CN110628942A - Colloidal gold test strip for detecting African swine fever virus and detection method - Google Patents
Colloidal gold test strip for detecting African swine fever virus and detection method Download PDFInfo
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- CN110628942A CN110628942A CN201910576695.0A CN201910576695A CN110628942A CN 110628942 A CN110628942 A CN 110628942A CN 201910576695 A CN201910576695 A CN 201910576695A CN 110628942 A CN110628942 A CN 110628942A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Abstract
The invention discloses a colloidal gold test strip for detecting African swine fever virus and a detection method, and relates to the technical field of virus detection. The colloidal gold test strip provided by the invention comprises a bottom plate, a nitrocellulose membrane, a combination pad, absorbent paper, a sample pad and AgNO3A pad and a reduction pad; the nitrocellulose membrane is bonded in the middle of the bottom plate; one end of the nitrocellulose membrane is pasted with a combination pad, the other end of the nitrocellulose membrane is pasted with absorbent paper, and the combination pad is pasted with a sample pad; a control line and a test line are sequentially arranged on the nitrocellulose membrane from the end close to the absorbent paper, a capture probe is sprayed on the test line, and a biotinylated goat polyclonal antibody is sprayed on the control line; the above-mentionedAnd the binding pad is fixed with colloidal gold labeled streptavidin.
Description
Technical Field
The invention relates to the technical field of virus detection. More particularly, relates to a colloidal gold test strip for detecting African swine fever and a detection method.
Background
African Swine Fever (ASF) is a highly-contact infectious disease of pigs, the death rate of the pigs infected by highly-pathogenic African Swine Fever Virus (ASFV) can reach 100%, the disease is popular in many African countries, 8 months and 1 day in 2018, the national foreign animal epidemic disease research center confirms the first ASF case in China in Shenbei area of Shenyang city, and then African swine fever cases occur in Henan Zheng Zhou, Jiangsu Liyuanchong, Jia Si City and Anhui Wen lake city, Xuan City and the like respectively. Because no vaccine is used for ASF epidemic prevention at present, the rapid and accurate detection of African swine fever virus is very important for quarantine and prevention and control of the spread of the disease.
Nucleic acid molecule detection typically requires two steps, the first step being amplification of the nucleic acid of interest and the second step being detection of the nucleic acid of interest. The PCR technology is firstly established and is the most commonly used amplification method at present, Doudna and Chinese academy of sciences royal gold subject group use one endonuclease Cas12a of CRISPR system, combines nucleic acid amplification and Cas12a detection reaction, establishes a DETECTR and HOLMES detection system, and can be used for rapid detection of DNA. The principle is that a large amount of fragments containing target sequences are obtained through PCR amplification, then the amplification products are added into a Cas12a detection system (including crRNA, Cas12a protein and reporter probe), once the Cas12a and a crRNA complex recognize and cut the target sequences, the non-specific single-strand cutting activity can be triggered, a single-strand reporter molecule in the detection system is cut, fluorescence is emitted, and therefore the existence of the target sequences is confirmed, and the method can be used for detecting virus DNA. However, the PCR method requires an expensive PCR instrument for temperature cycling. The LAMP detection technology is used as a new method based on nucleic acid amplification, provides great help for identifying microbial pathogens since the appearance of 2000, is simple in operation method and short in reaction time, can complete reaction only in a constant-temperature water bath kettle, greatly reduces detection cost, has good effects on different pathogenic microorganisms in different fields, and makes great contribution to the differential diagnosis and prevention control of diseases.
In addition, silver is easy to perform reduction reaction by taking gold particles as cores, and the silver staining technology is used for enhancing the nano gold mark detection result signals, so that the sensitivity can be effectively improved. The LAMP method and the Cas12a detection reaction are combined to be used for detecting the African swine fever virus, and the result is read quickly and intuitively by means of the colloidal gold test strip for enhancing the signal by the silver staining technology.
Disclosure of Invention
The invention aims at providing a colloidal gold test strip for detecting African swine fever virus, and also aims at providing a method for detecting African swine fever virus.
