CN114196788A - Method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology - Google Patents
Method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology Download PDFInfo
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Abstract
The invention discloses a method for rapidly detecting African swine fever virus by using a fluorescence in-situ detection technology, which comprises the steps of preparing a fluorescent probe, collecting and fixing cells of a sample to be detected, and hybridizing and dyeing the fluorescent probe and the cells; observing the stained hybrid cells, and identifying whether the sample to be detected is infected by the African swine fever virus; the method amplifies signals by a primer pool method, and signal points consist of thousands of DNA fragments, so that compared with the traditional in-situ hybridization method, the method has the advantages of stronger signals, easy signal capture, no need of a super-resolution imaging system, and capability of analyzing signal clusters by using a common fluorescence microscope, thereby reducing the experiment cost and the operation difficulty.
Description
Technical Field
The invention relates to the field of application of molecular biology technology, in particular to a method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology.
Background
The purpose of in situ hybridization is to determine the presence or absence of target DNA or RNA sequences and to localize these sequences to specific cellular or chromosomal sites. The specific sequence in the cell is identified by the combination of the probe and the target DNA sequence. Nucleic acid detection by In Situ Hybridization (ISH) was first reported since 1969. Making this approach an important tool in scientific and clinical research. In situ hybridization is a technique in which the complementary strand of a nucleotide probe is hybridized with a specific sequence. Depending on the probe used, there may be autoradiography, fluorescence microscopy or immunohistochemical visualization, respectively. This technique allows the localization and detection of specific DNA and RNA sequences in cells, tissue sections or tissues. One major advantage of in situ hybridization is that it maximizes the use of tissues that are difficult to obtain, and hundreds of hybridizations can be performed on the same tissue. The ISH has the defects of unstable probe, limited resolution, complex operation procedure and the like. Fluorescence in situ hybridization and chromogenic in situ hybridization are increasingly used as major alternative techniques for ISH. In situ hybridization is widely used in research and clinical applications such as pathogenic microbe diagnosis, gene localization, cytogenetics, gene expression, and prenatal diagnosis and development, particularly for tumor diagnosis.
Fluorescence In Situ Hybridization (FISH) is the design of probes to target regions to determine the relative location and content of target regions, such as tissues, cells, etc. The principle is that according to the base complementary pairing principle, fluorescent nucleic acid is used for directly labeling a probe, a fluorescent antibody is used for indirectly labeling the probe or the probe indirectly labeled by other methods is used; hybridizing with the target sequence, and observing the detection result by a fluorescence microscope. Can be used for detecting interphase and middle stage cells, and identifying specific normal and abnormal DNA sequences in chromosome. This technology was first developed in 1980 to replace ISH and identify chromosomal abnormalities. The high sensitivity and specificity of FISH and the rapid outcome make FISH a widely used technology. Has gained general acceptance in laboratories and clinical diagnostics. Chromosomal translocations, deletions, specific gene amplifications, and changes in chromosome number can all be detected by specific probes. One of the largest areas of influence is in the detection and diagnosis of human malignancies. FISH is highly sensitive and specific, and rapid. The FISH technique has the following advantages: the probe is safe, rapid and high in sensitivity, and can be stored for a long time; the method is simple and visual; the method can be used for analyzing metaphase chromosomes and interphase cells, and can also be used for detecting various substances such as fresh, frozen or paraffin embedded specimens, puncture objects, cast-off cells and the like.
The main target cell for the replication of the african swine fever virus is macrophages, which have great significance for pathogenic and immune escape mechanisms because the virus can regulate the function of the macrophages. The virus is in short of effective therapeutic drugs and preventive vaccines, so that epidemic prevention and control are hindered; meanwhile, because of recessive infection of wild boars in some areas, the epidemic situation is further complicated. The epidemic situation development of the African swine fever greatly affects the economy of all countries in the world, and particularly causes serious economic loss for the large pig-raising countries in China.
