CN115109859A - Multi-mode composite functional helicobacter pylori nucleic acid detection test paper - Google Patents
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Abstract
The invention belongs to the technical field of biological detection, and relates to a multi-mode composite functional test paper for detecting helicobacter pylori nucleic acid. The detection test paper, a DNA lysate, an initial detection reagent and a reinspection reagent form a detection kit, and the DNA lysate is used for cracking a sample in a nucleic acid extraction area to release helicobacter pylori nucleic acid; the primary detection reagent is used for reacting helicobacter pylori nucleic acid in the primary detection area of the detection test paper to release a fluorophore; and the rechecking reagent is used for carrying out enzyme digestion circulating amplification on the helicobacter pylori nucleic acid captured by the capture probe in the rechecking area of the detection test paper and simultaneously carrying out reaction to release a fluorophore. The invention integrates nucleic acid extraction and detection, sensitively detects helicobacter pylori nucleic acid in saliva samples, and semi-quantitatively screens according to results of different partitions.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to a multi-mode composite functional test paper for helicobacter pylori nucleic acid.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Because the infection of Helicobacter pylori (h. pylori) is universal and long-lasting, whether the infection is controlled, treated or prognosis-tracked, the infection needs to be detected frequently and in real time for a long time, which brings great burden to patients, and the convenient, household and cheap detection method has important significance for the prevention and treatment work. According to the research of the inventor, no household product capable of quickly, sensitively and accurately detecting H.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a multi-modal helicobacter pylori nucleic acid detection kit with complex functions, which integrates nucleic acid extraction and detection, sensitively detects helicobacter pylori nucleic acid in a saliva sample, and semi-quantitatively screens according to results of different partitions.
In order to achieve the purpose, the technical scheme of the invention is as follows:
on one hand, the multi-mode composite functional test paper for detecting the helicobacter pylori nucleic acid is sequentially provided with a sample area, a nucleic acid extraction area, a primary detection area and a secondary detection area according to the liquid flowing direction, wherein the sample area is used for dropwise adding a sample, and the secondary detection area is provided with a capture probe which can be hybridized with the helicobacter pylori nucleic acid.
In another aspect, the kit for detecting the multi-modal complex functional helicobacter pylori nucleic acid comprises:
the detection test paper is sequentially provided with a sample area, a nucleic acid extraction area, an initial detection area and a rechecking area according to the flowing direction of liquid, wherein the sample area is used for dripping a sample, and the rechecking area is provided with a capture probe which can be hybridized with helicobacter pylori nucleic acid;
DNA lysate for lysing the sample in the nucleic acid extraction region to release helicobacter pylori nucleic acid;
the primary detection reagent is used for reacting helicobacter pylori nucleic acid in the primary detection area of the detection test paper to release a fluorophore;
and the rechecking reagent is used for carrying out enzyme digestion circulating amplification on the helicobacter pylori nucleic acid captured by the capture probe in the rechecking area of the detection test paper and simultaneously carrying out reaction to release a fluorophore.
The invention simultaneously arranges a primary detection area and a secondary detection area in one detection test paper, when the nucleic acid quantity of the helicobacter pylori is more, the initial detection reagent reacts in the primary detection area, the helicobacter pylori can be visually detected through the released fluorophore, when the nucleic acid quantity of the helicobacter pylori is less, the fluorophore released only by the reaction in the primary detection area is less, the fluorescence signal can not be distinguished by naked eyes, at the moment, the secondary detection reagent circularly amplifies in the secondary detection area and simultaneously reacts to release the fluorophore, the release quantity of the fluorophore is increased after amplification, and the fluorescence signal which can be distinguished by naked eyes is generated, thereby realizing the screening of the helicobacter pylori with high concentration and the helicobacter pylori with low concentration.
