CN114480585A - Nucleic acid probe composition, pretreatment solution, nucleic acid detection kit and detection method - Google Patents
Nucleic acid probe composition, pretreatment solution, nucleic acid detection kit and detection method Download PDFInfo
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Abstract
The invention discloses a nucleic acid probe composition, which comprises: the kit comprises a detection probe, a quality control probe, a report probe, a first nucleic acid probe, a second nucleic acid probe and a signal amplification probe; the first nucleic acid probe comprises a first sequence region and a second sequence region, and the first sequence region is complementarily paired with a marker auxiliary sequence region of a nucleic acid to be detected; the second nucleic acid probe comprises a third sequence region and a fourth sequence region, the third sequence region is complementarily paired with the capture auxiliary sequence region of the nucleic acid to be detected, and the fourth sequence region is complementarily paired with the detection probe; the report probe and the quality control probe are complementarily paired; the signal amplification probe comprises an amplification chain preposing guide sequence region and a plurality of amplification sequence regions which are connected in sequence, wherein the amplification sequence regions are complementarily paired with the report probe, and the amplification chain preposing guide sequence region is complementarily paired with the second sequence region of the first nucleic acid probe. The invention also discloses a nucleic acid detection method and a kit.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a nucleic acid probe composition, a pretreatment solution, a nucleic acid detection kit and a detection method.
Background
The nucleic acid detection, namely the detection of DNA and RNA, is the most advanced and reliable detection method for viruses, bacteria and the like at present. It is used for detecting hepatitis B, hepatitis C, AIDS, respiratory tract pathogen, etc. At present, nucleic acid detection mainly adopts Polymerase Chain Reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP) and other nucleic acid amplification technologies, the technologies need to realize amplification of target nucleic acid fragments through accurate temperature control so as to detect, and the technologies have the advantages of improving detection specificity, reducing false positive probability and the like, but at the same time, the detection is long in time consumption, needs professional operation, highly depends on instrument and equipment, and cannot realize portable and rapid detection.
Chinese patent CN111593145A discloses a CRISPR/Cas12 one-step nucleic acid detection method and a novel coronavirus detection kit, and compared with the traditional RT-qPCR technology, the method can synchronously carry out amplification and detection, and realizes one-step and integrated nucleic acid detection. However, the method still depends on the nucleic acid amplification technology, i.e., needs professional instruments and equipment and personnel to operate, and cannot be applied to application scenes such as resource shortage areas or large-scale rapid detection and the like.
Disclosure of Invention
Accordingly, there is a need for a nucleic acid probe composition, a pretreatment solution, a nucleic acid detection kit, and a detection method that can detect nucleic acids in a sample to be detected with high sensitivity and low false positive without requiring PCR.
A nucleic acid probe composition comprising:
detecting the probe;
a quality control probe;
a first nucleic acid probe comprising a first sequence region and a second sequence region that do not overlap, the first sequence region for complementary pairing with a marker auxiliary sequence region of a nucleic acid to be detected;
a second nucleic acid probe comprising a third and a fourth non-overlapping sequence region, the third sequence region being for complementary pairing with a capture auxiliary sequence region of a nucleic acid to be detected, the tag auxiliary sequence region being different from or at least partially different in sequence from the capture auxiliary sequence region, and the fourth sequence region being for complementary pairing with the detection probe;
a report probe, comprising a fifth sequence region and a sixth sequence region which are not overlapped, wherein the fifth sequence region is used for complementary pairing with the quality control probe;
a signal amplification probe, wherein the signal amplification probe comprises a sequentially connected amplification chain preposition guide sequence region and a plurality of sequentially connected amplification sequence regions, and the amplification sequence regions are selected from a) or b);
a) said amplifying sequence region comprising a first pairing sequence for complementary pairing with said sixth sequence region of said reporter probe, respectively, said amplifying strand leader sequence region for complementary pairing with said second sequence region of said first nucleic acid probe;
b) the signal amplification probe comprises a plurality of levels, and can be paired step by step from a first level to a higher level according to the sequence of combination with the first nucleic acid probe, and the amplification sequence region of the signal amplification probe of the lower level at least comprises a second pairing sequence for complementary pairing with the amplification chain prepositive guide sequence region of the signal amplification probe of the higher level; the amplification strand leader sequence region of the first signal amplification probe is for complementary pairing with the second sequence region of the first nucleic acid probe, and the amplification sequence region of the highest signal amplification probe comprises at least one sequence for complementary pairing with the sixth sequence region of the reporter probe.
