acids that encode for histocompatibility studies. The target nucleic acid can be, but is not limited to, a nucleic acid comprising DNA, oligonucleotides, messenger RNA, or other types of RNA. The flow of transport liquid in the detector depends on gravity or is a result of capillary action or surface tension. The transport liquid may be applied by immersing the test strip in the transport liquid or the transport liquid may be maintained in a test housing for the test strip.

  The nucleic acid sequence used to detect the target nucleic acid sequence can be any length that is specific for the desired target. In a preferred embodiment, the sequence is about 10-25 nucleotides in length, but long and short sequences are possible as long as the sequence provides the required specificity and otherwise does not interfere with the assay. In some preferred embodiments, the sequence is about 15-17 nucleotides in length. When peptide nucleic acid sequences (PNAs) are used, the length can be as short as 10 or 11 nucleotides. When PCT primers are used, they are generally 16-24 nucleotides in length.

  The lateral flow nucleic acid detector of the present invention may be on a single plane with a single sheet on the test strip for the detection region. Alternatively, the detector may be multi-sided including multiple detection regions on multiple sheets in fluid communication with the simultaneous assay for the same or different target nucleic acids from the same or different samples. The test sample in the present invention may be any sample that is expected to potentially contain the target nucleic acid, including saliva, nasopharyngeal secretions, mucus, tissue, blood, urine, vaginal fluid, skin ulcer, and environmental water. Including but not limited to samples and soil samples. In most cases, it is preferred that a denaturant or lysing agent be added to the sample in situ to make the first and second complexes in the sample accessible to the nucleic acid. The denaturant or lysing agent may be added to the sample before the sample is applied to the test strip, but the denaturant or lysing agent is added to the test strip without the step of adding the denaturant or lysing agent to the sample. It is preferably pre-loaded in the area of the test strip as a dry modifier or solubilizer so that it is applied directly. Alternatively, the lysing agent may be pre-dried by freeze drying or lyophilization and then pre-loaded on the test strip. The lysing agent may be absorbed, adsorbed, embedded, or captured on the test strip. The dried or embedded denaturant or lysing agent is preliminarily deposited on the test strip at a location such that the denaturant or lysing agent releases the nucleic acid before the sample reaches the first complex on the test strip. Can be placed. The dried or embedded denaturant or solubilizer is preferably soluble in the transport liquid and is placed within the sample application area or pre-loaded with the sample application area and the first complex. Arranged between the area. In other embodiments, the weak solubilizer may be part of the buffer in use. In this scenario, there is no deleterious effect on the complex area downstream, and the sample may be either upstream or downstream of the complex area.

  If the collected sample is not lysed prior to collection and transfer to the sample analyzer, the number of steps required to collect and prepare the sample for analysis is reduced. In such an embodiment, after loading the sample, the sample that moves with the transport liquid (buffer) will encounter the lysing agent. The solubilizer is pre-loaded on the test strip, dried, and eluted by the transport liquid. The initially dried solubilizer is preferably localized between the sample application area and the composite area (application area of the first composite) or overlaps either area or both areas. ing. The lysing agent is preferably soluble in the sample transport liquid, and the lysing agent is solubilized and activated upon contact with the sample transport liquid. The sample transport liquid then contains both the solubilizer in solution or suspension and the sample components in suspension. Any lysis-sensitive component in the sample is then exposed to the lysis agent in suspension and lyses itself in situ. The buffer being used then carries the analyte containing the components released from any lysis to the detection area.

  The position where the solubilizer is pre-loaded and dried can be varied as needed. In order to maximize the time for the sample to interact with the lysing agent, similarly, to minimize the amount of lysing agent that reaches the detection area, It may be arranged just downstream. Alternatively, to minimize the distance that the dissolved product must travel before reaching the composite area, the solubilizer is placed closer to the composite area (application area of the first complex). Also good.

