CN112662820A - Kit for detecting porcine gata virus and detection method thereof - Google Patents
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Abstract
The invention belongs to the field of biomedical specialty, and particularly relates to a detection kit for porcine gata virus and a detection method thereof. The diagnostic kit comprises two pairs of nested PCR primers, wherein the first pair of primers is 5'-ATGGCGGACGTGTGACATCAC-3' and 5'-GTAACCTTCGCATGACACCACC-3'; the second pair of primers was 5'-GCTACTCCACATAGTGAGAG-3' and 5'-AACATGAATGGAGTGGTATC-3'. The kit can be used for early detection of the porcine gatifloxacin virus (GETV), is a quick, simple, specific and sensitive method for detecting the GETV, and has a very good application prospect.
Description
Technical Field
The invention belongs to the field of biomedical specialty, and particularly relates to a kit for detecting porcine gata virus and a detection method thereof.
Background
The porcine gata virus (GETV) is now an important arbovirus affecting the large-scale pig industry. The virus can cause the sow (especially in the early gestation period) to produce stillbirth, tremor of born piglets, skin redness, mental waste, brownish yellow diarrhea and higher death rate. When weaned piglets are stressed, GETV infection is easily caused, mixed infection often occurs with some bacteria or viruses, and the death rate can reach more than 50% in serious cases. Sick pigs, sick pigs and insect vectors are important sources of infection of the disease. In view of the serious harm of the virus to the animal husbandry in recent years, it is necessary to establish a fast, sensitive and accurate detection method for monitoring the virus so as to ensure the healthy development of the animal husbandry.
At present, an effective means for quickly and accurately detecting the Gatas virus (GETV) is not available, so that the development of a quick detection kit aiming at the virus becomes a problem to be solved urgently at present.
Disclosure of Invention
The invention aims to provide a detection kit for porcine gata virus and a detection method thereof. The inventor designs a nested PCR detection kit through earlier research, and the nested PCR detection kit can be used as a rapid, simple, convenient and specific GETV infection diagnosis method. The kit has the characteristics of simplicity, convenience, rapidness, high specificity, low cost and the like, and provides an important technical means for Gata virus detection and epidemiological investigation.
The invention provides the following technical scheme:
a kit for detecting NSP1 gene of porcine gatifloxacin comprises two pairs of nested PCR primers, wherein the first pair of primers is 5'-ATGGCGGACGTGTGACATCAC-3' and 5'-GTAACCTTCGCATGACACCACC-3'; the second pair of primers was 5'-GCTACTCCACATAGTGAGAG-3' and 5'-AACATGAATGGAGTGGTATC-3'.
The kit detects NSP1 gene of the porcine Gattea virus.
Further, other components of the kit may be selected as is conventional in the art, and for example, Probe qPCR MIX may be included, or PCR buffer, deoxynucleoside triphosphate mixture, and DNA polymerase may be used instead.
The use method of the kit comprises the following steps:
1) extracting DNA of sample genes or performing reverse transcription to obtain cDNA, and constructing a sample GETV-NSP1-pBM16 cloning plasmid;
2) performing a first round of PCR amplification by using a first pair of primers by taking GETV-NSP1-pBM16 clone plasmid as a template;
3) after the first round of PCR amplification is finished, taking a PCR product as a template, performing second round of PCR amplification by using a second pair of primers, and performing gel electrophoresis;
4) if a 326bp band appears in electrophoresis, the test proves to be positive.
Further, the PCR reaction conditions of step 2) are as follows: pre-denaturation at 95 ℃ for 5 min; 50s at 95 ℃, 30s at 50 ℃, 1min at 72 ℃, 30 cycles, and finally 10min at 72 ℃.
The PCR reaction conditions in 3) are as follows: pre-denaturation at 95 ℃ for 5 min; 50s at 95 ℃, 30s at 52 ℃, 1min at 72 ℃, 30 cycles, and finally 10min at 72 ℃.
The minimum nucleic acid detection amount in this method was 8 copies/. mu.L.
The invention designs specific nested PCR primers according to a non-structural protein NSP1 of a Getavirus. The selected gene sequence is highly conserved, so the gene sequence is used as an important basis for identifying the Gatas virus. The nested PCR kit established by the invention can be used for detecting virus particles with 8 copy numbers at the lowest energy, and has higher sensitivity.
Through specific tests, closely related entomoviruses PRRSV, PCV, PRV, PTOV, PKV, SVV and ASFV sharing mosquito vectors with GETV are not detected, and the GeV can amplify clear bands. Therefore, the nested PCR detection method established by the invention has high specificity, the whole detection can be completed within 2-3 h, expensive test equipment such as a fluorescent quantitative PCR instrument and an electron microscope is not needed, and the method is simple to operate and is more suitable for detecting large-batch samples. Provides important technical support for early discovery and prevention and control of the Gatas virus diseases.