The death rate of ASF can reach 100%, but no effective antiviral medicine and vaccine for preventing and treating the epidemic disease are developed so far, so that ASFV infection can be rapidly detected, and the ASF infection can become a key measure for preventing, controlling and eliminating diseases. Meanwhile, because ASF and swine fever (CSF) have great similarity in clinical expression, two diseases are extremely difficult to distinguish through clinical diagnosis and can be distinguished only through laboratory diagnosis, and therefore, the ASFV is very important for preventing and controlling the diseases through early accurate diagnosis.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for rapidly detecting African swine fever virus by using a fluorescence in-situ detection technology, which rapidly detects ASFV infection by designing and synthesizing a primer and a fluorescent probe in a rapid fluorescence in-situ hybridization mode and finally observes the result by a fluorescence microscope or a common microscope; the method can observe positive signals only by hybridizing for 15min, and overcomes the defects of complexity and radioactive pollution of the traditional in-situ hybridization; the cost is high; multiple hybridization, high imaging equipment requirement; poor probe specificity, etc. The detection technology of ASFV is numerous, but has certain limitations, such as PCR and FAT may be limited by expensive experimental equipment; ELISA is low in cost, rapid, poor in sensitivity and specificity and the like. Compared with other detection methods, the FISH has the characteristics of strong specificity, high sensitivity and safety, low cost, quick result and the like.
In order to achieve the purpose, the invention designs a method for rapidly detecting African swine fever virus by using a fluorescence in-situ detection technology, which comprises the following steps:
1) preparation of fluorescent probe:
2) cell collection and immobilization of samples to be tested
3) Hybridization of samples
Hybridizing a fluorescent probe with the cells, and staining;
4) identification of ASFV
Observing the stained hybrid cells, and identifying whether the sample to be detected is infected by African Swine Fever Virus (ASFV).
Further, in the step 1), the fluorescent probe is prepared as follows:
i. based on the African swine fever virus whole genome, n primer probes with the length of 120-150bp are designed and obtained, and the primer probes are a primer probe pool;
ii, adding a pair of universal primers to the front end and the rear end of each primer probe, carrying out amplification synthesis to obtain synthetic primer probes, and thus forming a synthetic primer probe pool containing n synthetic primer probes; wherein, the universal primers are respectively a T7 promoter sequence at the 5 'end and a reverse transcription primer sequence at the 3' end;
performing PCR amplification by using a synthetic primer probe pool as a template and using a T7-ASFV amplification primer pair to obtain an ASFV genome fragment; wherein the content of the first and second substances,
T7-ASFV-F：5’-TAATACGACTCACTATAGGGTAGATCTTGGCAAGAGGAAA-3’，
T7-ASFV-R：5’-GCGTGAATAGTCCGATCTGGAGCAGGCCTCGCGGTTTCGC-3’；
and iv, carrying out in-vitro transcription and reverse transcription on the ASFV genome fragment, and recovering to obtain the fluorescent probe.
Still further, in the step 1), the fluorescent probe is prepared as follows:
a. based on the African swine fever virus whole genome, n primer probes with the length of 120-150bp are designed and obtained, and the primer probes are a primer probe pool;
b. adding a pair of universal primers to the front end and the rear end of each primer probe for amplification synthesis to obtain synthetic primer probes, thereby forming a synthetic primer probe pool containing n synthetic primer probes; wherein, the universal primers are respectively a T7 promoter sequence at the 5 'end and a reverse transcription primer sequence at the 3' end;
c. performing PCR reaction by using a T7-ASFV amplification primer pair by using a synthetic primer probe pool as a template to obtain an ASFV genome library, purifying and recycling an amplification product by using a kit, and constructing a plasmid library by enzyme digestion; wherein the content of the first and second substances,
T7-ASFV-F：5’-TAATACGACTCACTATAGGG-TAGATCTTGGCAAGAGGAAA-3’，
T7-ASFV-R：5’-GCGTGAATAGTCCGATCTGG-AGCAGGCCTCGCGGTTTCGC-3’；
d. and carrying out PCR amplification by using the T7-ASFV amplification primer pair by using a plasmid library as a template to obtain an ASFV genome fragment, and carrying out in-vitro transcription and reverse transcription on the ASFV genome fragment to recover and obtain the fluorescent probe.