In one embodiment, the first pairing sequences in a) are identical;
or;
b) the second pair sequences on the signal amplification probes of the same order are identical.
3. The nucleic acid probe composition of claim 1, wherein the sequences of each of the amplification sequence regions are identical on the same signal amplification probe.
In one embodiment, in a), the entire amplification sequence region is capable of complementary pairing with the reporter probe;
alternatively, in b), the entire amplification sequence region of the signal amplification probe of the highest order is capable of complementary pairing with the reporter probe.
In one embodiment, in b), the signal amplification probe of the highest order, among the signal amplification probes of the non-highest order, comprises a sequence region complementarily paired with the amplification strand pre-leader sequence region of the signal amplification probe of the adjacent highest order and a sequence region complementarily paired with the reporter probe.
In one embodiment, the length of the signal amplification probe is 100bp to 600 bp.
In one embodiment, the number of the amplification sequence regions is 5-30.
In one embodiment, in b), the signal amplification probe has a 2-, 3-, 4-or 5-order.
In one embodiment, the first nucleic acid probe is a plurality of first nucleic acid probes, each of the first nucleic acid probes having a different first sequence region and a matching position that does not overlap with the tag auxiliary sequence region.
In one embodiment, the second nucleic acid probes are in a plurality, and each of the second nucleic acid probes has a different third sequence region and does not overlap with the capture auxiliary sequence region at the position where the capture auxiliary sequence region is paired.
In one embodiment, the reporter probe has a label attached thereto.
In one embodiment, the label is selected from any one or more of colloidal gold, alkaline phosphatase, and horseradish peroxidase.
A pretreatment solution comprising a solvent and a first nucleic acid probe, a second nucleic acid probe and a signal amplification probe as defined in said nucleic acid probe composition.
In one embodiment, the solvent is selected from at least one of sodium citrate solution, SSPE solution, Denhardt's solution, and phosphate solution.
In one embodiment, the molar ratio of the first nucleic acid probe, the second nucleic acid probe and the signal amplification probe in the pretreatment solution is (8-12): (0.8-1.2).
The utility model provides a nucleic acid detect reagent box, include nucleic acid detect test paper strip and pretreatment liquid, nucleic acid detect test paper strip is including lapped sample pad, connecting pad, reaction membrane and the pad that absorbs water in proper order, the package is gone up to the connecting pad and is had the report probe, be equipped with the detection line and the matter control line of mutual interval on the reaction membrane, the detection line is compared the matter control line is closer to the connecting pad, the package is gone up to the detection line has detect probe, the package is gone up to the matter control line has the matter control probe.
In one embodiment, the nucleic acid test strip is placed in a cartridge housing having a push-pull configuration.
A method for detecting nucleic acid, which adopts the nucleic acid detection kit and comprises the following steps:
mixing a sample to be detected with the pretreatment solution to obtain a solution to be detected;
and applying the liquid to be detected on the sample pad for detecting the reporter probe.
In one embodiment, the label attached to the reporter probe comprises an enzyme, further comprising: and applying the liquid to be detected on the sample pad for 0.5-30min, and then applying the chromogenic reaction substrate of the enzyme on the sample pad.
The nucleic acid detection system not only comprises a report probe, a detection probe and a quality control probe which can realize basic nucleic acid detection, but also comprises a signal amplification system, wherein the signal amplification effect of the nucleic acid to be detected, the first nucleic acid probe, the second nucleic acid probe and one or more signal amplification probes, the report probe, the detection probe and the quality control probe is realized through complex adaptation through the first nucleic acid probe, the second nucleic acid probe and one or more signal amplification probes. The amplification of signals is realized through the cooperation of various modes, and a nucleic acid to be detected is artificially divided into a marker auxiliary sequence area and a capture auxiliary area. The signal amplification probe comprises a plurality of amplification sequence regions which are sequentially connected, and each amplification sequence region in the signal amplification probe directly paired with the report probe can be complementarily paired with the report probe, so that one signal amplification probe can be paired with a plurality of report probes, the enrichment of the report probes is realized, and the report signals of the nucleic acids to be detected at a labeling end are amplified. The marking signal amplification and the capturing signal amplification are realized by forming a branched chain on the nucleic acid to be detected, the signal amplification of the whole nucleic acid detection is realized, and the detection sensitivity and the occurrence of false positive are greatly reduced.