Drawings
FIG. 1 is a diagram showing the results of the electrophoresis of nested PCR assays. M: DL2000 DNA Ladder; 1: GETV NSP1 gene 2: and (5) negative control.
FIG. 2 is a diagram showing the electrophoresis result of the nested PCR detection specificity test. DL2000 DNA Ladder, 1-8 get V, PRRSV, PCV, PRV, PTOV, PKV, SVV, ASFV, 9: and (5) negative control.
FIG. 3 is a diagram showing the results of electrophoresis in the sensitivity test of the nested PCR method. DL2000 DNA Ladder; 1-8: GETV NSP1 gene 8X 106-8×10-1copies/. mu.L; 9: negative control
Detailed Description
EXAMPLE 1 design and Synthesis of specific primers
6 GETV sequences (accession numbers are: KY363863.1, LC223132.1, EU015063.1, KY399029.1, MK693225.1 and AY702913.1 in sequence) are downloaded from GenBank, and after the gene sequences of the non-structural protein NSP1 are aligned by using SnapGene6.1, a conserved region is used as an identification region, and two pairs of primers are involved in the region.
A first pair of primers:
the sequence of the upstream primer is 5'-ATGGCGGACGTGTGACATCAC-3';
the sequence of the downstream primer is 5'-GTAACCTTCGCATGACACCACC-3';
a second pair of primers:
the sequence of the upstream primer is 5'-GCTACTCCACATAGTGAGAG-3';
the sequence of the downstream primer is 5'-AACATGAATGGAGTGGTATC-3'.
The amplified fragments were approximately 544bp and 326bp, respectively. Primers were synthesized from Shanghai.
Example 2
Firstly, experimental steps
1. Extraction of GETV genome RNA of Liaoning pig
3-5g of the diseased material determined to be infected with Getavirus (GETV) was taken and RNA extraction kit (MI20101) from Monad (Mona organism) was used.
Pre-cooling chloroform and isopropanol in a refrigerator at 4 deg.C, diluting anhydrous ethanol with DEPC water to 75% concentration, and pre-cooling. Adding liquid nitrogen to grind the pathological materials into powder. 1mL of Trizol reagent was added, and the powder was blown up and mixed well with a pipette. The suspension was transferred to a 1.5mL centrifuge tube for enzyme removal. Shake vigorously for 30sec, and bury in ice for 10 min. Add 200. mu.l chloroform, turn the mixture gently upside down for 30sec, and let stand on ice for 10 min. Centrifuge at 12000rpm at 4 deg.C for 13min, transfer 400. mu.l of colorless supernatant to a fresh precooled 1.5mL enzyme removal centrifuge tube, add 500. mu.l of isopropanol, gently invert, and stand on ice for 10 min. Centrifuging at 4 deg.C and 10000rpm for 10min, and discarding the supernatant. Adding prepared 75% ethanol, vortex oscillating, centrifuging at 8800rpm at 4 deg.C for 5min, removing all liquid, and air drying for 5 min. So that the ethanol is completely volatilized. Dissolved in 20. mu.l of RNase-freeddH2O, mixed well and the concentration of extracted RNA was measured using a UV spectrophotometer NanoDrop2000 c. After the RNA extraction, the RNA is stored at-80 ℃ for standby.
2. RNA reverse transcription to synthesize cDNA
The first step is as follows: reverse transcriptome removal as shown in table 1.
TABLE 1 reverse transcription genome removal System and conditions
Reaction conditions are as follows: mixing at 42 deg.C for 2min
And step two, directly adding 4 mu L of 5 XHiscript II qRT super MixII into the tube in the step one, uniformly mixing, reacting for 15min at 50 ℃, reacting for 5s at 85 ℃, and storing at-20 ℃ for later use after reverse transcription.
3. PCR amplification
The cDNA was used as a template, and the first pair of primers designed in example 1 was added, and the specific reaction system was 2uL of template cDNA, 0.8uL of each of the upstream and downstream primers, 10. mu.L of 2 XTaq PCR Mastermix, ddH206.4 uL, 20uL total reaction system, PCR amplification was performed according to the reaction system of Table 2. And 5 mu L of the amplification product is taken, electrophoresis is carried out on 1.2% agarose gel, a GETV band is detected by a gel imaging system, and the PCR positive product of the GETV-NSP1 is sent to Shanghai worker for sequencing.