The above object is achieved by:
firstly, a colloidal gold test strip for detecting African swine fever virus comprises a bottom plate, a nitrocellulose membrane, a combination pad, absorbent paper, a sample pad and AgNO3A pad and a reduction pad;
preferably, the nitrocellulose membrane is bonded in the middle of the bottom plate; one end of the nitrocellulose membrane is pasted with a combination pad, the other end of the nitrocellulose membrane is pasted with absorbent paper, and the combination pad is pasted with a sample pad; a control line and a test line are sequentially arranged on the nitrocellulose membrane from the end close to the absorbent paper, a capture probe is sprayed on the test line, and the capture probe is single-stranded DNA complementary with the reporter; the control line is sprayed with a biotinylated goat polyclonal antibody; fixing streptavidin marked by colloidal gold on the bonding pad;
preferably, the width of the sample pad and the end covering part of the combination pad is 1 mm, the width of the end covering part of the combination pad and the nitrocellulose membrane is 1 mm, and the width of the end covering part of the nitrocellulose membrane and the water-absorbing paper is 1 mm;
preferably, the bottom plate is a strip-shaped PVC bottom plate with the width of 4 mm; the lengths of the absorbent paper, the nitrocellulose membrane, the combination pad and the sample pad are respectively 18 mm, 25 mm, 8 mm and 18 mm;
preferably, the distance between the control line and the upper edge (close to the end of the absorbent paper) of the nitrocellulose membrane is 9 mm, and the distance between the control line and the test line is 8 mm;
preferably, the AgNO3The preparation method of the pad comprises the following steps: cutting the glass cellulose membrane into strips with the size of 1.3cm multiplied by 0.4cm, putting the glass cellulose membrane into physiological saline for fully rinsing, drying and uniformly coating AgNO3Sealing the solution at room temperature in dark, drying, and storingUsing;
preferably, the preparation method of the reduction pad comprises the following steps: cutting the glass cellulose membrane into strips with the size of 1.3cm multiplied by 0.4cm, putting the strips into physiological saline for fully rinsing, uniformly coating sodium citrate buffer solution containing hydroquinone after drying, airing the strips at room temperature in a dark place, and sealing the strips at room temperature in a dark place for drying and storing for later use;
preferably, the particle size of the nano-gold in the colloidal gold-labeled streptavidin is 15 ~ 20 nm;
preferably, the capture probe sequence is 3 '-AAAAAAAAAAAAAAAAAATTAAAAAAAAAAAAAAAAAATAA-5'.
Secondly, the invention provides a detection method for detecting African swine fever virus.
A. Constructing a detection reaction system: the reaction system comprises a LAMP amplification system and a Cas12a detection system; LAMP amplification system: four primers F3, B3, FIB, FIP, dNTP, MgSO4(ii) a 1 × isothermal amplification buffer; the Cas12a detection system includes a LbaCas12a-crRNA-activator mixture and a reporter probe. The specific operation method comprises the following steps: firstly, extracting target DNA of African swine fever virus, adding the target DNA into an LAMP amplification system, heating at 95 ℃ for 5min, cooling, adding 8U Bst DNA polymerase, preserving the heat at 65 ℃ for 15-30min, and then heating at 80 ℃ for 10min to terminate the reaction to obtain an amplification product dsDNA; next, a mixture of LbaCas12a-crRNA-activator (12 μ L1 Xsplit buffer (150 mM KCl, 10 mM MgCl)21% glycerol, 0.5 mM DTT, 20 nM HEPES (pH 7.5)), 3 μ L crRNA (300 nM), 1 μ L LbaCas12a (36 nM) and 5 μ L activator (40 nM)) are reacted at 37 ℃ for 30min, 2 μ L of biotinylation reporter (20 nM) is added, and after being uniformly mixed with 2 μ L of the amplification product, the reaction is carried out at 37 ℃ for 15min, and then heated at 98 ℃ for 2min to terminate the reaction;