Furthermore, in the primer probe pool, the GC content of each primer probe is 45-55% and the T content is less than 45-55%; n is 5000-9000;
still further, the T7 promoter sequence is:
5’-TAATACGACTCACTATAGGG-3’；
the reverse transcription primer sequence of the 3' end: 5'-GCGTGAATAGTCCGATCTGG-3' are provided.
Still further, the reverse transcription primer: 5'-GCGTGAATAGTCCGATCTGG-3' are provided.
Still further, the cell collecting and fixing steps of the sample to be detected are as follows:
(1) collecting cells, centrifuging at 1500rpm for 5min, precipitating cells, washing cells with PBS or physiological saline once
(2) Centrifuging at 1000rpm for 5min to remove supernatant, resuspending cells with 5ml hypotonic KCL, and incubating at 37 deg.C for 30 min;
(3) immediately adding 1ml of methanol/acetic acid stationary liquid after the incubation is finished, uniformly blowing, centrifuging at 1000rpm for 5min, and removing supernatant;
(4) adding 1ml methanol/acetic acid stationary liquid, blowing and beating uniformly, centrifuging at 1000rpm for 5min, and removing supernatant;
(5) adding 1ml methanol/acetic acid stationary liquid, blowing and beating uniformly, fixing at room temperature for 20min, and storing in a refrigerator at-20 deg.C; (the amount of fixative at this step varies with the amount of cells;)
Still further, the step of hybridizing the samples is as follows:
(1) dripping tablets, and drying in an oven for 20 min; marking a hybridization area by using an oil marker;
(2) boiling slices in 2XSSC solution at 65 deg.C for 30 min;
(3) gradient washing with 70-85-100% alcohol;
(4) air drying with ethanol, adding hybridization solution, sealing, and denaturing at 82 deg.C for 10 min;
(5) putting the slices into a wet box, and hybridizing for 2h at 45 ℃;
(6) after hybridization, removing mounting glue and slide (soaking water when cover glass is removed to prevent cell abrasion when slide is taken out by drying), preheating 0.3% NP40/0.4XSSC, and soaking for 4min at 65 ℃;
(7)ddH2after the O is performed once, the obtained product is placed into 10ul of DAPI aqueous solution for dyeing for 1 min;
(8) after the 100% alcohol is used for one time, the glass is placed on a piece of mirror wiping paper for drying.
The principle of the invention is as follows:
1. the design principle of the primer pool is as follows: comparing the GZ-201801-ASFV genome according to the nucleic acid sequences of ASFV classical strain and hypervariable strain published by GenBank; about 189kb of GZ-201801-ASFV whole genome is selected, about 5000 and 6000 primer probes with the length of about 150bp are designed, and the primers pool probes are synthesized in a large batch by the company. Each probe is divided into three parts, including a total library amplification region, an ASFV-sublibrary amplification region, and an ASFV target hybridization region (see FIG. 1).
2. The fluorescent probe is a cDNA probe, needs to combine T7 polymerase and NTP and the like by using sub-library DNA as a template, is placed in a metal bath at 37 ℃ for reaction for 2h to obtain mRNA, and then is subjected to reverse transcription to obtain a complementary single-stranded DNA probe, namely the cDNA probe. At the same time, CY3 fluorophore was added to the cDNA probe during reverse transcription, because CY3-dUTP takes part in the reverse transcription instead of TTP during reverse transcription, and then cDNA probe sequence was added to excite fluorescence, so that the synthesized probe sequence was fluorescent (FIG. 2).
