CN111505275A - Cas9 nucleic acid isothermal amplification-based immunochromatography multiple gene detection method - Google Patents
Cas9 nucleic acid isothermal amplification-based immunochromatography multiple gene detection method Download PDFInfo
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Abstract
The invention relates to the technical field of biotechnology and medical inspection, in particular to an immunochromatography multiple gene detection method based on Cas9 nucleic acid isothermal amplification, which comprises the following steps: (1) extracting genomic DNA to be detected from a sample to be detected; (2) performing multiple isothermal amplification by using the genomic DNA to be detected as a template to obtain multiple amplified nucleic acid products; (3) and (3) dripping the multiple amplified nucleic acid products on immunochromatographic test paper, wherein the immunochromatographic test paper is provided with a plurality of detection lines and a quality control line, observing the colors of the detection lines and the quality control line, and determining the detection result. The detection method can simultaneously detect a plurality of nucleic acid products, has simple and convenient operation, short detection time and instant and readable result, can be widely applied to basic level detection and family detection, and is not limited by instruments.
Description
Technical Field
The invention relates to the technical field of biotechnology and medical inspection, in particular to an immunochromatography multiple gene detection method based on Cas9 nucleic acid isothermal amplification.
Background
Nucleic acid detection has found widespread use in a variety of fields, including criminal investigation, detection of pathogenic microorganisms, disease diagnosis, and the like. Generally, the content of the target nucleic acid in the environmental sample or the physiological sample is very small, and in order to make the result more accurate, the target nucleic acid fragment is often required to be amplified. Among them, Polymerase Chain Reaction (PCR) is the most widely used nucleic acid amplification technique. However, this technique relies on thermal cycling, requires expensive specialized equipment, and is not suitable for testing in ordinary homes or remote locations.
Common isothermal nucleic acid amplification technologies include nucleic acid sequence-dependent amplification (NASBA), Strand Displacement Amplification (SDA), loop-mediated isothermal amplification (L AMP), Recombinase Polymerase Amplification (RPA), and the like.
Chinese patent literature discloses a constant-temperature nucleic acid detection and analysis method and a kit based on Cas9 nickase coupled DNA polymerase, and the application publication number is CN 110607355A, which is called Cas9nAR method for short. According to the invention, by using the Cas9 nickase with DNA nicking activity, the DNA polymerase with strand displacement activity and the carefully-selected primers, the specific amplification of genomes in biological samples such as microorganisms and cells under the normal temperature condition is realized, the detection sensitivity reaches 1 copy number DNA of a 10 microliter reaction system, the specificity of single base discrimination is realized, and the method has great potential in the precise and rapid detection application of disease-related genes (single nucleotide polymorphism), pathogens in blood, bacterial viruses and the like. However, this method uses fluorescence as an output signal, requires expensive instrument assistance, has high detection cost, can amplify only one nucleic acid fragment at a time, and has limitations in the field of gene multiplex detection.
Disclosure of Invention
In order to overcome the problems of high cost, single detection object and low universality of the traditional nucleic acid detection method, the invention provides the immunochromatography multiple gene detection method based on the isothermal amplification of the nucleic acid of Cas9, which has the advantages of low cost, no special requirements on instruments, capability of simultaneously detecting a plurality of nucleic acid products, rapidness, high efficiency and visualized detection results, and can be widely applied to basic level detection and family detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
an immunochromatography multiple gene detection method based on isothermal amplification of Cas9 nucleic acid, comprising the following steps:
(1) extracting genomic DNA (target DNA) to be detected from a sample to be detected;
(2) taking the genome DNA to be detected as a template, and performing multiple isothermal amplification under the action of Cas9 nickase, sgRNA, a labeled primer and DNA polymerase to obtain multiple amplified nucleic acid products;
(3) and (3) dripping the multiple amplified nucleic acid products on immunochromatographic test paper, wherein the immunochromatographic test paper is provided with a plurality of detection lines and a quality control line, observing the colors of the detection lines and the quality control line, and determining the detection result.