Because the helicobacter pylori bacteria concentration in the samples obtained at different time is different, when the primary detection concentration is lower, the samples are sampled again for rechecking, and errors caused by different samples exist, so that the invention also arranges the sample area and the nucleic acid extraction area when arranging the primary detection area and the rechecking area at the same time, realizes one-time sampling, and can simultaneously carry out the primary detection and the rechecking. The process is as follows: when a sample is placed in the sample area, the sample flows into the nucleic acid extraction area, helicobacter pylori nucleic acid in the sample is extracted through DNA lysate, and the extracted helicobacter pylori nucleic acid enters the primary detection area and the secondary detection area. Because the initial detection area carries out instant detection, therefore can be through carrying out the incubation after adding the initial detection reagent and can realize first detection. The re-detection area is used for re-detection after initial detection, so that a capture probe is arranged, the helicobacter pylori nucleic acid extracted from the nucleic acid extraction area sequentially enters the initial detection area and the re-detection area and is captured by the capture probe in the re-detection area, the helicobacter pylori nucleic acid extracted from the same sample is fixed in the re-detection area, and when re-detection is carried out, the re-detection reagent is added to realize re-detection of the helicobacter pylori nucleic acid in the same sample after initial detection, so that errors caused by re-sampling are avoided, and the detection is more accurate.
The invention has the beneficial effects that:
1. the invention arranges the sample area, the nucleic acid extraction area, the initial detection area and the reinspection area in the same test paper, can realize the completion of nucleic acid extraction and detection by one-time sample introduction, greatly shortens the time of sample pretreatment, is suitable for on-site instant detection, is convenient for household use, and can realize the detection of helicobacter pylori nucleic acid by utilizing the release of fluorophores in the initial detection area and the reinspection area.
2. The primary detection area and the secondary detection area of the invention are respectively matched with the primary detection reagent and the secondary detection reagent, detection of helicobacter pylori nucleic acid with different concentrations is completed through one-time signal conversion and circulating signal amplification, fluorescent signals in different areas can be used for preliminarily judging different stages of helicobacter pylori infection, the concentration of the helicobacter pylori nucleic acid is indicated by the strength of the fluorescent signals under the irradiation of exciting light, and helicobacter pylori in different concentration ranges can be sensitively detected.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of the detection principle of the embodiment of the present invention;
FIG. 2 is a diagram showing the result of feasibility verification of the embodiment of the present invention, wherein a is a fluorescence spectrum and b is a gel electrophoresis chart;
FIG. 3 is a graph showing the results of condition optimization of CRISPR/Cas12a detection in the primary detection region according to the embodiment of the present invention, where a is a histogram of the relationship between fluorescence signal intensity and temperature change, b is a curve of the effect of Cas12a enzyme concentration on fluorescence signal intensity, c is a curve of the change of fluorescence signal with time after the target strand (helicobacter pylori nucleic acid) is added, and d is a histogram of the relationship between fluorescence signal intensity and the concentration of reporter DNA (reporter DNA);
FIG. 4 is a graph showing the results of detection of target chains (helicobacter pylori nucleic acid) at different concentrations in the primary detection region and the secondary detection region in the example of the present invention; a is a fluorescence spectrum corresponding to a high-concentration target chain, b is high-concentration target chain detection standard curve fitting, c is a fluorescence spectrum corresponding to a low-concentration target chain, and d is low-concentration target chain detection standard curve fitting;
FIG. 5 shows the results of detection of nucleic acids of helicobacter pylori at different concentrations according to the present invention, wherein a is a high concentration (0.1nM) sample band and b is a low concentration (0.01nM) sample band.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The invention provides a multi-modal complex function helicobacter pylori nucleic acid detection kit, which is provided by the invention, in view of the fact that no household product capable of quickly, sensitively and accurately detecting H.pyrori is available at present.
According to a typical embodiment of the invention, a multi-mode composite functional test paper for detecting helicobacter pylori nucleic acid is provided, wherein a sample area, a nucleic acid extraction area, an initial detection area and a retest area are sequentially arranged in the liquid flowing direction, the sample area is used for dripping a sample, and the retest area is provided with a capture probe capable of hybridizing with the helicobacter pylori nucleic acid.