In addition, the invention does not limit the length of the nucleic acid to be detected, and can be widely applied to the detection of nucleic acid from any sources such as viruses, bacteria and the like. The longer the sequence of the nucleic acid to be detected, the more regions available for signal amplification, the more pronounced the signal amplification.
Drawings
FIG. 1 is a schematic structural diagram of a nucleic acid detection test strip according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a nucleic acid detection strip device according to an embodiment of the present invention;
FIG. 3 is a diagram showing the results of nucleic acid detection according to the embodiment of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides a nucleic acid probe composition, which comprises: the kit comprises a detection probe, a quality control probe, a report probe, a first nucleic acid probe, a second nucleic acid probe and one or more signal amplification probes.
A first nucleic acid probe comprising a first and second non-overlapping sequence region, the first sequence region for complementary pairing with a marker auxiliary sequence region of a nucleic acid to be detected.
A second nucleic acid probe comprising a third and a fourth non-overlapping sequence region, the third sequence region being for complementary pairing with a capture auxiliary sequence region of a nucleic acid to be detected, the tag auxiliary sequence region being different from or at least partially different in sequence from the capture auxiliary sequence region, and the fourth sequence region being for complementary pairing with the detection probe.
A reporter probe comprising a fifth and a sixth non-overlapping sequence region, the fifth sequence region for complementary pairing with the quality control probe.
The signal amplification probe comprises an amplification chain preposed guide sequence region and a plurality of amplification sequence regions which are connected in sequence.
The signal amplification probe is selected from a) or b).
a) The amplification sequence region comprises a first pairing sequence for complementary pairing with the sixth sequence region of the reporter probe, and the amplification strand leader sequence region is for complementary pairing with the second sequence region of the first nucleic acid probe.
b) The signal amplification probe comprises a plurality of levels, the signal amplification probe can be paired step by step from a first level to a higher level according to the sequence of combination with the first nucleic acid probe, and the amplification sequence region of the signal amplification probe of the lower level at least comprises a second pairing sequence for complementary pairing with the amplification chain prepositive guide sequence region of the signal amplification probe of the higher level; the amplification strand leader sequence region of the first signal amplification probe is for complementary pairing with the second sequence region of the first nucleic acid probe, and the amplification sequence region of the highest signal amplification probe comprises at least one sequence for complementary pairing with the sixth sequence region of the reporter probe.
The nucleic acid detection system not only comprises a report probe, a detection probe and a quality control probe which can realize basic nucleic acid detection, but also comprises a signal amplification system, wherein the signal amplification effect of the nucleic acid to be detected, the first nucleic acid probe, the second nucleic acid probe and one or more signal amplification probes, the report probe, the detection probe and the quality control probe is realized through complex adaptation through the first nucleic acid probe, the second nucleic acid probe and one or more signal amplification probes. The amplification of signals is realized through the cooperation of various modes, and a nucleic acid to be detected is artificially divided into a marker auxiliary sequence region and a capture auxiliary region. The signal amplification probe comprises a plurality of amplification sequence regions which are sequentially connected, and each amplification sequence region in the signal amplification probe directly paired with the report probe can be complementarily paired with the report probe, so that one signal amplification probe can be paired with a plurality of report probes, the enrichment of the report probes is realized, and the report signals of the nucleic acids to be detected at a labeling end are amplified. The marking signal amplification and the capturing signal amplification are realized by forming a branched chain on the nucleic acid to be detected, the signal amplification of the whole nucleic acid detection is realized, and the detection sensitivity and the occurrence of false positive are greatly reduced.
In addition, the invention does not limit the length of the nucleic acid to be detected, and can be widely applied to the detection of nucleic acid from any sources such as viruses, bacteria and the like. The longer the sequence of the nucleic acid to be detected, the more regions available for signal amplification, the more pronounced the signal amplification.