  In one embodiment, the sensitivity of the visually read out nucleic acid assay test is enhanced by adding a small amount of fluorescent dye or fluorescent latex bead complex to the initial complex material. When the visible spectrum inspection line is visible, the inspection results are observed and recorded. However, in the case of a weak positive that does not result in a clearly visible inspection line, an appropriate spectral ray, such as a UV spectrum, is cast on the inspection line to make the color visible on the inspection line vivid. Excites and emits fluorescent latex beads bound on the inspection line.

  Lateral flow devices are known and are described, for example, in US Published Patent Application Nos. 2005/0175992 and 2007/0059682. The contents of both applications are incorporated herein by reference. Other lateral flow devices known in the art can alternatively be used with the systems and methods of the present invention.

  US Published Patent Application No. 2007/0059682 discloses detecting samples and analytes that can also contain one or more interfering substances. This publication teaches separating an analyte from an interfering substance by acquiring an interfering substance on a chromatography carrier and detecting the analyte on the carrier separated from the interfering substance. .

  US Published Patent Application No. 2005/0175992 discloses a method for detecting targets such as pathogens and / or allergy-related components in human body fluids where body fluid samples are collected by a collection device such as a swab member. The sample is transferred from the swab member to a sample analyzer where the target is analyzed by immunochemical or enzymatic means. Inspection results can be displayed in a very short time and can be read directly by the user. This allows point-of-care testing to obtain results during patient visits. The invention disclosed in this co-pending application is particularly effective in the diagnosis of conjunctivitis.

  FIG. 1 shows a schematic top view of a test strip for a lateral flow binding assay for nucleic acid detection in an embodiment of the invention. While the test strip shown in FIG. 1 has a specific structure and other structures having different arrangements for sample application, the first and second composites, the arrangement of the modifier or solubilizer is the present invention. Within the spirit of The test strip (10) of this example includes a first portion (12), a second portion (14), a third portion (16), and optionally a fourth portion (18). Such a part is preferably mounted on the backing material (20). The flow occurs in the upward direction (22) in FIG. The fourth part (18) functions as a waste pad for collecting transport liquid and material flowing through the third part (16), also referred to herein as the detection region, preferably nitrocellulose Including membrane. The first complex described below rests on the complex area, which is the lower part (24) of the second part (14) of the figure. In one embodiment, the second complex described below is placed on the inspection area (26) of the detection area (16). In another embodiment, the second composite is placed on the upper portion (30) of the second portion (14). In this embodiment, the second complex includes an immobilizing agent such as biotin, and the test region (26) includes avidin, neutravidin, streptoid for binding the second complex to the test region (26). It has a fixed site such as avidin functionality. The third complex described below is preferably placed on the control area (28) of the detection area (16) on the examination area (26). The inspection area (26) and the control area (28) are preferably areas having a length longer than the width of the detection area (16).

  In one embodiment of the invention, the target nucleic acid in the sample to be tested moves faster than the first complex in the transport liquid. In this embodiment, the sample is placed on the first portion (12) below the first complex, and the transport liquid, such as the buffer in use, is below the place where the sample is placed to perform the assay. Of the first part (12). Alternatively, the sample solution itself may be used as the transport liquid in this embodiment. In another embodiment, the target nucleic acid in the sample to be tested moves slower than the first complex in the transport liquid. In this embodiment, the sample is placed on the upper part (30) of the second part (14) and the transport liquid is applied to the first part (12) of the test strip. In both embodiments, the assay is preferably performed before substantially all of the first complex not bound to the test line (26) reaches the control line (28). The position at which the sample is loaded and the position at which the first complex is loaded depend on the context of the assay, so that the sample can be bound to a complementary nucleic acid sequence on the labeled first complex. As long as the first complex is encountered on the test strip, the position may change. For example, the sample may be added directly over the first complex (complex) and other embedded material, or slightly downstream or slightly upstream of the first complex and other embedded material. May be added.