In some embodiments of this embodiment, the capture probe is a hairpin probe that is linked to the nanogold. The capture probe is fixed in the reinspection area through the nanogold, and the helicobacter pylori nucleic acid can be fixed in the reinspection area, so that the enzyme digestion circulation reaction is triggered, and the low-sensitivity detection is realized.
In some embodiments of this embodiment, the nucleic acid extraction zone, the primary detection zone, and the retest zone are located on a nitrocellulose membrane.
In one or more embodiments, the sample region overlaps the nucleic acid extraction region.
In another embodiment of the present invention, there is provided a kit for detecting a multi-modal complex function helicobacter pylori nucleic acid, comprising:
the detection test paper is sequentially provided with a sample area, a nucleic acid extraction area, an initial detection area and a re-detection area according to the flowing direction of liquid, wherein the sample area is used for dripping a sample, and the re-detection area is provided with a capture probe which can be hybridized with helicobacter pylori nucleic acid;
DNA lysate for lysing the sample in the nucleic acid extraction region to release helicobacter pylori nucleic acid;
the primary detection reagent is used for reacting helicobacter pylori nucleic acid in the primary detection area of the detection test paper to release a fluorophore;
and the rechecking reagent is used for carrying out enzyme digestion circulating amplification on the helicobacter pylori nucleic acid captured by the capture probe in the rechecking area of the detection test paper and simultaneously carrying out reaction to release a fluorophore.
The sample zone is configured for dropwise addition of a sample.
The nucleic acid extraction zone is configured to extract helicobacter pylori nucleic acid in the instilled sample.
The primary detection zone is configured for primary nucleic acid detection of helicobacter pylori nucleic acid of the sample.
The retest region is configured for a second nucleic acid detection of helicobacter pylori nucleic acid of the sample.
According to the invention, the sample area, the nucleic acid extraction area, the initial detection area and the rechecking area are simultaneously arranged on one detection test paper, so that one-time sampling is realized, the initial detection and the rechecking can be simultaneously carried out, the error caused by the re-sampling is avoided, and the detection is more accurate. The primary detection area and the secondary detection area are respectively matched with the primary detection reagent and the secondary detection reagent, so that the detection of the helicobacter pylori with different concentrations can be realized through the strength of a fluorescence signal at different stages for preliminarily judging the helicobacter pylori infection.
In some embodiments of this embodiment, the capture probe is a hairpin probe that is linked to the nanogold. The capture probe is fixed in the reinspection area through the nanogold, and the helicobacter pylori nucleic acid can be fixed in the reinspection area, so that the enzyme digestion circulation reaction is triggered, and the low-sensitivity detection is realized.
In some examples of this embodiment, the primary detection reagent comprises a reporter DNA linked to both a fluorophore and a quencher, a cas12a ribozyme, and a crRNA, and the reporter DNA is cleaved into a sequence containing the fluorophore and a sequence containing the quencher by the cas12a ribozyme, the crRNA, and the helicobacter pylori nucleic acid.
In one or more embodiments, the retest reagent comprises a retest additive, the retest additive is added to the primary detection reagent to form the retest reagent, the retest additive comprises a primer, a polymerase and a shear enzyme, and the capture probe, the polymerase and the shear enzyme are capable of performing an enzyme digestion-strand displacement cycle reaction on the helicobacter pylori nucleic acid to amplify the helicobacter pylori nucleic acid.
In some embodiments of this embodiment, the retesting reagents comprise primers, polymerase, cleaving enzyme, reporter DNA, cas12a ribozyme, crRNA; the primer, the capture probe, the polymerase and the cutting enzyme can perform enzyme cutting-chain substitution circulation reaction on the helicobacter pylori nucleic acid to amplify the helicobacter pylori nucleic acid, and the report DNA is cut into a sequence containing a fluorophore and a sequence containing a quenching group under the action of cas12a ribozyme, crRNA and the helicobacter pylori nucleic acid.