The above-mentioned "artificially dividing the nucleic acid to be detected into the labeling auxiliary region and the capturing auxiliary region" has no particular limitation on the division of the labeling auxiliary region and the capturing auxiliary region, and the basic condition is that the two auxiliary regions are not completely overlapped, and preferably, the sequence segments of the auxiliary regions and the capturing auxiliary regions which are not overlapped, so that the first nucleic acid probe and the second nucleic acid probe can be ensured to be respectively paired in different sequence regions of the nucleic acid to be detected, and the false negative phenomenon caused by the competitive pairing of the first nucleic acid probe and the second nucleic acid probe with the nucleic acid to be detected can be avoided. More preferably, the label auxiliary region and the capture auxiliary region are divided into 5 'and 3' ends, or sequence segments near the 5 'and 3' ends, respectively, of the nucleic acid molecule to be detected.
The signal amplification probe may have only one stage, or may have 2, 3, 4, 5 or more stages.
When the signal amplification probe has a primary level, the pre-leading sequence region of the amplification strand of the signal amplification probe is complementarily paired with the second sequence region of the first nucleic acid probe, and each amplification sequence region is complementarily paired with at least one reporter probe.
When the signal amplification probes have two or more levels, the amplification chain preposition guide sequence area of the first level signal amplification probe is complementarily paired with the second sequence area of the first nucleic acid probe, each amplification sequence area of the first level is complementarily paired with the amplification chain preposition guide sequence area of at least one second level signal amplification probe, the amplification chain preposition guide sequence area of the highest level signal amplification probe is complementarily paired with the amplification sequence area of the adjacent lower level signal amplification probe, each amplification sequence area of the highest level signal amplification probe is complementarily paired with at least one report probe, and the amplification chain preposition guide sequence areas of the other level signal amplification probes between the first level and the highest level are complementarily paired with the amplification sequence areas of the adjacent lower level signal amplification probes.
Specifically, when the signal amplification probe has two stages, the amplification strand leader sequence region of the first-stage signal amplification probe is complementarily paired with the second sequence region of the first nucleic acid probe, and the amplification sequence region of the first-stage signal amplification probe is complementarily paired with the amplification strand leader sequence region of the second-stage signal amplification probe; each amplification sequence region of the second signal amplification probe is complementary paired with at least one reporter probe.
The sequence of each amplification sequence region may not be identical on the same signal amplification probe, but at least each amplification sequence region should include a matching region to achieve complementary pairing with the reporter probe or an adjacent higher-order signal amplification probe.
In some embodiments, the pair of sequences of each amplification sequence region are identical on the same signal amplification probe. Namely, a plurality of pairing branches are formed in each amplification sequence area on the same signal amplification probe, except for a pairing area which is complementarily paired with a report probe or an adjacent high-order signal amplification probe, the sequences which do not participate in pairing in each amplification sequence area are also the same, so that the pairing branch structure is more orderly, the space utilization is improved, and the pairing positions of each amplification sequence area are fully paired. a) Wherein the first pair sequences are identical; or; b) the second pair sequences on the signal amplification probes of the same order are identical.
In some embodiments, the signal amplification probe is one, i.e., the entire amplification sequence region in a) is capable of complementary pairing with the reporter probe. In some embodiments, the signal amplification probe is a plurality, i.e., the entire amplification sequence region of the signal amplification probe of the highest order in b) is capable of complementary pairing with the reporter probe. That is, the entire amplification sequence region of the signal amplification probe is the mate sequence region.
Preferably, the sequences of the amplification sequence regions are identical on the same signal amplification probe. That is, the sense strand leader sequence region is sequentially linked to a plurality of repeated sense strand regions.
In some embodiments, the signal amplification probe is a plurality of, i.e., in the signal amplification probe of non-highest order in b), the amplification sequence region of the signal amplification probe of the highest order includes a sequence region complementarily paired with the amplification strand pre-leader sequence region of the signal amplification probe of an adjacent higher order and a sequence region complementarily paired with the reporter probe. That is, in the case of the signal amplification probes of the plurality of stages, the amplification sequence region of the signal amplification probe of each stage except for the highest stage can be complementarily paired with the signal amplification probe of the adjacent higher stage and also complementarily paired with the reporter probe.
In some embodiments, the amplification strand leader sequence region also includes a sequence region complementary to the reporter probe.
