CN219608936U - Magnaporthe grisea nucleic acid colloidal gold test strip based on RAA isothermal amplification reagent - Google Patents
Magnaporthe grisea nucleic acid colloidal gold test strip based on RAA isothermal amplification reagent Download PDFInfo
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- CN219608936U CN219608936U CN202320102736.4U CN202320102736U CN219608936U CN 219608936 U CN219608936 U CN 219608936U CN 202320102736 U CN202320102736 U CN 202320102736U CN 219608936 U CN219608936 U CN 219608936U
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- nucleic acid
- isothermal amplification
- test strip
- colloidal gold
- amplification reagent
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 24
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 19
- 238000011901 isothermal amplification Methods 0.000 title claims abstract description 18
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 18
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 18
- 241001344131 Magnaporthe grisea Species 0.000 title claims description 11
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 241000710778 Pestivirus Species 0.000 claims abstract description 11
- 239000010931 gold Substances 0.000 claims description 13
- 229910052737 gold Inorganic materials 0.000 claims description 13
- 238000010521 absorption reaction Methods 0.000 claims description 11
- 238000003908 quality control method Methods 0.000 claims description 10
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 8
- 241000282326 Felis catus Species 0.000 claims description 7
- 229960002685 biotin Drugs 0.000 claims description 5
- 239000011616 biotin Substances 0.000 claims description 5
- 235000020958 biotin Nutrition 0.000 claims description 4
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 claims description 4
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 claims description 3
- 229960005156 digoxin Drugs 0.000 claims description 3
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 claims description 3
- 239000002250 absorbent Substances 0.000 claims description 2
- 230000002745 absorbent Effects 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 241000282324 Felis Species 0.000 claims 1
- 239000002245 particle Substances 0.000 abstract description 4
- 239000003086 colorant Substances 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 108091008324 binding proteins Proteins 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000011897 real-time detection Methods 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- -1 PEG-biotin Chemical compound 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The utility model relates to the field of biological kits, and discloses a Mao pestivirus nucleic acid colloidal gold test strip based on an RAA isothermal amplification reagent, which is used for designing different primer sequences with different labels, specifically identifying Mao pestivirus nucleic acid sequences, amplifying nucleic acid sequences containing different labels, and respectively combining the different labels with colloidal gold particles and detection lines specifically to develop colors so as to achieve the aim of rapid detection. The device is simple to operate and high in detection efficiency, and can meet the timeliness requirements of standing and the like.
Description
Technical Field
The utility model relates to the field of biological kits, in particular to a Mao pestivirus nucleic acid colloidal gold test strip based on an RAA isothermal amplification reagent.
Background
The current detection method of the cat pestivirus mainly depends on molecular biological methods, such as PCR/qPCR/ELISA and the like, which are widely applied, but have the defects: 1. complicated operation and dependence on large-scale equipment; 2. the requirement on operators is high; 3. the detection efficiency is low, the time consumption is long, and the immediately-available timeliness requirement cannot be met. These deficiencies result in an inability to detect in real time in the field.
Disclosure of Invention
The utility model aims to provide a Mao pestivirus nucleic acid colloidal gold test strip based on an RAA isothermal amplification reagent, and aims to solve the problems that in the prior art, the operation is complicated, the requirements on instruments and operators are high, the detection efficiency is low, and the on-site real-time detection cannot be realized.
The utility model discloses a cat pestivirus nucleic acid colloidal gold test strip based on an RAA isothermal amplification reagent, which comprises a sample pad, a gold label release pad, an absorption pad, an NC film and a back lining, wherein the back lining is a strip-shaped flat plate and is positioned at the lowest layer, the sample pad, the gold label release pad and the NC film are sequentially stuck on the back lining from one end, the absorption pad is stuck at the other end, and the NC film is positioned between the gold label release pad and the absorption pad.
Preferably, the NC film is provided with a detection line and a quality control line.
Preferably, the detection line is close to the gold mark release pad, and the quality control line is close to the absorption pad.
Preferably, the RAA isothermal amplification reagent further comprises a first primer sequence and a second primer sequence, wherein the first primer sequence modifies the first tag, and the second primer sequence modifies the second tag.