FISH hybridization principle: the sample DNA double strand is decomposed into single strands by using the co-denaturation of the fluorescence-labeled nucleic acid probe and the sample at 82 ℃. After the denaturation is finished, the mixture is placed in a water bath kettle at 45 ℃ for co-incubation for 90min, so that the probe and the target sequence are hybridized. Finally, the detection results were observed using a fluorescence microscope (fig. 3).
The invention has the beneficial effects that:
(1) the specificity is strong: the invention hybridizes different African swine fever genome fragments in a probe pool mode, thereby reducing non-specific amplification signals; the signal to noise ratio is high.
(2) Is quick and simple: the invention can observe the result after hybridizing for 15min, thereby reducing the time cost
(3) The imaging condition universality is high: the signal spots can be observed with a conventional fluorescence microscope.
(4) Can be used for early diagnosis: the detection sample can detect positive signals within 4h after ASFV infected cells, thereby further indicating that the method is extremely sensitive to early diagnosis of virus infection.
(5) High accuracy, and single copy detection
In conclusion, the signal is amplified by the primer pool method, the signal point is composed of thousands of DNA fragments, and compared with the traditional in-situ hybridization method, the signal point is stronger, is easy to capture, does not need a super-resolution imaging system, can analyze the signal cluster by using a common fluorescence microscope, and reduces the experiment cost and the operation difficulty.
Drawings
FIG. 1 is a schematic diagram of primer pool design;
FIG. 2 is a schematic diagram of probe preparation;
FIG. 3 is a FISH schematic diagram;
FIG. 4 is a diagram of the effect of ASFV infection imaging by fast FISH detection;
in the figure, A: experimental groups: ASFV infection of PAM cells, fast FISH results, B: control group: normal PAM cells, fast FISH results, C and D: blank group: ASFV-infected PAM cells
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
The method for rapidly detecting the African swine fever virus by using the fluorescence in-situ detection technology comprises the following steps:
1) preparation of fluorescent probe:
i. based on the African swine fever virus whole genome, n primer probes with the length of 120-150bp are designed and obtained, and the primer probes are a primer probe pool; in the primer probe pool, the GC content of each primer probe is 45-55% and the T content is less than 45-55%; n is 5000-9000;
ii, adding a pair of universal primers to the front end and the rear end of each primer probe, carrying out amplification synthesis to obtain synthetic primer probes, and thus forming a synthetic primer probe pool containing n synthetic primer probes; wherein the universal primers are respectively a T7 promoter sequence at the 5 ' end and are 5'-TAATACGACTCACTATAGGG-3';
the reverse transcription primer sequence at the 3 ' end is 5'-GCGTGAATAGTCCGATCTGG-3'.
Performing PCR amplification by using a synthetic primer probe pool as a template and using a T7-ASFV amplification primer pair to obtain an ASFV genome fragment; wherein the content of the first and second substances,
T7-ASFV-F：5’-TAATACGACTCACTATAGGGTAGATCTTGGCAAGAGGAAA-3’，
T7-ASFV-R：5’-GCGTGAATAGTCCGATCTGGAGCAGGCCTCGCGGTTTCGC-3’；
iv, performing in vitro transcription and reverse transcription by utilizing the ASFV genome fragment, and recovering to obtain a fluorescent probe; the reverse transcription primer comprises the following components: 5'-GCGTGAATAGTCCGATCTGG-3' are provided.