The immunochromatography technology is a rapid diagnosis technology, and the technology uses color-developing particles such as colloidal gold and the like as tracers to be applied to antigen-antibody reaction, and is most commonly used as an immune colloidal gold chromatography test strip which consists of a water absorption pad, a combination pad, a nitrocellulose membrane and a sample pad. The sample containing the substance to be detected is moved up along the membrane by the capillary action of the nitrocellulose membrane, and the receptor immobilized on the membrane binds thereto to cause a specific reaction. The antigen-antibody complex is accumulated continuously, and the chromogenic particles mark the precipitate for color development, thereby achieving the purpose of detection. The amount of the marker is in direct proportion to the amount of the antigen in the sample, and the quantitative detection of the substance to be detected is realized by detecting the amount of the marker. The antigen (antibody) labeling technology is combined with the immunochromatography technology, the detection result can be obtained within 15min, and the purpose of rapid detection is realized. Nucleic acid molecules can be detected by immunochromatographic test strips by labeling small molecule antigens.
The invention provides a method for amplifying nucleic acid in multiple ways, which is suitable for immunochromatography test paper, on the basis of a Cas9nAR method. The method realizes simultaneous amplification of multiple sequences under the combined action of Cas9 nickase, specially designed sgRNA, primers and DNA polymerase, and uses an immunochromatography test strip to read detection results, thereby realizing multiple detection of genes. The method can detect various nucleic acid products simultaneously, has simple and convenient operation, short detection time (5-10 minutes) and instant and readable result, can be widely applied to basic level detection and family detection, and is not limited by instruments.
Preferably, in step (2), the multiple isothermal amplification process comprises the following steps:
(a) taking the genome DNA to be detected as a template, and respectively reacting the genomic DNA to be detected with a Cas9 nickase and sgRNA aiming at the genomic DNA to be detected in a water bath at 20-42 ℃ for 20-30 min to obtain a mixed solution; the water bath temperature is more preferably 37 ℃ and the water bath reaction time is more preferably 20 min.
(b) Adding DNA polymerase, a labeled primer, NEBuffer 2 and dNTP into the mixed solution, uniformly mixing, and reacting in a water bath at 20-42 ℃ for 1-1.5 h to obtain the multiple amplified nucleic acid product. The bath temperature is more preferably 37 ℃ and the bath reaction time is more preferably 1 h.
Preferably, in step (2), the labeled primers comprise a first primer and a second primer, and the first primer is labeled with a biotin group; the second primer is labeled with a small molecule compound with a specific antibody, such as a digoxigenin group, a FITC group, or other compound group.
Preferably, in the step (3), the detection line is coated with a specific antibody corresponding to the small molecule compound marked by the second primer; the quality control line is coated with a biotin-modified goat anti-mouse IgG antibody.
The detection principle of the invention is as follows: the invention designs a multiple immunochromatographic test strip based on the principle of antigen-antibody specific binding. The invention respectively designs a pair of specific primers aiming at target DNAs of different samples to be detected, wherein the first primer is labeled with biotin groups, and the second primer is labeled with different specific groups (such as digoxin groups, FITC groups or other compound groups) respectively for distinguishing. Specific products corresponding to the target are amplified through Cas9nAR, and different products simultaneously carry biotin groups (from the first primer) and specific groups (from the second primer). Dropwise adding the amplification product solution onto a test strip, enabling the solution of the product to be detected to migrate on the chromatographic strip through capillary action, combining biotin groups carried by the product with streptavidin-modified latex microsphere particles coated on a latex microsphere pad in the migration process to form a compound, specifically combining the compound with antibodies corresponding to specific groups coated on different detection lines when the compound migrates to a detection area again, enabling the specific product to be enriched on a chromatographic material of the detection area of the chromatographic strip, and obtaining a macroscopic reaction compound through the color of the latex microsphere particles, so that visual color development is formed. The product which is not enriched continuously flows forwards and finally migrates to the quality control area, and forms visual color development by immunological combination with the goat anti-mouse IgG antibody modified by the biotin.
Preferably, in the step (3), the immunochromatographic test strip comprises a base plate, and a sample pad, a latex microsphere pad, an NC membrane and a water absorption pad which are sequentially overlapped and bonded on the base plate, wherein adjacent parts of the sample pad, the latex microsphere pad, the NC membrane and the water absorption pad are partially overlapped, the NC membrane is coated with a plurality of parallel detection lines and a quality control line, the detection lines are close to one side of the latex microsphere pad, and the quality control line is far away from the latex microsphere pad and close to one side of the water absorption pad, during detection, a multiple amplified nucleic acid product is dripped on the sample pad, and the sample pad is partially immersed in a flowing buffer solution (4 × sodium citrate buffer solution containing 2% BSA and 0.05% Tween20 in volume concentration, and the pH is 7.0).