  In an embodiment of the invention, at least one first nucleic acid sequence is conjugated to a label to form a first complex. As used herein, the term “label” refers to an atom, a plurality of atoms, or a molecule, such as a fluorescent tag, that is attached to or bound to a nucleic acid and is detectable. And is preferably used to provide a quantified signal. The nucleic acid sequence may be appropriately coupled to the label via a non-covalent bond such as biotin-avidin. Similarly, the label may be a fluorescent bead or fluorescent bead or a microbead such as colloidal gold particles mixed with dyed latex beads, in which case the dye may be a red dye, fluorescent dye, phosphorescent dye, Alternatively, a visible dye such as a chemiluminescent dye may be used. Such a first complex is used in the mobile phase of lateral flow chromatography. Label detection methods include, but are not limited to, fluorescence, chemiluminescence, radiation, colorimetry, gravimetry, X-ray diffraction, X-ray absorption, magnetism, and enzyme activity. The visible spectrum inspection line may be interpreted by a spectrometer to produce a quantified inspection result. The first complex is placed on the lateral flow strip in a shape that is fixed by a transport liquid, such as a buffer in use, or by a sample applied to the lateral flow strip.

  A qualitative interpretation is that in the example where the visible red dose, which is made visually by observing the intensity and shade of the test line, is used as a label, the analyte concentration is equal to or below the lower limit of detection. If slightly above, the inspection line can be seen faintly and the shade is pink. As the analyte concentration increases, the intensity of the test line increases correspondingly, and the hue changes from pink to fresh blood. A quantitative interpretation is developed using a spectrometer operating in the visible spectrum. Either absorption measurements or reflectance measurements may be used in the visible spectrum to develop quantification of the inspection line. A first set of characteristic concentrations of the analyte is developed. Each concentration is applied to the sample application area and the inspection is developed. The spectrometer is used to measure either the absorption or reflectance of the inspection line. A standard curve is calculated from the measured values of the spectrometer. A standard curve is usually linear. In other embodiments, if fluorescent tags are used, a similar set of known concentrations of analyte may be developed. The unknown concentration of the analyte that is examined and quantified by the spectrometer yields a value that can be correlated with the concentration of the analyte when shown on a standard curve.

  In one embodiment, the second nucleic acid sequence is immobilized in the second complex on a lateral flow test strip in the test area. The second nucleic acid sequence may be immobilized by various methods to form the second complex within the spirit of the invention. If immobilization is achieved by combining a nucleic acid sequence with at least one chemical, immobilization may be direct and the immobilization may be physically performed with at least one chemical or other component. Immobilization may be indirect, if achieved by capture. Immobilization includes, but is not limited to, binding to polylysine, binding to microbeads, and incorporation into a hydrogel. Microbeads such as latex beads may be fixed directly or captured in the lumen of a medium such as nitrocellulose membrane, nylon, or polyester. The hydrogel may be immobilized directly in a suitable medium or may be immobilized by a capture mechanism.

  In one embodiment, different portions of a single contiguous sequence of target nucleic acids can be complementary to both the first and second nucleic acid sequences. As an example, 25 nucleotide peptide nucleic acid sequences (PNAs) are complementary to a portion of the target nucleic acid. About half of such nucleotides (eg, 11 or 12 nucleotides) are used as the first nucleic acid sequence in the first complex and the other half of nucleotides (eg, 12 or 13 nucleotides) are used in the second complex. Used as the second nucleic acid sequence in the middle. In this example, the first nucleic acid sequence binds to one part of the single sequence of the target nucleic acid and the second nucleic acid sequence binds to the second part of the single sequence of the target nucleic acid.