wherein the African swine fever virus DNA sequence is 5'-CTGCTCATGGTATCAATCTTATCGATAAGTTTCCATCAAAGTTCTGCAGCTCTTACATACCCTTCCACTACGGAGGCAATGCAATTAAAACCCCCGATGATCCGGGTGCGATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATACCAACCCAGTGGTCATATTAACGTATCCAGAGCAAGAGAATTTTATATTAGTTGGGACACGGATTACGTGGGGTCTATCACTACGGCTGATCTTGTGGTATC-3'
The sequence of the primer pair is
F3 TCTGTGATCGAAAGGCTG
B3 TCGAGGCGGGATAGCATC
FIP(F1c+F2) GCCTGGATTGCCGCACCACATGTCTGGCTGGTCAAG
BIP (B1c + B2) CTCTGCGATGTACGACTCAAGGTGGTTGTTATCCATGTACC biotinylated Reporter sequence was Bio-5'-TTTTTTTTTTTTATT-3'
The Activator sequence is TTTACCAAGCGCGCGGGTGAGCGTG
The sequence of CrRNA is AGCAGATACGACCCCAAAAACGAAGGGGACUAAAACAGCACGCUCACCCGCGGGUUGCCU
B. Detection of African swine fever virus by using colloidal gold test strip for detecting African swine fever virus
And (C) taking the solution obtained by the reaction in the step A as a detection sample, and dropwise adding the detection sample to a sample pad of the colloidal gold test strip for detecting the African swine fever virus. AgNO is added when red strips appear on the control line due to aggregation of colloidal gold3The pad and the reduction pad are sequentially covered on the control line and the test line, and a proper amount of distilled water is dripped on the reduction pad, and the detection result is observed after 10 minutes.
Compared with the prior art, the colloidal gold test strip for detecting African swine fever virus has the following advantages:
(1) the colloidal gold test strip for detecting the African swine fever virus provided by the invention has strong specificity, and other viruses do not generate interference on detection signals. Only when African swine fever virus exists, the reporter single-stranded reporter molecule is cut and can not be captured by the capture probe coated on the test line.
(2) The colloidal gold test strip for detecting the African swine fever virus provided by the invention enhances signals by means of a silver staining technology, and can remarkably improve the detection sensitivity of the test strip.
(3) The colloidal gold test strip for detecting African swine fever virus provided by the invention is simple in detection method, a sample can be directly detected on the test strip after LAMP amplification and Cas12a detection, the detection result is visual and accurate, and the requirement of on-site detection can be met.
Drawings
FIG. 1 is a schematic structural diagram of a colloidal gold test strip for detecting African swine fever virus prepared by the present invention;
FIG. 2 is a schematic diagram showing the specific detection result of the colloidal gold test strip for detecting African swine fever virus prepared by the present invention.
Detailed description of the preferred embodiments
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Preparation of colloidal gold-labeled streptavidin
A. Adding 50 mL of 1 mM chloroauric acid solution into the flask, and heating and stirring until the solution is boiled; and then adding 2g of sodium citrate into the solution, stopping heating after the solution turns red, continuously stirring for 30min to obtain a colloidal gold AuNP solution, and storing the colloidal gold AuNP solution at 4 ℃ in a dark place.
B. To 1mL of the colloidal gold solution prepared in step A, 200 μ L of suspension buffer (10 mM Na) was added3PO42.5% BSA, 0.25% Tween-20, 10% sucrose, and 0.1% NaN3) After stirring and mixing, 2mL of SA (streptavidin) was added, and shaking and mixing were performed at 4 ℃. Blocking with BSA was added for 30min, centrifuged at 4 ℃ and washed with the suspension buffer to remove unbound SA, yielding AuNP-SA (colloidal gold-labeled streptavidin). Discard the supernatant and resuspend the AuNP-SA with suspension buffer.