Specifically, the immunochromatographic test paper is a strip. The front end of the sample pad is stuck on the front end of the bottom plate, the tail end of the sample pad is lapped on the latex microsphere pad, the latex microsphere pad is positioned between the sample pad and the NC membrane, the front end of the latex microsphere pad is lapped and stuck on the bottom plate, and the rear end of the latex microsphere pad extends out of the upper part of the front end of the NC membrane by a certain length; the NC film comprises a detection area and a quality control area, is arranged in the middle of the bottom plate, and is partially overlapped with the latex microsphere pad. The front end of the NC membrane is inserted between the latex microsphere cushion and the bottom plate and is adhered to the bottom plate; the water absorption pad is lapped on the NC membrane and is adhered to the rear end of the bottom plate; the detection lines and the quality control lines are respectively coated on the NC membrane correspondingly to form parallel belts which are respectively immune coatings capable of combining with the detection object markers.
Specifically, during detection, the multiple amplified nucleic acid products are dripped on a sample pad, the immunochromatographic test paper is placed in a flowing buffer solution in a mode that the sample pad faces downwards, and the multiple amplified nucleic acid products flow through the latex microsphere pad from the sample pad, are specifically combined with the latex microsphere on the latex microsphere pad, flow through a detection line and a quality control line on an NC (numerical control) membrane and finally flow to a water absorption pad. As the complex continues to flow through the NC membrane, it will specifically bind to the corresponding antibody on it to form a sandwich complex.
Preferably, the sample pad is a glass cellulose membrane or a polyester cellulose membrane; the NC membrane is a nitrocellulose membrane.
Preferably, the latex microsphere pad comprises a membrane body and a streptavidin-labeled latex microsphere particle coating uniformly coated on the surface of the membrane body; the membrane body is a glass cellulose membrane or a polyester cellulose membrane.
Preferably, in step (3), the detection result includes:
when the quality control line and the detection line are simultaneously colored or the quality control line and any detection line are colored, judging that the sample to be detected contains a corresponding object to be detected; and when only the quality control line is developed, judging that the sample to be detected does not contain the object to be detected. If the quality control line and the detection line are not colored, the detection fails, and the test paper needs to be replaced for re-detection.
Preferably, in step (1), the sample to be detected contains at least two specific gene sequences.
Preferably, in step (1), the sample to be detected is derived from one or more of animal cells, plant cells, microorganisms and viruses.
Therefore, the invention has the following beneficial effects: the method is based on a Cas9nAR multiple isothermal amplification technology to carry out rapid and specific amplification on a sample to be detected containing multiple substances to be detected, and an immunochromatographic test paper is used for obtaining a visual detection result; the object to be detected can be semi-quantitatively detected by judging the color depth of the detection line, and the use place is not limited. The method can be used for simultaneously detecting various nucleic acid products, is simple and convenient to operate, short in detection time (5-10 min), and immediately readable in result, can be widely applied to basic level detection and family detection, and is not limited by instruments.
Drawings
FIG. 1 is a schematic diagram showing the principle of detection of Escherichia coli and Salmonella by immunochromatography in example 1.
FIG. 2 is a graph showing the feasibility results of the immunochromatographic multiplex gene detection of E.coli and Salmonella target DNA in example 1.
FIG. 3 is a graph showing the results of the specificity of the immunochromatographic multiplex gene E.coli and Salmonella target DNA detection in example 1.
FIG. 4 is a graph showing the results of sensitivity in the immunochromatographic multiplex gene detection of E.coli and Salmonella target DNAs in example 1.
FIG. 5 is a graph showing the results of specificity of the detection of target DNA of Escherichia coli and Salmonella mixed in milk in example 3.
FIG. 6 is a graph showing the sensitivity results of the detection of target DNA of Escherichia coli and Salmonella mixed in milk in example 3.
Detailed Description
The technical solution of the present invention is further specifically described below by using specific embodiments and with reference to the accompanying drawings.
In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.
Example 1 detection of Escherichia coli and Salmonella by multiplex immunochromatographic diagnostic test strip
In this example, the DNA amplification template is derived from a genome or plasmid extracted from the kit, the primers are synthesized by biotechnology companies, the sgRNA is obtained by in vitro transcription, and 20 bases at the 5' end of the sgRNA are a seed sequence complementary to the pre-spacer.