In some embodiments, the signal amplification probe may be 100bp to 600bp in length. Here the length of a single signal amplification probe.
In some embodiments, the length of the amplification strand leader sequence of the signal amplification probe may be 10bp to 20 bp.
In some embodiments, the length of the single amplification sequence region of the signal amplification probe may be 10bp to 30 bp.
In some embodiments, the number of the amplification sequence regions per signal amplification probe may be 5 to 30.
In some embodiments, the first nucleic acid probe is a plurality of first nucleic acid probes, each of the first nucleic acid probes having a different first sequence region and a matching position that does not overlap with the tag helper sequence region. That is, the nucleic acid to be detected is divided into a plurality of labeled auxiliary regions, each labeled auxiliary region is complementary paired with a first nucleic acid probe, and a plurality of labeled auxiliary sequence region paired branches are formed on one nucleic acid molecule to be detected, so that the signal amplification degree is further improved.
In some embodiments, the second nucleic acid probe is a plurality, and the third sequence region of each of the second nucleic acid probes is different and does not overlap with the capture helper sequence region at the position of the pair. That is, the nucleic acid to be detected is divided into a plurality of capture auxiliary regions, each label auxiliary region is complementary paired with a second nucleic acid probe, and a plurality of paired branches of capture auxiliary sequence regions are formed on one nucleic acid molecule to be detected, so that the signal amplification degree is further improved.
In some embodiments, the reporter probe has a label attached thereto for signal detection.
The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid or protein. Labels include, but are not limited to, dyes; radiolabels, e.g.32P; binding moieties such as biotin; haptens such as digoxin; a luminescent, phosphorescent, or fluorescent moiety; and a fluorescent dye alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like. Labels can be charged moieties (positive or negative) or alternatively, can be charge neutral. The label may comprise or be combined with a nucleic acid or protein sequence, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is detected directly (e.g., direct sequence reading) without a label.
In some preferred embodiments, the label is a fluorophore, a colorimetric label, a quantum dot, biotin, and other label molecules that can be used for detection (e.g., alkyne groups for raman diffraction imaging, cyclic olefins for click reactions, priming groups for polymer labeling), and can also be selected from polypeptide/protein molecules, LNA/PNA, unnatural amino acids and their analogs (e.g., peptidomimetics), unnatural nucleic acids and their analogs (nucleotide mimetics), and nanostructures (including inorganic nanoparticles, NV-centers, aggregation/assembly-induced emission molecules, rare earth ion ligand molecules, polyoxometalate clusters, etc.). More preferably, the label is selected from any one or more of colloidal gold, alkaline phosphatase and horseradish peroxidase.
The embodiment of the present invention also provides a pretreatment solution comprising a solvent and the first nucleic acid probe, the second nucleic acid probe and the signal amplification probe as defined in the nucleic acid probe composition of any of the above embodiments.
The pretreatment solution is used for detecting a target nucleic acid by coordinating with the detection probe, the quality control probe and the reporter probe defined in the nucleic acid probe composition of any of the above embodiments.
The pretreatment solution is used as a premixed solution of a sample to be detected when a test strip is used for detecting nucleic acid.
In some embodiments, the solvent is selected from at least one of Sodium citrate solution, SSPE solution (SSPE: salt-Sodium Phosphate-EDTA), Denhardt's solution, and Phosphate solution.
In some embodiments, the molar ratio of the first nucleic acid probe, the second nucleic acid probe, and the signal amplification probe in the pretreatment solution may be (8-12): (0.8-1.2). Preferably 10:1: 1.
The embodiment of the invention also provides a nucleic acid detection kit, which comprises a nucleic acid detection test strip and the pretreatment solution, wherein the nucleic acid detection test strip comprises a sample pad, a connecting pad, a reaction membrane and a water absorption pad which are sequentially overlapped, the connecting pad is coated with a report probe defined in the nucleic acid probe composition, the reaction membrane is provided with a detection line and a quality control line which are mutually spaced, the detection line is closer to the connecting pad than the quality control line, the detection line is coated with the detection probe defined in the nucleic acid probe composition, and the quality control line is coated with the quality control probe defined in the nucleic acid probe composition.
The reaction membrane may be a nitrocellulose membrane or the like.