Example 2
Preparing colloidal gold test strip for detecting African swine fever virus
A colloidal gold test strip for detecting African swine fever virus comprises a base plate, a nitrocellulose membrane, a combination pad, absorbent paper, a sample pad and AgNO3A pad and a reduction pad;
adhering the nitrocellulose membrane to the middle part of the bottom plate; pasting a combination pad on one end of the nitrocellulose membrane, pasting absorbent paper on the other end of the nitrocellulose membrane, and pasting the sample pad on the combination pad;
a control line and a test line are sequentially arranged on the nitrocellulose membrane from the end close to the absorbent paper, a capture probe is sprayed on the test line, and the capture probe is single-stranded DNA complementary with the reporter; the control line is sprayed with a biotinylated goat polyclonal antibody; immobilizing the colloidal gold-labeled streptavidin prepared in example 1 on the binding pad;
the width of the covered part of the end parts of the sample pad and the combination pad is 1 mm, the width of the covered part of the end parts of the combination pad and the nitrocellulose membrane is 1 mm, and the width of the covered part of the end parts of the nitrocellulose membrane and the absorbent paper is 1 mm;
the bottom plate is a strip-shaped PVC bottom plate with the width of 4 mm; the lengths of the absorbent paper, the nitrocellulose membrane, the combination pad and the sample pad are respectively 18 mm, 25 mm, 8 mm and 18 mm;
the distance between the control line and the upper edge (close to the end of the absorbent paper) of the nitrocellulose membrane is 9 mm, and the distance between the control line and the test line is 8 mm;
the AgNO3The preparation method of the pad comprises the following steps: cutting the glass cellulose membrane into strips with the size of 1.3cm multiplied by 0.4cm, putting the glass cellulose membrane into physiological saline for fully rinsing, drying and uniformly coating AgNO3Sealing the solution at room temperature, keeping out of the sun, and drying for later use;
the preparation method of the reduction pad comprises the following steps: cutting the glass cellulose membrane into strips with the size of 1.3cm multiplied by 0.4cm, putting the strips into physiological saline for fully rinsing, uniformly coating buffer solution containing sodium citrate of hydroquinone after drying, airing the strips at the room temperature in a dark place, and sealing the strips at the room temperature in the dark place for drying and storing for later use;
the particle size of nano gold in the colloidal gold labeled streptavidin is 15 ~ 20 nm;
the capture probe sequence is
3’-AAAAAAAAAAAAAAAAAATTAAAAAAAAAAAAAAAAAATAA-5’
Example 3
Detection method of African swine fever virus
A. Constructing a detection reaction system: the reaction system comprises a LAMP amplification system and a Cas12a detection system; LAMP amplification system: four primers F3, B3, FIB, FIP, dNTP, MgSO4(ii) a 1 × isothermal amplification buffer; the Cas12a detection system includes a LbaCas12a-crRNA-activator mixture and a reporter probe. The specific operation method comprises the following steps: firstly, extracting target DNA of African swine fever virus, adding the target DNA into an LAMP amplification system, heating at 95 ℃ for 5min, cooling, adding 8U Bst DNA polymerase, preserving the heat at 65 ℃ for 15-30min, and then heating at 80 ℃ for 10min to terminate the reaction to obtain an amplification product dsDNA; next, a mixture of LbaCas12a-crRNA-activator (12 μ L1 Xsplit buffer (150 mM KCl, 10 mM MgCl)2After 1% glycerol, 0.5 mM DTT, 20 nM HEPES (pH 7.5)), 3 μ L crRNA (300 nM), 1 μ L LbaCas12a (36 nM) and 5 μ L activator (40 nM)) are reacted at 37 ℃ for 30min, 2 μ L of the biotinylated reporter (20 nM) and 2 μ L of the amplification product are added, and after uniform mixing, the reaction is reacted at 37 ℃ for 15min, and then heated at 98 ℃ for 2min to terminate the reaction;