The detection is carried out according to the detection principle of the immunochromatography multiple gene escherichia coli and salmonella shown in figure 1:
(1) extracting genome DNA to be detected from a sample to be detected, namely shaking and culturing Escherichia coli ATCC35128 and salmonella AS L1174 in L B liquid medium until exponential growth phase (OD is 0.4-0.6), extracting bacterial DNA (target DNA) from 1 ml of bacterial liquid,
(2) performing multiple isothermal amplification by using a target DNA as a template:
(a) reacting Cas9 nickase and two pairs of sgRNAs aiming at escherichia coli and salmonella target genes in a water bath at 37 ℃ for 20 minutes to form a Cas9n/sgRNA complex; the amplified template sequence is a uidA target gene sequence (SEQ, ID, NO.1) in an Escherichia coli genome; the amplification template sequence is an invA target gene sequence (SEQ, ID, NO.2) in a salmonella genome;
(b) mixing the two pairs of the total four Cas9n/sgRNA complexes obtained in step (a), DNA polymerase, labeled primers, NEBuffer 2 and dNTPs, reacting for 1h in a 37 ℃ water bath to obtain a dual amplified nucleic acid product of Escherichia coli and Salmonella characteristic genes, as shown in Table 1, sgRNA for locating Escherichia coli uidA gene includes sgRNA1-1(SEQ, ID, NO.3) and sgRNA1-2(SEQ, ID, NO.4), sgRNA for locating Salmonella invA gene includes sgRNA2-1(SEQ, ID, NO.5) and sgRNA2-2(SEQ ID, NO.6), wherein underlined sequences are complementary to the sequence of the midregion of the target, as shown in Table 3, primers for amplifying Escherichia coli uidA gene include digoxin labeled primers 1-1(SEQ ID, NO.13) and biotin labeled primers 1-2(SEQ ID, NO.14) for detecting the concentration of Escherichia coli uidA gene amplified nucleic acid sample including PCR primer for detecting the PCR amplification of Escherichia coli uidA gene including PCR amplification of PCR products, PCR amplification of PCR amplification products, PCR amplification of PCR products.
The immunochromatographic test paper comprises a bottom plate, and a sample pad, a latex microsphere pad, an NC membrane and a water absorption pad which are sequentially overlapped and bonded on the bottom plate; the adjacent parts of the sample pad, the latex microsphere pad, the NC membrane and the water absorption pad are partially overlapped; the NC membrane is coated with two mutually parallel detection lines and a quality control line, the detection lines are close to one side of the latex microsphere pad, and the quality control line is far away from the latex microsphere pad and is close to one side of the water absorption pad; the front end of the sample pad is adhered to the front end of the bottom plate, the tail end of the sample pad is lapped on the latex microsphere pad, and the latex microsphere pad comprises a membrane body and a streptavidin-labeled latex microsphere particle coating which is uniformly coated on the surface of the membrane body; the latex microsphere pad is positioned between the sample pad and the NC membrane, the front end of the latex microsphere pad is lapped and stuck on the bottom plate, and the rear end of the latex microsphere pad extends out of the upper part of the front end of the NC membrane by a certain length; the front end of the NC membrane is inserted between the latex microsphere cushion and the bottom plate and is adhered to the bottom plate; the water absorption pad is lapped on the NC membrane and is adhered to the rear end of the bottom plate; the two detection lines and one quality control line are respectively coated on an NC membrane correspondingly to form parallel bands which are respectively immune coatings capable of being combined with a detection object marker: the detection line 1 is coated with digoxin antibody, the detection line 2 is coated with FITC antibody, and the quality control line is coated with biotin-modified goat anti-mouse IgG antibody. Wherein, the sample pad adopts a glass cellulose membrane, the latex microsphere pad adopts a glass cellulose membrane, and the NC membrane adopts a nitrocellulose membrane.