Alternatively, the fluorescent probe is prepared as follows:
i. based on the African swine fever virus whole genome, n primer probes with the length of 120-150bp are designed and obtained, namely a primer probe pool; in the primer probe pool, the GC content of each primer probe is 45-55% and the T content is less than 45-55%; n is 5000-9000;
ii, adding a pair of universal primers to the front end and the rear end of each primer probe, carrying out amplification synthesis to obtain synthetic primer probes, and thus forming a synthetic primer probe pool containing n synthetic primer probes; wherein the universal primers are respectively a T7 promoter sequence at the 5 ' end and are 5'-TAATACGACTCACTATAGGG-3',
the reverse transcription primer sequence of the 3 ' end is 5'-GCGTGAATAGTCCGATCTGG-3';
taking a synthetic primer probe pool as a template, carrying out PCR reaction by using a T7-ASFV amplification primer pair to obtain an ASFV genome library, purifying and recycling an amplification product by using the kit, and constructing a plasmid library by enzyme digestion; wherein the content of the first and second substances,
T7-ASFV-F：5’-TAATACGACTCACTATAGGG-TAGATCTTGGCAAGAGGAAA-3’，
T7-ASFV-R：5’-GCGTGAATAGTCCGATCTGG-AGCAGGCCTCGCGGTTTCGC-3’；
carrying out PCR amplification by using the T7-ASFV amplification primer pair by using a plasmid library as a template to obtain an ASFV genome fragment, carrying out in vitro transcription and reverse transcription on the ASFV genome fragment, and recovering to obtain a fluorescent probe, wherein the reverse transcription primer: 5'-GCGTGAATAGTCCGATCTGG-3', respectively;
2) cell collection and immobilization of samples to be tested
(1) Collecting cells, centrifuging at 1500rpm for 5min, precipitating cells, washing cells with PBS or physiological saline once
(2) Centrifuging at 1000rpm for 5min to remove supernatant, resuspending cells with 5ml hypotonic KCL, and incubating at 37 deg.C for 30 min;
(3) immediately adding 1ml of methanol/acetic acid stationary liquid after the incubation is finished, uniformly blowing, centrifuging at 1000rpm for 5min, and removing supernatant;
(4) adding 1ml methanol/acetic acid stationary liquid, blowing and beating uniformly, centrifuging at 1000rpm for 5min, and removing supernatant;
(5) adding 1ml methanol/acetic acid stationary liquid, blowing and beating uniformly, fixing at room temperature for 20min, and storing in a refrigerator at-20 deg.C; (the amount of fixative at this step varies with the amount of cells;)
3) Hybridization of samples
(1) Dripping tablets, and drying in an oven for 20 min; marking a hybridization area by using an oil marker;
(2) boiling slices in 2XSSC solution at 65 deg.C for 30 min;
(3) gradient washing with 70-85-100% alcohol;
(4) air drying with ethanol, adding hybridization solution, sealing, and denaturing at 82 deg.C for 10 min;
(5) putting the slices into a wet box, and hybridizing for 2h at 45 ℃;
(6) after hybridization, removing mounting glue and slide (soaking water when cover glass is removed to prevent cell abrasion when slide is taken out by drying), preheating 0.3% NP40/0.4XSSC, and soaking for 4min at 65 ℃;
(7)ddH2after the O is performed once, the obtained product is placed into 10ul of DAPI aqueous solution for dyeing for 1 min;
(8) after the 100% alcohol is used for one time, putting the glass cleaning paper on the glass cleaning paper for drying;
4) identification of ASFV
Adding 10ul of mounting agent, mounting and microscopic examination; observing the stained hybrid cells, and identifying whether the sample to be detected is infected by African Swine Fever Virus (ASFV).
Based on the method, the following detection is carried out according to the actual situation:
example 1
The method for rapidly detecting the African swine fever virus by using the fluorescence in-situ detection technology comprises the following steps:
1) preparation of fluorescent Probe
i. Based on the African swine fever virus whole genome, according to the design requirement: 5000 primer probes with the length of 120-150bp are synthesized by a gene company with the GC content of 45-55% and the T content of less than 45-55% to form a primer probe pool;
ii, adding a pair of universal primers to the front end and the rear end of each primer probe, carrying out amplification synthesis to obtain synthetic primer probes, and thus forming a synthetic primer probe pool containing 5000 synthetic primer probes; wherein, the universal primers are respectively a T7 promoter sequence at the 5' end: 5'-TAATACGACTCACTATAGGG-3' and reverse transcription primer sequence at the 3 ' end: 5'-GCGTGAATAGTCCGATCTGG-3', respectively;
amplifying the DNA library by PCR by using a T7-ASFV primer to obtain an ASFV genome fragment; after amplification, identifying the target fragment by gel electrophoresis; after the PCR amplification reaction is finished, identifying by using gel electrophoresis, wherein,
T7-ASFV-F：5’-TAATACGACTCACTATAGGG-TAGATCTTGGCAAGAGGAAA-3’，
T7-ASFV-R：5’-GCGTGAATAGTCCGATCTGG-AGCAGGCCTCGCGGTTTCGC-3’；
in vitro transcription
Reacting the purified and recovered ASFV genome fragment for 2h at 37 ℃ in a metal bath; and identifying by using low-melting-point gel electrophoresis after the reaction is finished.