wherein the African swine fever virus target DNA sequence is 5'-CTGCTCATGGTATCAATCTTATCGATAAGTTTCCATCAAAGTTCTGCAGCTCTTACATACCCTTCCACTACGGAGGCAATGCAATTAAAACCCCCGATGATCCGGGTGCGATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATACCAACCCAGTGGTCATATTAACGTATCCAGAGCAAGAGAATTTTATATTAGTTGGGACACGGATTACGTGGGGTCTATCACTACGGCTGATCTTGTGGTATC-3'
The sequence of the primer pair is
F3 TCTGTGATCGAAAGGCTG
B3 TCGAGGCGGGATAGCATC
FIP(F1c+F2) GCCTGGATTGCCGCACCACATGTCTGGCTGGTCAAG
BIP(B1c+B2) CTCTGCGATGTACGACTCAAGGTGGTTGTTATCCATGTACC
The biotinylated Reporter sequence was Bio-5'-TTTTTTTTTTTTATT-3'
The Activator sequence is TTTACCAAGCGCGCGGGTGAGCGTG
The sequence of CrRNA is AGCAGATACGACCCCAAAAACGAAGGGGACUAAAACAGCACGCUCACCCGCGGGUUGCCU
B. Detection of African swine fever virus by using colloidal gold test strip for detecting African swine fever virus
And (D) taking the solution obtained by the reaction in the step (A) as a detection sample, and dropwise adding the detection sample to a sample pad of a test strip. AgNO is added when red strips appear on the control line due to aggregation of colloidal gold3The pad and the reduction pad are sequentially covered on the control line and the test line, and a proper amount of distilled water is dripped on the reduction pad, and the detection result is observed after 10 minutes.
The detection principle is as follows: the LAMP amplification product is added into a Cas12a detection system, and once a cleavage target sequence is recognized by a Cas12a and CrRNA complex, the cleavage activity of the reporter single-stranded reporter molecule can be triggered, so that the reporter single-stranded reporter molecule can be cleaved. The higher the DNA content of the African swine fever virus, the lower the content of the biotinylated reporter in the solution to be tested after cutting.
After the liquid to be detected is dripped into the sample pad, the liquid to be detected is chromatographed towards the direction of absorbent paper due to the chromatography action of the test paper, when biotinylation reporter reaches the combination pad, the liquid is combined with the colloidal gold labeled streptavidin coated on the combination pad to form a compound which is diffused to a test line (T), the compound can be captured by a capture probe coated on the test line and is fixed on the test line, when a certain amount of compound is gathered at the test line, a red strip visible to naked eyes can appear, and the uncombined compound and excessive nano gold labeled streptavidin reach a control line (C) through the test line and are combined with the biotinylated goat polyclonal antibody coated on the control line to form a red strip visible to naked eyes. When the sample had no biotinylated reporter or very low levels, a macroscopic red band could not be formed. By mixing AgNO3The pad and the reduction pad are sequentially covered on the control line and the test line, and a proper amount of distilled water is dripped on the reduction pad, so that signals on the test line are amplified due to the large accumulation of reduced silver around the gold particles, and a red strip formed by the accumulation of the colloidal gold is dyed into black.
Negative results: the test line and the control line are black;
positive results: the test line does not develop color, and the control line develops black;
and (4) invalidation: no matter the test line is colored or not, the control line is not colored.
Example 4
Specificity detection
In order to examine the specificity of the test strip, the method described in example 3 was performed using inactivated swine fever and respiratory syndrome virus, porcine circovirus, and porcine pseudorabies virus as negative samples, and the detection results are shown in fig. 2 (wherein 1 is african swine fever virus, 2 is porcine reproduction and respiratory syndrome virus, 3 is porcine circovirus, and 4 is porcine pseudorabies virus), while the detection results of other viruses are positive, and only the detection results of the african swine fever virus sample are negative.