  In another embodiment, the second nucleic acid sequence is combined with an immobilizing agent to form a second complex. In this embodiment, the second composite is placed on the upper portion (30) of the second portion (14) of the test strip. The receptor for the immobilizing agent is immobilized at an immobilization site on the examination area (26). When the immobilizing agent reaches the test area during the assay, the immobilizing agent binds to the receptor to immobilize the second complex on the test area (26). The immobilizing agent and receptor may be any pair with a strong specific interaction that does not interfere with the desired nucleic acid interaction of the assay. The immobilizing agent may be biotin and the receptor may be adivine, neutravidin, or streptavidin. Alternatively, the immobilizing agent may be a lectin and the immobilization site may be a glycosyl moiety. For example, in some embodiments, the lectin is a pea lectin and the glycosyl moiety is an erythrocyte glycosyl unit. The immobilization agent and the immobilization site may be reversed within the spirit of the present invention. For example, avidin may be conjugated to a second nucleic acid sequence to form a second complex having biotin that acts as a receptor at an immobilization site in the test region (26).

  In a preferred embodiment of the assay, a third nucleic acid sequence complementary to the first nucleic acid sequence is immobilized with a third complex on the control region. The third nucleic acid may be immobilized by any of the various methods discussed above with respect to the second nucleic acid sequence within the spirit of the invention. During the performance of the assay, the third complex is designed such that when any of the first complexes that do not bind to the target nucleic acid in the sample reach the control region, it binds to the third complex through the test region. Is done. In this way, any first complex that does not bind to the target nucleic acid is detected and, in some embodiments, quantified. Although the assay of the present invention may be performed without including a control region, a control region is preferred for confirming a negative test result and for comparing a test region with a positive test result.

  The target nucleic acid may be any nucleic acid sequence, which is a nucleic acid from a virus, bacterium, fungus, plant, animal, artificially created sequence, genetically modified organism (GMO), or fossil specimen Including but not limited to sequences. The sample is applied to the strip in the sample application area. The sample application area may come before the first composite, overlap the first composite, or may come next to the first composite on the test strip. When the sample is applied after the first complex, the transport complex is used to mobilize the first complex and transport the first complex to expose the first complex to any of the target nucleic acid samples. To do. In one embodiment, the test region consists of at least one immobilized nucleic acid sequence, which is a sequence used in a conventional polymerase chain reaction (PCR) assay. The sequence is preferably made from deoxyribonucleotides, ribonucleotides or peptide nucleotides. In this embodiment, the first complex comprises such a PCR type primer of the target nucleic acid complexed to a label. Such a primer is preferably an antisense sequence, which complements the corresponding “sense” sequence of nucleic acids from organisms such as viruses, bacteria, and fungi. Sense and antisense primers may be interchanged in lateral flow assays within the spirit of the invention.

  Although only one inspection line is shown in the drawing, multiple inspection lines are within the spirit of the invention. In some embodiments where there are multiple targets, the presence of each target preferably corresponds to a separate test line. In other embodiments where there are multiple targets, the presence of multiple targets may be displayed on the same test line so that the presence of one or more targets has different characteristics than the presence of a single target . For example, the presence of multiple targets on the same inspection line may be visibly displayed with a different color than the presence of each target alone.

  FIG. 2 shows a complex of a target nucleic acid capable of positive detection in the first embodiment of the present invention. In this embodiment, the target nucleic acid (40) in the sample is a single stranded nucleic acid sequence. The first complex (42) comprises at least one first nucleic acid sequence (44) that is complementary to an internal portion of the sequence of the target nucleic acid (40). The first nucleic acid sequence (44) is bound to a label (46). The second complex includes at least one second nucleic acid sequence (48) that is complementary to the outer portion of the sequence of the target nucleic acid (40). Using two sequences that are complementary to different portions of the target nucleic acid in a lateral flow assay improves the specificity of the assay. In some embodiments, the first nucleic acid sequence (44) is preferably an internal PCR primer and the second nucleic acid sequence (48) is preferably an external PCR primer. Alternatively, the first nucleic acid sequence (44) is an external PCT primer and the second nucleic acid sequence (48) is an internal PCR primer. Although one first nucleic acid sequence and one label are sufficient for each first complex, the first complex preferably includes multiple copies of the first nucleic acid sequence shown in FIG. And a plurality of labeling units indicated by an asterisk. Although eight nucleic acid sequences are shown in FIG. 2, any number that facilitates binding of the first complex to the target nucleic acid may be used within the spirit of the invention.