v. reverse transcription
After the in vitro transcription is finished, PCR reaction is utilized; after the reaction is finished, adding the same volume of 0.25M EDTA/0.5M NaOH, mixing uniformly, and reacting at 98 ℃ for 10 min. Purification and recovery are carried out, and the recovery probe is verified by FISH.
vi. purification recovery of probes and characterization
1) After the above procedure was completed, 80ul of olive Binding buffer, 320ul of 100% ethanol was added to the reaction product.
2) After mixing uniformly, transferring to an adsorption column at 13000rpm/min for 1min, discarding the liquid in the tube, and repeating once.
3) Adding DNA wash buffer 13000rpm/min for 1min into the adsorption column, and repeating once.
4) Discarding the liquid in the tube, repeating the steps once, and centrifuging the tube for 2min at 13000rpm
5) Transferring the adsorption column to a clean 1.5ml centrifuge tube, air drying for 5min
6) Add 15. mu.L of dd H to the adsorption column2And O, standing at room temperature for 2min, centrifuging at 13000rpm/min for 2min, and storing at-20 ℃ for later use after the probe is prepared.
2) Cell collection and immobilization of samples to be tested
(1) Collecting cells, centrifuging at 1500rpm for 5min, precipitating cells, washing cells with PBS or physiological saline once
(2) Centrifuging at 1000rpm for 5min to remove supernatant, resuspending cells with 5ml hypotonic KCL, and incubating at 37 deg.C for 30 min;
(3) immediately adding 1ml of methanol/acetic acid stationary liquid after the incubation is finished, uniformly blowing, centrifuging at 1000rpm for 5min, and removing supernatant;
(4) adding 1ml methanol/acetic acid stationary liquid, blowing and beating uniformly, centrifuging at 1000rpm for 5min, and removing supernatant;
(5) adding 1ml methanol/acetic acid stationary liquid, blowing and beating uniformly, fixing at room temperature for 20min, and storing in a refrigerator at-20 deg.C; (the amount of fixative at this step varies with the amount of cells;)
3) Hybridization of samples
Fixing sample cells to be detected, and then directly preparing a hybridization sample by a dropping mode:
(1) dripping tablets, and drying in an oven for 20 min; marking a hybridization area by using an oil marker;
(2) boiling slices in 2XSSC solution at 65 deg.C for 30 min;
(3) gradient washing with 70-85-100% alcohol;
(4) air drying with alcohol, adding hybridization solution mixed with fluorescent probe, sealing (sealing), and denaturing at 82 deg.C for 10 min;
(5) putting the slices into a wet box, and hybridizing for 2h at 45 ℃;
(6) after hybridization, removing mounting glue and slide (soaking water when cover glass is removed to prevent cell abrasion when slide is taken out by drying), preheating 0.3% NP40/0.4XSSC, and soaking for 4min at 65 ℃;
(7)ddH2after one time in O, 10ul of DAPI are addedDyeing for 1min in aqueous solution;
(8) after the 100% alcohol is used for one time, putting the glass cleaning paper on the glass cleaning paper for drying;
(9) add 10ul mounting agent, mount, examine under microscope.