In one or more embodiments, the reinspection reagent further comprises deoxyribonucleoside triphosphates (dntps).
In some embodiments of this embodiment, the nucleic acid extraction zone, the primary detection zone, and the retest zone are located on a nitrocellulose membrane.
In one or more embodiments, the sample region overlaps the nucleic acid extraction region.
In some examples of this embodiment, the kit further comprises a buffer solution. Used for preparing an initial detection reagent and/or a retest reagent.
In some examples of this embodiment, the kit further comprises purified water and absorbent paper. Pure water was used to soak the sample and absorbent paper was used for drainage.
The method for detecting by using the kit for detecting the multi-modal helicobacter pylori nucleic acid with compound functions comprises the following steps:
dropwise adding a sample to be detected to a sample area, infiltrating with pure water, and then conducting drainage with absorbent paper to enter a nucleic acid extraction area;
adding a DNA lysate to the nucleic acid extraction zone to free helicobacter pylori nucleic acid in the sample in the nucleic acid extraction zone;
dissociating helicobacter pylori nucleic acid in the nucleic acid extraction area into an initial detection area and a re-detection area;
adding the prepared primary detection reagent solution into the primary detection area, incubating, performing fluorescence detection, and judging whether to perform reinspection according to a fluorescence signal of the primary detection area;
when the fluorescence signal exists in the initial detection area, the retest is not needed; and when the fluorescence signal does not exist in the primary detection area, adding the prepared reinspection reagent solution into the reinspection area, incubating, and performing fluorescence detection.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Examples
A kit for detecting multi-modal helicobacter pylori nucleic acid with complex functions comprises detection test paper, DNA lysate, a primary detection reagent and a secondary detection reagent.
The detection test paper is sequentially provided with a sample area, a nucleic acid extraction area, a primary detection area and a secondary detection area, and the secondary detection area contains a capture probe connected with nanogold.
The primary detection reagent mainly comprises reporter DNA, cas12a ribozyme and crRNA.
The reinspection reagent mainly comprises a primer, a polymerase, a shear enzyme, a report DNA, a cas12a ribozyme and crRNA.
The detection principle of the detection kit is shown in figure 1: collecting saliva sample, dripping into the sample region, diffusing into the nucleic acid extraction region, and completing the extraction of nucleic acid from helicobacter pylori after DNA lysate. The extracted helicobacter pylori nucleic acid flows through the absorbent paper along with the solution and enters the initial detection area and the re-detection area. In the initial detection region, the CRISPR/Cas12a detection is carried out on helicobacter pylori nucleic acid, report DNA, Cas12a ribozyme and crRNA, so that the detection on the high-concentration helicobacter pylori nucleic acid is realized. In the retest area, the capture probe is hybridized with the helicobacter pylori nucleic acid, target circulation amplification reaction is carried out under the action of a primer, polymerase and a cutting enzyme to realize the amplification of the helicobacter pylori nucleic acid, and the amplified helicobacter pylori nucleic acid, the report DNA, the Cas12a ribozyme and the crRNA are subjected to CRISPR/Cas12a detection, so that the detection of the low-concentration helicobacter pylori nucleic acid is realized. The extraction and detection of the helicobacter pylori nucleic acid are finished by one-time sample injection, and the time for sample pretreatment is greatly shortened.
Sample treatment: and pre-dripping the DNA lysate in the area, dripping 10 mu L of the collected saliva sample in the area, and incubating at room temperature for 3min to finish the nucleic acid extraction.
CRISPR/Cas12a detection: after mixing and incubating cas12a ribozyme and crRNA with certain concentration for 1h, adding prepared reporter DNA (final concentration is 500nM) and target with different concentrations, fixing the volume to 100 mu L with DEPC water, incubating for 90min at 37 ℃, terminating the reaction at 65 ℃ for 10min, and taking the solution to scan a standard curve by using a fluorescence spectrophotometer.