Example 5
Sensitivity detection
The concentration is respectively 10-10M，10-11M，10-12M，10-13M，10-14M，10-15M，10-16M，10-17M African swine fever virus is detected according to the method described in the embodiment 3, and the detection result shows that the lowest detection limit of the test strip is 10 before silver staining-13M, but the detection sensitivity of the test strip after silver staining can reach 10-15M。
Example 6
Stability test
The stability of different batches of immunochromatographic test paper is tested in 1, 3, 5, 7 and 9 weeks by 10-13M African swine fever virus was detected as described in example 3 using test strips stored at 4 ℃ for 1, 3, 5, 7, 9 weeks. The stability test results of different batches are the same and all negative.
List of orders of description
<110> Guangdong Langyuan Biotechnology Co., Ltd
<120> colloidal gold test strip for detecting African swine fever virus and detection method
<140>201910576695.0
<141>2019-06-28
<160>1
<210>1
<211>250
<212>DNA
<213> Artificial sequence
<220>
<223> African swine fever virus DNA sequence
<400>1
ctgctcatgg tatcaatctt atcgataagt ttccatcaaa gttctgcagc tcttacatac
ccttccacta cggaggcaat gcaattaaaa cccccgatga tccgggtgcg atgatgatta
cctttgcttt gaagccacgg gaggaatacc aacccagtgg tcatattaac gtatccagag
caagagaatt ttatattagt tgggacacgg attacgtggg gtctatcact acggctgatc
ttgtggtatc
Claims (3)
1. A colloidal gold test strip for detecting African swine fever virus is characterized in that the test strip comprises a bottom plate, a nitrocellulose membrane, a combination pad, absorbent paper, a sample pad and AgNO3A pad and a reduction pad;
the nitrocellulose membrane is bonded in the middle of the bottom plate; one end of the nitrocellulose membrane is pasted with a combination pad, the other end of the nitrocellulose membrane is pasted with absorbent paper, and the combination pad is pasted with a sample pad; a control line and a test line are sequentially arranged on the nitrocellulose membrane from the end close to the absorbent paper, a capture probe is sprayed on the test line, and the capture probe is single-stranded DNA complementary with the reporter; the control line is sprayed with a biotinylated goat polyclonal antibody; fixing streptavidin marked by colloidal gold on the bonding pad;
the width of the covered part of the end parts of the sample pad and the combination pad is 1 mm, the width of the covered part of the end parts of the combination pad and the nitrocellulose membrane is 1 mm, and the width of the covered part of the end parts of the nitrocellulose membrane and the absorbent paper is 1 mm;
the bottom plate is a strip-shaped PVC bottom plate with the width of 4 mm; the lengths of the absorbent paper, the nitrocellulose membrane, the combination pad and the sample pad are respectively 18 mm, 25 mm, 8 mm and 18 mm;
the distance between the control line and the upper edge (close to the end of the absorbent paper) of the nitrocellulose membrane is 9 mm, and the distance between the control line and the test line is 8 mm;
the AgNO3The preparation method of the pad comprises the following steps: cutting the glass cellulose membrane into strips with the size of 1.3cm multiplied by 0.4cm, putting the glass cellulose membrane into physiological saline for fully rinsing, drying and uniformly coating AgNO3Sealing the solution at room temperature, keeping out of the sun, and drying for later use;
the preparation method of the reduction pad comprises the following steps: cutting the glass cellulose membrane into strips with the size of 1.3cm multiplied by 0.4cm, putting the strips into physiological saline for fully rinsing, uniformly coating sodium citrate buffer solution containing hydroquinone after drying, airing the strips at room temperature in a dark place, and sealing the strips at room temperature in a dark place for drying and storing for later use;
the particle size of nano gold in the colloidal gold labeled streptavidin is 15 ~ 20 nm;
the capture probe sequence is 3 '-AAAAAAAAAAAAAAAAAATTAAAAAAAAAAAAAAAAAATAA-5'.