Preferably, the primer sequence one is:
5'-TGGAGGTAAAACAGGAATTAACTATACTAA-3',5' -end modification tag one
Preferably, the primer sequence II is:
5'-CTTGGTTTTAAGTCAGTATCAAATTCTTTATCC-3',5' end modification tag two.
Preferably, tag one is a biotin tag and tag two is a digoxin tag.
Compared with the prior art, the utility model can specifically identify the nucleic acid sequence of the Magnaporthe grisea by designing different primer sequences and carrying different labels on the different primer sequences, amplify the nucleic acid sequence containing the different labels, and the different labels are respectively combined with the colloidal gold particles and the detection line specifically to develop color, thereby achieving the purpose of rapid detection. The device is simple to operate and high in detection efficiency, can meet the immediately-available timeliness requirement, and can realize on-site real-time detection.
Drawings
FIG. 1 is a schematic diagram of a Magnaporthe grisea nucleic acid colloidal gold test strip based on an RAA isothermal amplification reagent according to an embodiment of the present utility model.
Reference numerals:
1-sample pad; 2-gold mark release pad; 3-detecting lines; 4-a quality inspection line; 5-an absorbent pad; 6-NC film; 7-backing.
Detailed Description
The present utility model will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present utility model more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the utility model.
The same or similar reference numerals in the drawings of the present embodiment correspond to the same or similar components; in the description of the present utility model, it should be understood that, if there is an azimuth or positional relationship indicated by terms such as "upper", "lower", "left", "right", etc., based on the azimuth or positional relationship shown in the drawings, it is only for convenience of describing the present utility model and simplifying the description, but it is not indicated or implied that the apparatus or element referred to must have a specific azimuth, be constructed and operated in a specific azimuth, and thus terms describing the positional relationship in the drawings are merely illustrative and should not be construed as limitations of the present patent, and specific meanings of the terms described above may be understood by those skilled in the art according to specific circumstances.
The implementation of the present utility model will be described in detail below with reference to specific embodiments.
Referring to FIG. 1, a preferred embodiment of the present utility model is provided.
A cat pestivirus nucleic acid colloidal gold test strip based on RAA isothermal amplification reagent comprises a sample pad 1, a gold label release pad 2, an absorption pad 5, an NC membrane 6 and a back lining 7. The backing 7 is a strip-shaped flat plate, the backing 7 is positioned at the lowest layer, the sample pad 1, the gold mark release pad 2 and the NC film 6 are sequentially stuck on the backing 7 from one end, the absorption pad 5 is stuck on the other end, and the NC film 6 is positioned between the gold mark release pad 2 and the absorption pad 5.
Wherein, the NC film 6 is provided with a detection line 3 and a quality control line 4.
Further, the detection line 3 is close to the gold mark release pad 2, and the quality control line 4 is close to the absorption pad 5.
The RAA isothermal amplification refers to a recombinase-mediated isothermal nucleic acid amplification technology, namely a technology for amplifying nucleic acid under isothermal conditions (such as 37 ℃) by utilizing recombinase, single-stranded binding protein and DNA polymerase. The specific principle is as follows: the recombination enzyme, single-chain binding protein (SSB) and primer form a complex to scan double-chain DNA, the double-chain DNA is unwound at a sequence homologous to the primer, the single-chain binding protein prevents the double-chain DNA from renaturation, and under the condition of energy and dNTP, the DNA polymerase completes the chain extension, so that the instrumentalless amplification can be realized within 30 minutes. The colloidal gold test strip is specifically combined with the Magnaporthe grisea nucleic acid amplified by the RAA to form a visual red straight line, so that the purpose of judging whether the sample to be tested contains Magnaporthe grisea or not is achieved.
The primers in the utility model have two strips, and are respectively provided with different labels, namely a first label and a second label.
Further, the primer sequence one is: 5
' TGGAGGTAAAACAGGAATTAACTATACTAA-3',5' end modified tags-i.e., biotin tags, include, but are not limited to, analogs of biotin such as PEG-biotin, avidin, rhodamine, fluorescein Isothiocyanate (FITC).
Further, the primer sequence II is as follows: 5
The' -CTTGGTTTTAAGTCAGTATCAAATTCTTTATCC-3',5' end modifies the tag two, namely the digoxin tag, including but not limited to digoxin analogs such as the common fluorophores FAM, VIC, ROX, JOE, HEX, cy5.