  3 to 9 show a complex of a target nucleic acid that can be positively detected in the second embodiment of the present invention. As shown in FIG. 3, in this embodiment, the target nucleic acid (50) in the sample is a double-stranded nucleic acid. The target nucleic acid (50) in the sample is preferably DNAzole Direct, formamide, or urea in order to open at least a portion of the double strand into a first strand (52) and a second strand (54). Denature in situ with a denaturant or solubilizer such as This can also be done by adding a denaturant or lysing agent to the sample solution before applying the sample to the test strip, or the denaturant or lysing agent, as discussed above, can be It may be placed in advance in the area. Alternatively, nucleic acid modifications may be made by adjusting or controlling assay toxicity, temperature, or pH within the spirit of the invention. The assays of the invention may be performed by either partially denaturing or completely denaturing nucleic acids in the sample, although partial opening is preferred for detection in lateral flow assays.

  In a preferred embodiment, the denaturant or lysing agent is placed in the sample application area so that the nucleic acid is denatured and / or lysed immediately after it is added to the test strip. In other embodiments, the denaturant or lysing agent is on the test strip such that the target nucleic acid is at least partially denatured and / or lysed before or during encountering the first labeled complex (56). Arranged at an arbitrary position.

  As shown in FIG. 4, the first complex (56) comprises at least one first nucleic acid sequence (58), which is preferably a primer for a single strand (52) of the target nucleic acid sequence or The antisense sequence (58) of the oligonucleotide. The first nucleic acid sequence (58) is bound to a label (60). The label may be any atom, multiple atoms, or molecule, such as a visible tag or a fluorescent tag, that is attached or bound to the nucleic acid and is detectable and preferably Used to provide a quantified signal. The nucleic acid sequence may be appropriately coupled to the label via a non-covalent bond such as biotin-avidin. Similarly, the label may be a fluorescent bead or fluorescent bead or a microbead such as a colloidal gold particle mixed with a dyed latex bead, in which case the dye is a red dye, fluorescent dye, phosphorescent dye Or a visible dye such as a chemiluminescent dye.

  FIG. 5 shows a second complex (62), which preferably comprises at least one second nucleic acid sequence (64), which is an antisense primer or oligonucleotide, and an immobilizing agent (66, such as biotin). ). The second nucleic acid sequence (64) is complementary to the Y binding site of the double strand (54) of the target nucleic acid (50). In this embodiment, the second composite (62) is preferably disposed on the upper portion (30) of the second portion (14) of the test strip (10) shown in FIG. 1 and shown in FIG. A receptor, such as an avidin-bound probe (70), is immobilized on the test area (26) (preferably a nitrocellulose membrane) of the test strip (10) at the start of the assay. The second complex may alternatively penetrate on the part of the test strip on which the sample is placed, or any in the test strip where it encounters the sample before it reaches the detection area It may penetrate in places. In addition, the second complex does not necessarily have to be arranged downstream of the first complex. For example, the sample pad may be infiltrated with a biotinylated (B) viral primer or oligonucleotide antisense to single stranded Y DNA of the sample. Adibin (72) and biotin (66) are shown as immobilizing agents in these figures, but other immobilizing agents described herein or other known in the art An immobilizing agent can alternatively be used.

  In the embodiment of FIGS. 3-9, when lateral flow is initiated by immersing the absorbent tip in the transport liquid, the first complex (56) is mobilized and the first complex (56). The upper primer or oligonucleotide sequence (58) is conjugated to a single strand (52) of nucleic acid opened at the X binding site shown in FIG. The first complex (56) and sample nucleic acid (50) may be placed anywhere on the test strip so that they encounter each other while they elute through the test strip.