The nucleic acid detection test strip also comprises a bottom plate, a sample pad, a connecting pad, a reaction membrane and a water absorption pad which are lapped on the bottom plate.
In some embodiments, the sample pad is prepared by: soaking the glass fiber membrane in solution containing polyethylene glycol octyl phenyl ether and/or triton X-100 for 0.1-12h, drying and storing in a dryer. The method can promote the flow of the sample to be detected and reduce the retention and blockage of the sample.
The specific form of the reporter probe labeled with the label can be one of colloidal gold labeled with the reporter probe, colloidal gold labeled with the reporter probe and horseradish peroxidase together, a horseradish peroxidase modified reporter probe or an alkaline phosphatase modified reporter probe.
In one embodiment, the preparation of the colloidal gold labeled with the reporter probe comprises the following steps:
preparing colloidal gold (for example, by a sodium citrate method), then modifying a report probe on the surface of the colloidal gold, centrifuging the prepared solution at the rotating speed of 5000-30000rpm for 5-60min, and removing the free report probe to obtain the colloidal gold modified by the report probe.
In one embodiment, the preparation of the colloidal gold labeled with both the reporter probe and horseradish peroxidase comprises the following steps:
preparing colloidal gold, then modifying a report probe on the surface of the colloidal gold, centrifuging the prepared solution at the rotating speed of 5000-30000rpm for 5-60min, and removing the free report probe to obtain the colloidal gold modified by the report probe. And then adding horseradish peroxidase to promote the horseradish peroxidase to replace a part of the report probe, centrifuging for 5-60min at the rotating speed of 5000-.
In one embodiment, the preparation of horseradish peroxidase-modified reporter probes comprises the following steps:
and (3) mixing the biotin-modified report probe with streptavidin-modified horse radish peroxidase for reaction to obtain the horse radish peroxidase-modified report probe.
In one embodiment, the preparation of the alkaline phosphatase modified reporter probe comprises the steps of:
and mixing the report probe and alkaline phosphatase for reaction to obtain the report probe modified by the alkaline phosphatase.
In some embodiments, the method for preparing the connection pad comprises the following steps: the connecting pad is soaked in a buffer solution containing bovine serum albumin and disodium hydrogen phosphate for 1-1000 minutes, and then dried and stored in a drier. The reporter probe labeled with the labeling substance is sprayed on the connecting pad and is stored in a dry state at room temperature.
In some embodiments, a method of preparing a reactive membrane comprises the steps of: the detection probe and the quality control probe are respectively modified on a reaction membrane substrate to form a detection line (T line) and a quality control line (C line), and the detection line (T line) and the quality control line (C line) are soaked in triethanolamine buffer saline solution containing skimmed milk, then dried at room temperature and stored in a dryer.
In some embodiments, streptavidin is applied to the reaction membrane substrate. The 5' end of the detection probe is modified with biotin. The 3' end of the quality control probe is modified with biotin. The quality control probe and the detection probe are modified on the reaction membrane through streptavidin on a matrix of the reaction membrane.
In some embodiments, the nucleic acid detection kit further includes a card housing, the nucleic acid detection strip is mounted in the card housing, and the card housing may have a housing and a push-pull structure, the push-pull structure is used to pull the nucleic acid detection strip into or out of the housing of the card housing, and the arrangement of the card housing structure is more convenient for the use of the test strip in detection.
The embodiment of the invention also provides a detection method of nucleic acid, which adopts the nucleic acid detection kit and comprises the following steps:
mixing a sample to be detected with the pretreatment solution to obtain a solution to be detected;
and applying the liquid to be detected on the sample pad for detecting the reporter probe.
The manner in which the reporter probe is detected varies depending on the label on the reporter probe.
The reporter probe detection comprises a naked eye detection part or an electronic detection part. The naked eye detection is that the test strip color development result, namely the detection result, is directly observed by the naked eye. The electronic detection is to collect bright field or fluorescence signals of a detection line and a quality control line after horseradish peroxidase or alkaline phosphatase catalyzed substrate color development or enhanced chemiluminescence is carried out for signal amplification by using an optical method, and obtain a detection result through image analysis.
In some embodiments, the label attached to the reporter probe comprises an enzyme, further comprising: and applying the liquid to be detected on the sample pad for 0.5-30min, and then applying the chromogenic reaction substrate of the enzyme on the sample pad.