Before hybridization detection, a fluorescent probe is prepared in advance, ASFV-infected PAM cells (namely, sample cells to be detected) and normal porcine alveolar macrophages (PAM cells) are hybridized, and the hybridization result is shown in a fluorescence diagram of FIG. 4: the experimental group (sample cells to be detected) can see that the signal points surround the cell nucleus for a circle, and the signal points in the cell nucleus are fewer; this is because after the ASFV invades the body, it first replicates and proliferates in the nucleus in the first 3 hours, and the virus gradually packages and transfers to the cytoplasm after 4 hours of infection, further proliferating and amplifying the infection.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology
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Claims (8)
1. A method for rapidly detecting African swine fever virus by using a fluorescence in-situ detection technology is characterized by comprising the following steps: the method comprises the following steps:
1) preparing a fluorescent probe;
2) collecting and fixing cells of a sample to be detected;
3) hybridizing a fluorescent probe with the cells, and staining;
4) observing the stained hybrid cells, and identifying whether the sample to be detected is infected by the African swine fever virus.
2. The method for rapidly detecting African swine fever virus by using fluorescence in situ detection technology according to claim 1, which is characterized in that: in the step 1), the fluorescent probe is prepared as follows:
i. based on the African swine fever virus whole genome, n primer probes with the length of 120-150bp are designed and obtained, and the primer probes are a primer probe pool;
ii, adding a pair of universal primers to the front end and the rear end of each primer probe, carrying out amplification synthesis to obtain synthetic primer probes, and thus forming a synthetic primer probe pool containing n synthetic primer probes; wherein, the universal primers are respectively a T7 promoter sequence at the 5 'end and a reverse transcription primer sequence at the 3' end;
performing PCR amplification by using a synthetic primer probe pool as a template and using a T7-ASFV amplification primer pair to obtain an ASFV genome fragment; wherein the content of the first and second substances,
T7-ASFV-F：5’-TAATACGACTCACTATAGGGTAGATCTTGGCAAGAGGAAA-3’，
T7-ASFV-R：5’-GCGTGAATAGTCCGATCTGG-AGCAGGCCTCGCGGTTTCGC-3’；
and iv, carrying out in-vitro transcription and reverse transcription on the ASFV genome fragment, and recovering to obtain the fluorescent probe.
3. The method for rapidly detecting African swine fever virus by using fluorescence in situ detection technology according to claim 1, which is characterized in that: in the step 1), the fluorescent probe is prepared as follows:
a. based on the African swine fever virus whole genome, n primer probes with the length of 120-150bp are designed and obtained, and the primer probes are a primer probe pool;
b. adding a pair of universal primers to the front end and the rear end of each primer probe for amplification synthesis to obtain synthetic primer probes, thereby forming a synthetic primer probe pool containing n synthetic primer probes; wherein, the universal primers are respectively a T7 promoter sequence at the 5 'end and a reverse transcription primer sequence at the 3' end;
c. performing PCR reaction by using a T7-ASFV amplification primer pair by using a synthetic primer probe pool as a template to obtain an ASFV genome library, purifying and recycling an amplification product by using a kit, and constructing a plasmid library by enzyme digestion; wherein the content of the first and second substances,
T7-ASFV-F：5’-TAATACGACTCACTATAGGG-TAGATCTTGGCAAGAGGAAA-3’，
T7-ASFV-R：5’-GCGTGAATAGTCCGATCTGG-AGCAGGCCTCGCGGTTTCGC-3’；
d. and carrying out PCR amplification by using the T7-ASFV amplification primer pair by using a plasmid library as a template to obtain an ASFV genome fragment, and carrying out in-vitro transcription and reverse transcription on the ASFV genome fragment to recover and obtain the fluorescent probe.
4. The method for rapidly detecting African swine fever virus by using fluorescence in situ detection technology according to claim 2 or 3, wherein the method comprises the following steps: in the primer probe pool, the GC content of each primer probe is 45-55% and the T content is less than 45-55%; n is 5000 to 9000.