2. A detection method for detecting African swine fever virus is characterized by comprising the following steps:
A. constructing a detection reaction system: the reaction system comprises a LAMP amplification system and a Cas12a detection system; LAMP amplification system: four primers F3, B3, FIB, FIP, dNTP, MgSO4(ii) a 1 × isothermal amplification buffer; the Cas12a detection system comprises a LbaCas12a-crRNA-activator mixture and a reporter probe; the specific operation method comprises the following steps: firstly, extracting target DNA of African swine fever virus, adding the target DNA into an LAMP amplification system, heating at 95 ℃ for 5min, cooling, adding 8U Bst DNA polymerase, preserving the heat at 65 ℃ for 15-30min, and then heating at 80 ℃ for 10min to terminate the reaction to obtain an amplification product dsDNA; next, a mixture of LbaCas12a-crRNA-activator (12 μ L1 Xsplit buffer (150 mM KCl, 10 mM MgCl)21% Glycerol, 0.5 mM DTT, 20 nM HEPES (pH 7.5)), 3. mu.L crRNA (300 nM), 1. mu.L LbaCas12a (36 nM) and 5. mu.L activator (40 nM)) reacted at 37 ℃ for 30 reactionsAfter min, adding 2 mu L of biotinylation reporter (20 nM) and 2 mu L of the amplification product, uniformly mixing, reacting for 15min at 37 ℃, and then heating for 2min at 98 ℃ to terminate the reaction;
wherein the African swine fever virus DNA sequence is 5'-CTGCTCATGGTATCAATCTTATCGATAAGTTTCCATCAAAGTTCTGCAGCTCTTACATACCCTTCCACTACGGAGGCAATGCAATTAAAACCCCCGATGATCCGGGTGCGATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATACCAACCCAGTGGTCATATTAACGTATCCAGAGCAAGAGAATTTTATATTAGTTGGGACACGGATTACGTGGGGTCTATCACTACGGCTGATCTTGTGGTATC-3'
The sequence of the primer pair is
F3 TCTGTGATCGAAAGGCTG
B3 TCGAGGCGGGATAGCATC
FIP(F1c+F2) GCCTGGATTGCCGCACCACATGTCTGGCTGGTCAAG
BIP(B1c+B2) CTCTGCGATGTACGACTCAAGGTGGTTGTTATCCATGTACC
The biotinylated Reporter sequence was Bio-5'-TTTTTTTTTTTTATT-3'
The Activator sequence is TTTACCAAGCGCGCGGGTGAGCGTG
The sequence of CrRNA is AGCAGATACGACCCCAAAAACGAAGGGGACUAAAACAGCACGCUCACCCGCGGGUUGCCU
B. Detection of African swine fever virus by using colloidal gold test strip for detecting African swine fever virus
Taking the solution obtained by the reaction in the step A as a detection sample, and dropwise adding the detection sample to a sample pad of the colloidal gold test strip in claim 1; AgNO is added when red strips appear on the control line due to aggregation of colloidal gold3The pad and the reduction pad are sequentially covered on the control line and the test line, and a proper amount of distilled water is dripped on the reduction pad, and the detection result is observed after 10 minutes.
3. A colloidal gold test strip for detecting African swine fever virus is characterized in that the test strip is used for detecting the African swine fever virus.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951920A (en) * | 2019-12-26 | 2020-04-03 | 武汉博杰生物医学科技有限公司 | Detection kit and detection method for African swine fever virus |
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| CN110951920A (en) * | 2019-12-26 | 2020-04-03 | 武汉博杰生物医学科技有限公司 | Detection kit and detection method for African swine fever virus |
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| CN111647642B (en) * | 2020-05-19 | 2023-10-20 | 深圳市众循精准医学研究院 | Nucleic acid detection method and nucleic acid detection kit based on detection test paper display |
| CN112782400A (en) * | 2020-12-30 | 2021-05-11 | 上海振诚生物科技有限公司 | Preparation method of reagent card for rapidly detecting pet virus colloidal gold |
| CN112980929A (en) * | 2021-04-13 | 2021-06-18 | 广州海思医疗科技有限公司 | Kit for rapidly detecting HLA-B1502 |
| CN112980929B (en) * | 2021-04-13 | 2021-07-20 | 广州海思医疗科技有限公司 | Kit for rapidly detecting HLA-B1502 |
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