TABLE 1 sgRNA sequences
Wherein, the synthesis of sgRNA is carried out in three steps: as shown in table 2, in the first step, a T7 promoter sequence was inserted into a sgRNA plasmid template with a primer T7-F-sgRNA (SEQ, ID, No.7) and a primer R (SEQ, ID, No.8) to obtain a template with a promoter; secondly, synthesizing a sgRNA template by using the template, wherein a common primer R and a characteristic primer F-sgRNA1-1(SEQ, ID, NO.9), 1-2(SEQ, ID, NO.10), 2-1(SEQ, ID, NO.11) and 2-2(SEQ, ID, NO.12) are needed in the synthesis process; and thirdly, transcribing the sgRNA template in vitro by using a transcription kit.
TABLE 2 primers for sgRNA in vitro transcription
TABLE 3 primer sequences
As shown in fig. 2, when detecting target DNA of escherichia coli and salmonella, only in the presence of DNA polymerase, two pairs of Cas9n/sgRNA complexes, and two pairs of primers, both detection lines can be colored, neither primer or Cas9n/sgRNA complex can be colored, neither DNA polymerase can be used for amplification reaction, and both detection lines can be colored.
As shown in FIG. 3, if the amplified product contains DNA of Escherichia coli and Salmonella, the two detection lines and the quality control line will be colored; if only contains Escherichia coli, only the detection line 1 and the quality control line are developed; if the salmonella is contained, only the detection line 2 and the quality control line are developed; if the two bacteria are not contained, neither detection line develops color, and only the quality control line develops color; if the quality control line and the detection line are not colored, the detection fails, and the test paper needs to be replaced for re-detection.
The sensitivity of this method was evaluated as shown in FIG. 4, with a detection limit of 100 copies/microliter.
Example 2
The difference between the example 2 and the example 1 is that the immunochromatographic test paper is different, the sample pad and the latex microsphere pad adopt a polyester cellulose membrane, the temperature of a water bath in the multiple isothermal amplification process is 42 ℃, and the rest processes are completely the same. The detection result of this embodiment is equivalent to that of embodiment 1, and will not be described herein.
Example 3
Escherichia coli and Salmonella were mixed with milk and stored overnight at room temperature, and then used as samples for experimental detection in the same manner as in example 1. As shown in fig. 5, the method still has good specificity for the bacteria in the food sample. As shown in fig. 6, the limit of detection of bacteria in the food sample by this method was 100 colonies/ml.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Sequence listing
<110> Zhejiang industrial university
<120> immunochromatography multiple gene detection method based on isothermal amplification of Cas9 nucleic acid
<160>16
<170>SIPOSequenceListing 1.0
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<213> Escherichia coli (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400>1
agcatcaggg cggctataca ccgtttgaag ccgatgtcac gccgtatgtt attgccggga 60
aaagtgtgcg tatcaccgtt tgtgtgaaca acgaactgaa ctggcagact atcccgccgg 120
gaatggtgat taccgacgaa aacggcaaga aaaagcagtc ttacttccat gatttcttta 180
attatgccgg gatccatcgc agcgtaatgc tctacaccac gccgaacacc tgggtggacg 240
atatcaccgt ggtgacgcat gtcgcgcaag actgtaacca cgcgtctgtt gactggcagg 300
tggtggccaa tggtgatgtc agcgttgaac tgcgtgatgc ggatcaacag gtggttgcaa 360
ctggacaagg cactagcggg actttgcaag tggtgaatcc acacctctgg caaccgggtg 420
aaggttatct ctatgaactg tgcgtcacag ccaaaagcca gacagagtgt gatatctacc 480
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tttaatatta acaggatacc tatagtgctg ctttctctac ttaacagtgc tcgtttacga 120
cctgaattac tgattctggt actaatggtg atgatcattt ctatgttcgt cattccatta 180
cctacctatc tggttgattt cctgatcgca ctgaatatcg tactggcgat attggtgttt 240
atggggtcgt tctacattga cagaatcctc agtttttcaa cgtttcctgc ggtactgtta 300
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gccgatgccg gtgaaattat cgccacgttc gggcaattcg ttattggcga tagcctggcg 420
gtgggttttg ttgtcttctc tattgtcacc gtggtccagt ttatcgttat taccaaaggt 480
tcagaacgtg tcgcggaagt cgcggcccga ttttctctgg atggtatgcc cggtaaacag 540
atgagtattg atgccgattt gaaggccggt attattgatg cggatgccgc gcgcgaacgg 600
cgaagcgtac tggaaaggga aagccagctt tacggttcct ttgacggtgc gatgaagttt 660