5. The method for rapidly detecting African swine fever virus by using fluorescence in situ detection technology according to claim 2 or 3, wherein the method comprises the following steps: the T7 promoter sequence is: 5'-TAATACGACTCACTATAGGG-3', respectively;
the reverse transcription primer sequence of the 3' end: 5'-GCGTGAATAGTCCGATCTGG-3' are provided.
6. The method for rapidly detecting African swine fever virus by using fluorescence in situ detection technology according to claim 2 or 3, wherein the method comprises the following steps: the reverse transcription primer comprises the following components: 5'-GCGTGAATAGTCCGATCTGG-3' are provided.
7. The method for rapidly detecting African swine fever virus by using fluorescence in situ detection technology according to claim 1, which is characterized in that: the cell collecting and fixing steps of the sample to be detected are as follows:
(1) collecting cells, centrifuging at 1500rpm for 5min, precipitating cells, washing cells with PBS or physiological saline once
(2) Centrifuging at 1000rpm for 5min to remove supernatant, resuspending cells with 5ml hypotonic KCL, and incubating at 37 deg.C for 30 min;
(3) immediately adding 1ml of methanol/acetic acid stationary liquid after the incubation is finished, uniformly blowing, centrifuging at 1000rpm for 5min, and removing supernatant;
(4) adding 1ml methanol/acetic acid stationary liquid, blowing and beating uniformly, centrifuging at 1000rpm for 5min, and removing supernatant;
(5) adding 1ml methanol/acetic acid fixing solution, blowing, fixing at room temperature for 20min, and storing in refrigerator at-20 deg.C.
8. The method for rapidly detecting African swine fever virus by using fluorescence in situ detection technology according to claim 1, which is characterized in that: the sample hybridization steps are as follows:
(1) dripping tablets, and drying in an oven for 20 min; marking a hybridization area by using an oil marker;
(2) boiling slices in 2XSSC solution at 65 deg.C for 30 min;
(3) gradient washing with 70-85-100% alcohol;
(4) air drying with ethanol, adding hybridization solution, sealing, and denaturing at 82 deg.C for 10 min;
(5) putting the slices into a wet box, and hybridizing for 2h at 45 ℃;
(6) after hybridization, removing mounting glue and glass slides, preheating 0.3% NP40/0.4XSSC, and soaking for 4min at 65 ℃;
(7)ddH2after the O is performed once, the obtained product is placed into 10ul of DAPI aqueous solution for dyeing for 1 min;
(8) after the 100% alcohol is used for one time, the glass is placed on a piece of mirror wiping paper for drying.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1840698A (en) * | 2006-01-06 | 2006-10-04 | 云南出入境检验检疫局检验检疫技术中心 | Fluorescence quantitative PCR detection reagent for Asf virus and preparation method and use thereof |
| CN107603971A (en) * | 2017-10-24 | 2018-01-19 | 厦门龙进生物科技有限公司 | A kind of preparation method of in situ hybridization probe |
| CN109161542A (en) * | 2018-02-23 | 2019-01-08 | 广州简册生物技术有限公司 | fluorescence in situ hybridization probe and its preparation method and application |
| CN112176103A (en) * | 2020-09-28 | 2021-01-05 | 山东绿都生物科技有限公司 | African swine fever virus fluorescence ERA constant temperature rapid detection kit |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1840698A (en) * | 2006-01-06 | 2006-10-04 | 云南出入境检验检疫局检验检疫技术中心 | Fluorescence quantitative PCR detection reagent for Asf virus and preparation method and use thereof |
| CN107603971A (en) * | 2017-10-24 | 2018-01-19 | 厦门龙进生物科技有限公司 | A kind of preparation method of in situ hybridization probe |
| CN109161542A (en) * | 2018-02-23 | 2019-01-08 | 广州简册生物技术有限公司 | fluorescence in situ hybridization probe and its preparation method and application |
| CN112176103A (en) * | 2020-09-28 | 2021-01-05 | 山东绿都生物科技有限公司 | African swine fever virus fluorescence ERA constant temperature rapid detection kit |
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