A detection process of the Magnaporthe grisea nucleic acid colloidal gold test strip based on the RAA isothermal amplification reagent comprises the following steps:
1. taking a whole blood sample of a cat to be tested, uniformly mixing 20 mu L of whole blood with 200 mu L of the reagent A solution without extraction, standing for 2min, adding 50 mu L of the reagent B solution without extraction, and uniformly mixing to obtain a sample nucleic acid solution;
2. adding a proper amount of sample nucleic acid solution into an RAA isothermal amplification reagent, incubating for 30min at a constant temperature of 39 ℃, wherein two designed labeled primers (a primer sequence I and a primer sequence II) can specifically identify a nucleic acid sequence of the Magnaporthe grisea, and only in the presence of the Magnaporthe grisea, amplifying a nucleic acid sequence containing two labels (a label I and a label II);
3. inserting the DNA molecule label I and the colloidal gold particles into the incubated solution, wherein the DNA molecule label I and the colloidal gold particles are specifically combined and colored red, the solution reaches a detection line 3 through chromatography, the detection line 3 can be specifically combined with the DNA molecule label II to display a red straight line, and if the DNA molecule does not contain the DNA molecule label II, the detection line 3 does not display red;
4. after standing for 15min, directly observing the color development conditions of the test strip detection line 3 and the quality control line 4 by naked eyes: if the detection line 3 is colorless and the quality control line 4 is red, the sample result is judged to be negative (namely, the cat pestivirus is not contained); if the detection line 3 is red and the quality control line 4 is red, the sample result is judged to be positive (namely, the cat pestivirus is contained); if the quality control line 4 is colorless, whether the detection line 3 is colorless or red, whether the detection line 3 is invalid (i.e. the test strip is invalid) is judged, a new test strip is needed to be replaced, and the step 1 is repeated for re-detection.
Compared with the prior art, the utility model has the following advantages:
1. the operation is simple, the operation of an operator is convenient, and the on-site real-time detection can be realized;
2. the detection efficiency is high, the time consumption is short, and the immediately-available timeliness requirement can be met.
The foregoing description of the preferred embodiments of the utility model is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the utility model.
Claims (5)
1. The cat pestivirus nucleic acid colloidal gold test strip based on the RAA isothermal amplification reagent is characterized by comprising a sample pad, a gold mark release pad, an absorption pad, an NC film and a back lining, wherein the back lining is a strip-shaped flat plate and is positioned at the lowest layer, the sample pad, the gold mark release pad and the NC film are sequentially stuck on the back lining from one end, the absorption pad is stuck on the other end, and the NC film is positioned between the gold mark release pad and the absorption pad.
2. The Magnaporthe grisea nucleic acid colloidal gold test strip based on the RAA isothermal amplification reagent according to claim 1, wherein a detection line and a quality control line are arranged on the NC membrane.
3. The RAA isothermal amplification reagent based feline pestivirus nucleic acid colloidal gold test strip according to claim 2, wherein said detection line is adjacent to said gold label release pad and said quality control line is adjacent to said absorbent pad.
4. The Magnaporthe grisea nucleic acid colloidal gold test strip based on the RAA isothermal amplification reagent according to claim 3, wherein the RAA isothermal amplification reagent comprises a first primer sequence and a second primer sequence, wherein the first primer sequence modifies the first tag, and the second primer sequence modifies the second tag.
5. The RAA isothermal amplification reagent based colloidal gold test strip according to claim 4, wherein the first label is a biotin label and the second label is a digoxin label.
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|---|---|---|---|
| CN202320102736.4U CN219608936U (en) | 2023-02-02 | 2023-02-02 | Magnaporthe grisea nucleic acid colloidal gold test strip based on RAA isothermal amplification reagent |
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| CN219608936U true CN219608936U (en) | 2023-08-29 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117051173A (en) * | 2023-10-12 | 2023-11-14 | 上海基灵生物科技有限公司 | Cat digestive tract virus PCR detection method and application |
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2023
- 2023-02-02 CN CN202320102736.4U patent/CN219608936U/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117051173A (en) * | 2023-10-12 | 2023-11-14 | 上海基灵生物科技有限公司 | Cat digestive tract virus PCR detection method and application |
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