The target nucleic acid of the first complex is then exposed to the second complex (62). The second sequence (64) is conjugated to the second strand at the Y binding site shown in FIG. The overall complex shown in FIG. 8 then moves to the test area, where biotin (66) is immobilized in the test area via binding of biotin (66) to avidin (72). Captured by activated avidin (72). This results in a visible readout line. The test area is only visible when the target nucleic acid sequence is present in the sample, indicating that the sample is positive for the target nucleic acid. In negative samples where the target nucleic acid sequence is not present, the test area remains colorless. The intensity of the examination area may be increased by either fluorescence bound with visiblely tagged composite particles or fluorescence mixed with fluorescent composite particles.
Since the first and second nucleic acid sequences are both preferably complementary antisense sequences of the target nucleic acid, the X and Y binding sites are preferably non-overlapping sequences. If the first and second nucleic acid sequences are antisense primers, the quality of the engineered primer is determined by the potential of the first and second sequences, which in the absence of the target nucleic acid will result in a false positive test result. Important to prevent any direct binding.

  In another embodiment of the invention, the lateral flow assay tests for one or more target nucleic acids in a sample. In this embodiment, another combination of first complex and second complex is used for each target nucleic acid to be detected. In this embodiment, each first complex has a different label that is detectable by different methods so that each target nucleic acid is detected and quantified separately without interference from other target nucleic acids. Have

  The analytical tests discussed herein preferably allow results while the patient is still being examined by the practitioner. In a preferred embodiment, test results are obtained in less than 10 minutes after applying the sample to the device, and the test results are preferably read out for about 10 minutes. In high positive samples, the readout of the test area (preferably the test line) is visible within 1 to 5 minutes.

  Accordingly, it is to be understood that the embodiments described herein are merely illustrative of the application of the principles of the present invention. References herein to details of the illustrated embodiments are not intended to limit the scope of the claims enumerating features that are considered essential to this invention.

Claims (29)