Specifically, when the report probe is modified with the colloidal gold, a detection result can be obtained through naked eye detection. The detection line and the quality control line are red, so that the target nucleic acid sequence is contained in the sample to be detected; the detection line is colorless, the quality control line is red, and the sample to be detected has no target nucleic acid sequence.
Specifically, when the connection pad is a colloidal gold jointly modified by a report probe and horseradish peroxidase, the detection method may be:
dripping the solution to be detected on the sample pad, dripping 10-300 μ l buffer solution (such as sodium citrate buffer solution) on the sample pad after 0.5-30min, and waiting for 0.5-30 min;
dripping a chromogenic reaction substrate of horseradish peroxidase on the sample pad, and obtaining a detection result through naked eye detection or electronization detection after 0.5-30 min;
in the naked eye detection, the detection line and the quality control line are dark brown, and a sample to be detected contains a target nucleic acid sequence; the detection line is colorless, the quality control line is dark brown, and the sample to be detected has no target nucleic acid sequence;
the electronic detection is that after the signals at the detection line and the quality control line are subjected to image processing, the signals are obviously different from the signals at other positions of the test strip, and then the sample to be detected contains a target nucleic acid sequence; after image processing is carried out on signals at the detection line, the signals are not obviously different from signals at other positions of the test strip, and a target nucleic acid sequence does not exist in a sample to be detected;
specifically, when the connection pad is a horseradish peroxidase-modified reporter probe, the detection method may be:
dripping the solution to be detected on a sample pad, dripping 10-150 μ l of chemiluminescent reaction solution on the sample pad after 0.5-30min, and waiting for 5-30min to obtain a detection result by electronization detection;
the electronic detection is that after the signals at the detection line and the quality control line are subjected to image processing, the signals are obviously different from the signals at other positions of the test strip, and then the sample to be detected contains a target nucleic acid sequence; after the signals at the detection line are subjected to image processing, the signals are not obviously different from the signals at other positions of the test strip, and the target nucleic acid sequence is not contained in the sample to be detected.
Specifically, when the connecting pad is an alkaline phosphatase modified reporter probe, the detection method may be:
dripping the solution to be detected on a sample pad, dripping 10-150 μ l of chromogenic reaction substrate on the sample pad after 0.5-30min, and obtaining a detection result through electronization detection after waiting for 5-30 min;
the electronic detection is that after the signals at the detection line and the quality control line are subjected to image processing, the signals are obviously different from the signals at other positions of the test strip, and then the sample to be detected contains a target nucleic acid sequence; after the signals at the detection line are subjected to image processing, the signals are not obviously different from the signals at other positions of the test strip, and the target nucleic acid sequence is not contained in the sample to be detected.
Preferably, the concentration of the sodium citrate buffer solution can be 0.1 mol/L-10 mol/L.
The following are specific examples.
Example 1 (taking SARS-CoV-2 as an example)
Referring to fig. 1 and 2, a nucleic acid detecting apparatus and a detecting method thereof, the detecting method uses a nucleic acid detecting strip 1 as a reaction unit, the nucleic acid detecting strip 1 is disposed in a card housing 2, and the card housing 2 has a push-pull structure 3. The nucleic acid detection test strip 1 comprises a bottom plate 4, and a sample pad 5, a connecting pad 6, a nitrocellulose membrane and a water absorption pad 10 which are sequentially overlapped and fixed on the bottom plate 4;
a reporting group is fixed on the connecting pad 6, and the reporting group is colloidal gold marked by a probe; the reaction membrane 7 has a base membrane of nitrocellulose membrane, and is provided with a detection line 8 and a quality control line 9; a detection probe is fixed on the detection line 8; and a quality control probe is fixed on the quality control line 9.
The 5 'end of the detection probe is modified by biotin, the 3' end of the quality control probe is modified by biotin, the nitrocellulose membrane is attached with streptavidin, and the detection probe and the quality control probe are fixed on the reaction membrane 7 by the combination of biotin-streptavidin.
The sample pad 5, the connecting pad 6, the nitrocellulose membrane and the absorbent pad 10 are overlapped by 2mm, the interval between the detection line 8 and the quality control line 9 is 4mm, and the width of the bottom plate 4 is 4 mm.