atcaaaggtg acgctattgc cggcatcatt attatctttg tgaactttat tggcggtatt 720
tcggtgggga tgactcgcca tggtatggat ttgtcctccg ccctgtctac ttataccatg 780
ctgaccattg gtgatggtct tgtcgcccag atccccgcat tgttgattgc gattagtgcc 840
ggttttatcg tgacccgcgt aaatggcgat acggataata tggggcggaa tatcatgacg 900
cagctgttga acaacccatt tgtattggtt gttacggcta ttttgaccat ttcaatggga 960
actctgccgg gattcccact gccggttttt gttattttat cggtggtttt aagcgtactc 1020
ttctatttta aattccgtga agcaaaacgt agcgccgcca aacctaaaac cagcaaaggc 1080
gagcagccgc tcagtattga ggaaaaagaa gggtcgtcgt taggactgat tggcgatctc 1140
gataaagtct ctacagagac cgtaccgttg atattacttg tgccgaagag ccggcgtgaa 1200
gatctggaaa aagctcaact tgcggagcgt ctacgtagtc agttctttat tgattatggc 1260
gtgcgcctgc cggaagtatt gttacgagat ggcgagggcc tggacgataa cagcatcgta 1320
ttgttgatta atgagatccg tgttgaacaa tttacggtct attttgattt gatgcgagtg 1380
gtaaattatt ccgatgaagt cgtgtccttt ggtattaatc caacaatcca tcagcaaggt 1440
agcagtcagt atttctgggt aacgcatgaa gagggggaga aactccggga gcttggctat 1500
gtgttgcgga acgcgcttga tgagctttac cactgtctgg cggtgaccgt ggcgcgcaac 1560
gtcaatgaat atttcggtat tcaggaaaca aaacatatgc tggaccaact ggaagcgaaa 1620
tttcctgatt tacttaaaga agtgctcaga catgccacgg tacaacgtat atctgaagtt 1680
ttgcagcgtt tgttaagcga acgtgtttcc gtgcgtaata tgaagttaat tatggaagcg 1740
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gtatctgctg aagttgagga tgttattcgc aaagggatcc gtcagacctc tggcagtacc 1920
ttcctcagcc ttgacccgga agcctccgct aatttgatgg atctcattac acttaagttg 1980
gatgatttat tgattgcaca taaagatctt gtcctcctta cgtctgtcga tgtccgtcga 2040
tttattaaga aaatgattga aggtcgtttt ccggatctgg aggttttatc tttcggtgag 2100
atagcagata gcaagtcagt gaatgttata aaaacaatat aagggcttaa ttaaggaaaa 2160
gatctatgca acattt 2176
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<211>80
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
aauaacauac ggcgugacau uuuuagagcu agaaauagca aguuaaaaua aggcuagucc 60
guuaucaacu ugaaaaagug 80
<210>4
<211>80
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ucauagagau aaccuucacc uuuuagagcu agaaauagca aguuaaaaua aggcuagucc 60
guuaucaacu ugaaaaagug 80
<210>5
<211>80
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
aucugguuga uuuccugauc uuuuagagcu agaaauagca aguuaaaaua aggcuagucc 60
guuaucaacu ugaaaaagug 80
<210>6
<211>80
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
ucgauuuauu aagaaaauga uuuuagagcu agaaauagca aguuaaaaua aggcuagucc 60
guuaucaacu ugaaaaagug 80
<210>8
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
gatcccgcga aattaatacg actcactata 30
<210>8
<211>35
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
ataaaaaacg cccggcggca accgagcgtt ctgaa 35
<210>9
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
taatacgact cactatagaa taacatacgg cgtgacatgt tttagagcta gaaatagc 58
<210>10
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
taatacgact cactatagtc atagagataa ccttcaccgt tttagagcta gaaatagc 58
<210>11
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
taatacgact cactatagat ctggttgatt tcctgatcgt tttagagcta gaaatagc 58
<210>12
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
taatacgact cactatagtc gatttattaa gaaaatgagt tttagagcta gaaatagc 58
<210>13
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
gactagttat cccatgtcac gccgtatgtt attgccggga aaagdgdtgt g 51
<210>14
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gactagttat cccggtgaag gttatctcta tgaacccggt bdttgccaga ggt 53
<210>15
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
gactagttat cccatctggt tgatttcctg atcgcactcd ttgaatatcg 50
<210>16
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
gactagttat ccctcgattt attaagaaaa tgacgacgga cabdttcgac aga 53
Claims (10)
1. An immunochromatography multiple gene detection method based on isothermal amplification of Cas9 nucleic acid, which is characterized by comprising the following steps:
(1) extracting genomic DNA to be detected from a sample to be detected;
(2) taking the genome DNA to be detected as a template, and performing multiple isothermal amplification under the action of Cas9 nickase, sgRNA, a labeled primer and DNA polymerase to obtain multiple amplified nucleic acid products;
(3) and (3) dripping the multiple amplified nucleic acid products on immunochromatographic test paper, wherein the immunochromatographic test paper is provided with a plurality of detection lines and a quality control line, observing the colors of the detection lines and the quality control line, and determining the detection result.