A test strip for detecting at least one target nucleic acid in a sample comprising:
The test strip is
At least one detection area comprising at least one examination area;
A sample application area for applying the sample to the test strip;
Comprising at least one first complex comprising at least one first nucleic acid sequence coupled to at least one label;
The first composite is placed in a first composite application area; and
The first nucleic acid sequence comprises a sequence complementary to a first portion of the sequence of the target nucleic acid;
The test strip is
Comprising at least one second complex comprising at least one second nucleic acid sequence coupled with at least one immobilizing agent;
The immobilizing agent is immobilizable in the test area of the assay; and
The second nucleic acid sequence comprises a sequence complementary to a second portion of the sequence of the target nucleic acid;
The sample, the first complex, and the second complex are all in an assay for detecting the target nucleic acid in the sample while the sample is in the first complex and the second complex. A test strip, characterized in that it is placed on a test strip in a position where both are encountered.   The test strip according to claim 1, wherein the target nucleic acid is a single-stranded nucleic acid.   The test strip according to claim 1, wherein the target nucleic acid is a double-stranded nucleic acid.   The detection region further includes a control region disposed beyond the inspection region in order to immobilize an arbitrary portion of the first complex that does not bind to the target nucleic acid. Inspected strip. A receptor that recognizes and immobilizes the immobilizing agent;
The test strip according to claim 1, wherein the second complex is placed at an upstream position of the test area, and an immobilization site is arranged in the test area. A receptor that recognizes and immobilizes the immobilizing agent;
The test strip according to claim 1, wherein the second complex is placed on the test area, and the receptor is placed at an upstream position of the test area. The sign is
(A) at least one colloidal gold particle mixed with at least one fluorescent microbead;
(B) at least one dye latex microbead;
The test strip of claim 1, wherein the test strip is selected from the group consisting of: The first nucleic acid sequence and the second nucleic acid sequence are respectively
(A) an internal primer;
(B) an external primer;
(C) a sense primer, and
The test strip according to claim 1, which is a polymerase chain reaction (PCR) type primer of the target nucleic acid selected from the group consisting of (d) an antisense primer.   The test strip of claim 1 further comprising a solubilizer or denaturant.   A polymerase chain reaction primer coupled to at least one label.   Nucleic acid immobilized in the detection region of a test strip for point-of-care assays.   The nucleic acid according to claim 11, wherein the nucleic acid is a polymerase chain reaction primer. A method for detecting at least one target nucleic acid in a sample using a point-of-care assay, comprising:
The method
(A) exposing the sample to at least one first complex comprising at least one first nucleic acid sequence coupled to at least one label;
(B) exposing the sample to at least one second complex comprising at least one second nucleic acid sequence;
When the target nucleic acid is present in the sample, the first nucleic acid sequence and the second nucleic acid sequence are the first complex, the target nucleic acid, and the second complex are in the test region upon completion of the assay. A method comprising binding to the target nucleic acid so that the target nucleic acid is immobilized. The second complex binds to at least one immobilizing agent;
14. The method of claim 13, wherein the immobilizing agent is immobilizable within the test area of the assay.   14. A method according to claim 13, characterized in that the second complex is located in the examination area of the assay. The first nucleic acid sequence and the second nucleic acid sequence are respectively
(A) an internal primer;
(B) an external primer;
(C) a sense primer, and
14. The method according to claim 13, wherein the target nucleic acid is a polymerase chain reaction (PCR) type primer selected from the group consisting of (d) an antisense primer.   14. The method of claim 13, further comprising collecting the sample from a patient. The sample is
(A) saliva;
(B) mucus,
(C) the organization;
(D) tears, (e) cerebrospinal fluid,
(F) vaginal fluid;
(G) penile fluid;
(H) nasopharyngeal secretions;
(I) a skin ulcer;
(J) blood and
14. The method of claim 13, wherein the method is from a body fluid selected from the group consisting of (k) urine. Further comprising exposing the sample to a solubilizer or denaturant;
The method according to claim 13, wherein the target nucleic acid is a double-stranded nucleic acid.   14. The method of claim 13, further comprising detecting the presence or absence of the label in the examination area.   The step of detecting the label includes the step of using a method selected from the group consisting of fluorescence, chemiluminescence, radiation, colorimetry, gravimetry, X-ray diffraction, X-ray absorption, magnetism, and enzyme activity. 21. The method according to claim 20, wherein: The sign is
(A) at least one colloidal gold particle mixed with at least one fluorescent microbead;
(B) at least one dye latex bead;
14. The method of claim 13, wherein the method is selected from the group consisting of:   14. The method of claim 13, wherein the sample is used in the assay without an amplification step that amplifies the target nucleic acid.   14. The method of claim 13, wherein the assay is performed on a chromatographic test strip. A test strip for detecting at least one target nucleic acid in a sample comprising:
The test strip is
At least one detection area comprising at least one examination area;
A sample application area for applying the sample to the test strip;
Comprising at least one first complex comprising at least one first nucleic acid sequence coupled to at least one label;
The first composite is placed on the first composite application area,
The test strip further comprises:
Comprising at least one second complex immobilized at a test region of the assay, comprising at least one second nucleic acid sequence;
The sample and the first complex are disposed on a test strip at a location such that the sample encounters the first complex; and
The target nucleic acid includes at least one first target nucleic acid having a sequence complementary to the first nucleic acid sequence, and at least one second target nucleic acid having a sequence complementary to the second nucleic acid sequence, To test strip.   The test strip according to claim 25, wherein the target nucleic acid is a single-stranded nucleic acid.   The test strip according to claim 25, wherein the target nucleic acid is a double-stranded nucleic acid.   26. The control region further comprises a control region disposed beyond the test region in order to immobilize any part of the first complex that does not bind to the target nucleic acid. Inspected strip. The first nucleic acid sequence and the second nucleic acid sequence are respectively
(A) an internal primer;
(B) an external primer;
(C) a sense primer, and
26. The test strip of claim 25, wherein the test strip is a polymerase chain reaction (PCR) type primer of the target nucleic acid selected from the group consisting of (d) an antisense primer.