Wherein,
the sequence of the detection probe is:
5’-CCCCCCCCCCCC-3’。
the sequence of the quality control probe is as follows:
5’-CGTTGTCCCTAG-3’。
the sequence of the second nucleic acid probe is:
5’-TGAGGAACGAGAAGAGGGGGGGGGGGG-3’。
the sequence of the first nucleic acid probe is:
5’-TTTGTATGCGTCAATCCTCGGAAGTGTGCT-3’。
the first-stage signal amplification probe consists of a section of amplification chain prepositive guide sequence and 5 connected repeated amplification sequence regions, wherein the amplification chain prepositive guide sequence is as follows:
5’-GCACACTTCCGAGG-3’。
the sequence of the amplification sequence region of the first-stage signal amplification probe is as follows:
5’-AAAACGACAGGACCGA-3’。
the second-stage signal amplification probe consists of a section of amplification chain prepositive guide sequence and 5 connected repeated amplification sequence regions, wherein the amplification chain prepositive guide sequence is as follows:
5’-TAATCGGTCCTGTCG-3’。
the sequence of the amplification sequence region of the second-stage signal amplification probe is as follows:
5’-AAATGCCGTTGTCCCTAG-3’。
the sequence of the reporter probe is:
5’-CTAGGGACAACGAAAGCACTTGGTACGGGCGCTGACT-3’。
the target nucleic acid sequence to be detected is as follows:
5’-TCTTCTCGTTCCTCAAAACGTGGTTGACCTACAAAAATTGACGCATACAAA-3’。
the specific detection method comprises the following steps:
dispersing 40 mu l of a sample to be detected with the concentration of 10pmol in 60 mu l of sodium citrate buffer solution containing 10nmol of a first nucleic acid probe, a second nucleic acid probe, a first-stage signal amplification chain and a second-stage signal amplification chain to obtain a solution to be detected;
dripping the solution to be detected on the sample pad 5, and obtaining a test strip color development result after 5-30 min; the detection line 8 is colorless, the quality control line 9 is red, and the sample to be detected has no target nucleic acid sequence to be detected. As shown in fig. 3.
Example 2 (taking SARS-CoV-2 as an example)
The difference between this example and the preparation method is the same as example 1, in that the reporter group fixed on the connecting pad 6 is colloidal gold labeled by a reporter probe and horseradish peroxidase.
The specific detection method comprises the following steps:
dispersing 40 mu l of a sample to be detected with the concentration of 10pmol in 60 mu l of sodium citrate buffer solution containing 10nmol of the complementary primer of the labeling auxiliary region, the primary signal amplification chain and the secondary signal amplification chain to obtain a solution to be detected;
dripping the solution to be detected on the sample pad 5, dripping 60 mul of sodium citrate buffer solution on the sample pad 5 after 5-30min, dripping 60 mul of chromogenic substrate on the sample pad 5 after 1-15min, and obtaining the test strip chromogenic result after 1-30 min;
through naked eye detection, the detection line 8 and the quality control line 9 are dark brown, and a sample to be detected contains a target nucleic acid sequence. As shown in fig. 3.
The control group was substantially the same as in example 2 except that the test sample was replaced with a sodium citrate buffer. As shown in fig. 3.
Comparative example 1
The samples of example 2 were tested directly using the commercially available colloidal gold test strips (AJP10-100) of nucleic acid product. The first nucleic acid probe, the second nucleic acid probe, and the one or more signal amplification probes of example 2 are not included in the kit. The reporter probe can be complementarily paired with the nucleic acid sequence to be detected and the detection line and the quality control line.
Using the above-described kits of example 2 and comparative example 1, tests were performed under the same conditions as in Table 1 below.
TABLE 1
By using the above-mentioned kits of example 2 and comparative example 1, 100 identical samples containing the target nucleic acid sequence to be detected were detected, and as a result, the kit of example 2 could achieve 98 positive detections, while the kit of comparative example 1 could only detect 58 positive results.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (19)
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| CN116656786A (en) * | 2023-03-14 | 2023-08-29 | 广州医科大学附属第一医院（广州呼吸中心） | CRISPR-lateral flow nucleic acid detection test paper based on strand hybridization and its detection probe |
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