2. The immunochromatography multiplex gene detection method based on isothermal amplification of Cas9 nucleic acids according to claim 1, wherein in step (2), the multiplex isothermal amplification process comprises the following steps:
(a) taking the genome DNA to be detected as a template, and respectively reacting the genomic DNA to be detected with a Cas9 nickase and sgRNA aiming at the genomic DNA to be detected in a water bath at 20-42 ℃ for 20-30 min to obtain a mixed solution;
(b) adding DNA polymerase, a labeled primer, NEBuffer 2 and dNTP into the mixed solution, uniformly mixing, and reacting in a water bath at 20-42 ℃ for 1-1.5 h to obtain the multiple amplified nucleic acid product.
3. The method for immunochromatographic multiplex gene detection based on isothermal amplification of Cas9 nucleic acids according to claim 1, wherein in step (2), the labeled primers comprise a first primer and a second primer, and the first primer is labeled with a biotin group; the second primer is labeled with a small molecule compound having a specific antibody.
4. The immunochromatographic multiplex gene detection method based on isothermal amplification of Cas9 nucleic acids according to claim 3, wherein in step (3), the detection line is coated with a specific antibody corresponding to the small molecule compound labeled by the second primer; the quality control line is coated with a biotin-modified goat anti-mouse IgG antibody.
5. The immunochromatographic multiplex gene detection method based on isothermal amplification of Cas9 nucleic acid according to claim 1, wherein in step (3), the immunochromatographic test strip comprises a base plate, and a sample pad, a latex microsphere pad, an NC membrane and a water absorption pad which are sequentially bonded on the base plate in an overlapping manner; the adjacent parts of the sample pad, the latex microsphere pad, the NC membrane and the water absorption pad are partially overlapped; the NC membrane is coated with a plurality of mutually parallel detection lines and a quality control line, the detection lines are close to one side of the latex microsphere pad, and the quality control line is far away from the latex microsphere pad and is close to one side of the water absorption pad; for detection, the multiple amplified nucleic acid products are dropped onto the sample pad, and the sample pad is partially immersed in the running buffer.
6. The method for immunochromatography multiplex gene detection based on isothermal amplification of Cas9 nucleic acids according to claim 5, wherein the sample pad is a glass cellulose membrane or a polyester cellulose membrane; the NC membrane is a nitrocellulose membrane.
7. The immunochromatographic multiplex gene detection method based on isothermal amplification of Cas9 nucleic acids according to claim 5, wherein the latex microsphere pad comprises a membrane body and a streptavidin-labeled latex microsphere particle coating uniformly coated on the surface of the membrane body; the membrane body is a glass cellulose membrane or a polyester cellulose membrane.
8. The immunochromatographic multiplex gene detection method based on isothermal amplification of Cas9 nucleic acids according to claim 1, wherein in step (3), the detection result comprises:
when the quality control line and the detection line are simultaneously colored or the quality control line and any detection line are colored, judging that the sample to be detected contains a corresponding object to be detected; and when only the quality control line is developed, judging that the sample to be detected does not contain the object to be detected.
9. The method for immunochromatography multiplex gene detection based on isothermal amplification of Cas9 nucleic acids according to claim 1, wherein in step (1), the sample to be detected contains at least two specific gene sequences.
10. The method for immunochromatography multiplex gene detection based on isothermal amplification of Cas9 nucleic acids according to any one of claims 1-9, wherein in step (1), the sample to be detected is derived from one or more of animal cells, plant cells, microorganisms and viruses.
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