Target cycle amplification reaction: first, after mixing a certain concentration of hairpin, primer and target strands, annealing was carried out at 95 ℃ for 10min, and then 1. mu.L of the product, 1.8. mu.L of dNTP (10mM), 1. mu.L of ATP (10mM), 1. mu.L of Klenow (500U/mL), 1. mu.L of Nb.BbvCI (1000U/mL), 10 XNEBuffer 2, 1. mu.L of DTT (10mM) were added to a volume of 10. mu.L with pure water. The mixed solution was incubated at 37 ℃ for 1 hour in a constant temperature shaker, and then at 95 ℃ for 10min, the enzymatic reaction was terminated. The reaction product after termination is subjected to signal readout according to the procedure of CRISPR/Cas12a detection described above.
Visual detection of test paper: after a saliva sample is directly dripped on paper, the paper is soaked by pure water, CRISPR/Cas12a detection reaction liquid is dripped in an initial detection area, and nanogold fixed hairprobe nucleic acid chains are dripped in a rechecking area and used for capturing target chains. And (3) dropwise adding the target molecule solution into the nucleic acid extraction area, draining the sample from the other end by using absorbent paper, allowing the sample to flow through the detection area, and photographing under the irradiation of an excitation lamp to test the fluorescence intensity. When the concentration of the target molecules is higher, green strips appear in the primary detection area and the secondary detection area, and the brightness of the primary detection area is obviously higher than that of the secondary detection area; when the concentration of the target molecule is low, the initial detection area is not bright, enzyme digestion-strand replacement cycle and CRISPR/Cas12a detection reaction liquid are added into the re-detection area to carry out target cycle amplification reaction, and obvious green bands can appear in the re-detection area through signal amplification.
FIG. 2 shows that only when crRNA, cas12a and the target strand are present at the same time in the primary detection region, target will activate cas12a enzyme to transect the reporter DNA sequence by recognizing crRNA, so that the fluorescent group is far away from the quencher group, and the fluorescent signal is recovered. The gel electrophoresis of the rechecking area can see that a band with the molecular weight consistent with that of the target strand nucleic acid is generated through enzyme digestion circulation, which shows that the experiments of the rechecking area and the primary detection area have feasibility
FIG. 3 shows that the optimized cas12a enzyme concentration is 80nM, the reporter concentration is 500nM, the temperature is 37 deg.C, and the reaction time is 90min, the experimental conditions are optimized.
FIG. 4 shows that the response was found by fluorescence signal measurement for various concentrations of H.pylori nucleic acid.
FIG. 5 shows that two different concentrations of H.pylori nucleic acid can appear as a green band visible to the naked eye after detection by the test strip of the present invention.
The nucleic acid sequence used in this example was (5 'to 3'):
UAAUU UCUAC UAAGU GUAGA U GCAAG ACGGA AAGAC CCCGU-3 in SEQ ID NO. 1.
hairprobe (hairpin probe):
TCTGCAAGACGGAAAGACCCCGTCCTCAGCTCTTGCAGA, see SEQ ID NO. 2.
target: ACGGGGTCTTTCCGTCTTGC, see SEQ ID NO. 3.
primer (primer): TCTGCAAG, see SEQ ID NO. 4.
reporter DNA (reporter DNA): BHQ1-TTATT-FAM, see SEQ ID NO. 5.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
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Claims (10)
1. A multi-mode composite functional test paper for detecting helicobacter pylori nucleic acid is characterized in that a sample area, a nucleic acid extraction area, an initial detection area and a re-detection area are sequentially arranged according to a liquid flowing direction, the sample area is used for dropwise adding a sample, the re-detection area is provided with a capture probe, and the capture probe can be hybridized with the helicobacter pylori nucleic acid.
2. The test paper for detecting helicobacter pylori nucleic acid as claimed in claim 1, wherein the capture probe is a hairpin probe, and the hairpin probe is connected with the nano-gold.
3. The multi-modal complex function test paper for detecting helicobacter pylori nucleic acid as claimed in claim 1, wherein the nucleic acid extraction area, the initial detection area and the re-detection area are positioned on a nitrocellulose membrane;
preferably, the sample region overlaps the nucleic acid extraction region.
4. A kit for detecting multi-modal helicobacter pylori nucleic acid with compound functions is characterized by comprising:
the detection test paper is sequentially provided with a sample area, a nucleic acid extraction area, an initial detection area and a rechecking area according to the flowing direction of liquid, wherein the sample area is used for dripping a sample, and the rechecking area is provided with a capture probe which can be hybridized with helicobacter pylori nucleic acid;
DNA lysate for lysing the sample in the nucleic acid extraction region to release helicobacter pylori nucleic acid;
the primary detection reagent is used for reacting helicobacter pylori nucleic acid in the primary detection area of the detection test paper to release a fluorophore;
and the rechecking reagent is used for carrying out enzyme digestion circulating amplification on the helicobacter pylori nucleic acid captured by the capture probe in the rechecking area of the detection test paper and simultaneously carrying out reaction to release a fluorophore.
5. The kit for detecting the nucleic acid of helicobacter pylori with multi-modal complex functions as claimed in claim 4, wherein the capture probe is a hairpin probe, and the hairpin probe is connected with the nanogold;
or the nucleic acid extraction area, the primary detection area and the secondary detection area are positioned on the nitrocellulose membrane; preferably, the sample region overlaps the nucleic acid extraction region.
6. The multi-modal complex function helicobacter pylori nucleic acid detection kit according to claim 4, wherein the primary detection reagent comprises reporter DNA, cas12a ribozyme and crRNA, the reporter DNA is linked with a fluorophore and a quencher, and the reporter DNA is cleaved into a sequence containing the fluorophore and a sequence containing the quencher by the cas12a ribozyme, the crRNA and the helicobacter pylori nucleic acid.
7. The kit of claim 6, wherein the reinspection reagent comprises an reinspection additive, the reinspection additive is added to the primary detection reagent to form an reinspection reagent, the reinspection additive comprises a primer, a polymerase and a cleavage enzyme, and the primer, the capture probe, the polymerase and the cleavage enzyme can perform an enzyme digestion-strand displacement cycle reaction on the helicobacter pylori nucleic acid to amplify the helicobacter pylori nucleic acid.
8. The kit for detecting the multi-modal complex function helicobacter pylori nucleic acid as claimed in claim 4, wherein the retest reagent comprises primers, polymerase, a shear enzyme, a reporter DNA, cas12a ribozyme, crRNA; the primer, the capture probe, the polymerase and the cutting enzyme can perform enzyme digestion-chain substitution circulation reaction on the helicobacter pylori nucleic acid to amplify the helicobacter pylori nucleic acid, and the report DNA is cut into a sequence containing a fluorophore and a sequence containing a quenching group under the action of cas12a ribozyme, crRNA and the helicobacter pylori nucleic acid;
preferably, the retest reagent further comprises deoxyribonucleoside triphosphates.
9. The kit for detecting a multimodal complex function helicobacter pylori nucleic acid according to claim 4, wherein the kit further comprises a buffer solution.
10. The kit for detecting the nucleic acid of helicobacter pylori with multi-modal complex functions as claimed in claim 4, wherein the kit further comprises pure water and absorbent paper.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115820889A (en) * | 2022-12-27 | 2023-03-21 | 广州启源生物医药有限公司 | CrRNA and CRISPR Cas12a system for detecting helicobacter pylori and application thereof |
| 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 chain hybridization and detection probe thereof |
-
2022
- 2022-06-16 CN CN202210680140.2A patent/CN115109859A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115820889A (en) * | 2022-12-27 | 2023-03-21 | 广州启源生物医药有限公司 | CrRNA and CRISPR Cas12a system for detecting helicobacter pylori and application thereof |
| 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 chain hybridization and detection probe thereof |
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