TABLE 2 PCR reaction System
4. GETV-NSP1 gene purification
The GETV-NSP1 gene is determined by comparing the sequencing result with GenBank. Under UV-302nm UV irradiation, the strips were cut and weighed. PCR product purification was performed using a gel recovery kit. The method comprises the following steps:
(1) GSH solution was added to completely immerse the gel pieces in the liquid and water bath at 55 deg.C. Mix by inversion at intervals of minutes until the gel is completely thawed. The solution is put into a refrigerator with the temperature of 4 ℃ to be quickly cooled to about room temperature, and the liquid is completely transferred into a centrifugal column and is kept stand for 2 min.
(2) Centrifuging at 10000 Xg for 1min, discarding the filtrate, and repeatedly loading the gel solution on the column. 650. mu.L of WB solution was added along the tube wall to wash the impurities. Centrifuging at 10000 Xg for 1min, and discarding the filtrate. Centrifugation at 10000 Xg for 2min completely removed the residual WB solution.
(3) Placing the column into a new enzyme-removing centrifuge tube, opening the cover, and standing at room temperature for 5min to completely volatilize the residual ethanol. 40 mul ddH2O was suspended and dropped into the center of the inner membrane of the adsorption column, and after standing for 2min, centrifugation was carried out at 10000 Xg for 1 min. Collecting the filtrate to obtain DNA solution, and storing at-20 deg.C.
5. Construction of GETV-NSP1 gene and pBM16A recombinant plasmid
(1) DNA fragment 5 u L, pBM16AVector1 u L, 10 XToposmant 1 u L and water 3 u L, adding reagent, gently mixing with the tip of the gun, centrifuging at constant speed to the bottom of the tube, and concentrating the solution at the bottom of the tube.
(2) The PCR instrument was set at 25 ℃ for 15 minutes. Loading the sample into agarose gel electrophoresis, wherein the strip is single and bright, and a proper amount of PCR product can be directly taken for subsequent cloning test.
6. Transferring the recombinant plasmid GETV-NSP1-pBM16A into competent cells
Adding 10 μ L of recombinant vector GETV-NSP1-pBM16A into 50 μ L of DH5 α chemically competent cells, ice-bathing for 30min, heat-shocking in 42 deg.C water bath for 90sec, ice-bathing again for 5min, adding 500 μ L of LB medium, and placing in a shaker at 37 deg.C and 200rpm for 1.5 h; centrifuging at 4000rpm for 5min, discarding 400. mu.L of the supernatant, mixing the remaining solution, and adding dropwise to LB plate medium containing AMP resistance. The smear stick is smeared evenly and cultivated overnight at 37 ℃ by inversion.
7. Screening, identification and preservation of positive clonal strains
(1) And taking the overnight flat plate out of the incubator after 14h, observing and recording the distribution condition of escherichia coli colonies on the flat plate, picking single colonies with a bacteria picking rod in an aseptic operation table on the premise that the colonies grow well and are distributed uniformly, transferring the single colonies into 5mL of resistance-free LB liquid culture medium subjected to autoclaving in advance, adding 5 mu LAMP antibiotics, and placing the mixture in a constant-temperature shaking table at 37 ℃ and 200r for overnight culture until the bacteria liquid is turbid.
(2) The shake-mixed culture broth was used as a template for ordinary RT-PCR (Table 3-4). 10. mu.L of enzyme, 6.4. mu. L, DNA 2. mu.L of water, and 0.8. mu.L of each of the upstream and downstream primers.
(3) After the PCR is finished. The PCR product was analyzed and detected by electrophoresis using 1.2% agarose gel, and the bacterial solution was sent to Beijing Bomaide for sequencing.
(4) The bacteria preservation is carried out in a sterile operating platform. Taking 2mL of the enzyme-removed centrifuge tube after high pressure, adding 200 mu L of the identified error-free positive bacteria liquid into glycerol subjected to high-pressure sterilization, and storing the bacteria liquid in a refrigerator at the temperature of-80 ℃.
8. Plasmid extraction
Used herein is a high purity plasmid miniprep kit (MI 13101).
The CP4 preparative column was placed in a collection tube, treated with 500. mu.L of BL equilibration solution dropwise at the center of the inner membrane, and centrifuged at 12000rpm for 1 min. 5mL of the bacterial solution is added into a centrifuge tube, centrifuged at 12000rpm for 1min, and the supernatant is discarded. Add 500. mu. l P1 solution to the tube and mix it with shaking. mu.L of cell lysate P2 was added to the tube, and after 8 times gentle inversion, 700. mu.L of solution P3 was added, and immediately, 8 times gentle inversion was carried out. At this time, white floc appeared in the liquid, which was centrifuged at 12000rpm for 10 min. The supernatant was added in portions to a treated CP4 preparation column without aspirating the pellet, centrifuged at 12000rpm for 1min, and the filtrate was discarded. Add 500. mu.L of deproteinized solution PD to the center of the preparative column and centrifuge at 12000rpm for 1 min. Adding 600 μ L of rinsing solution PW to the center of the preparation column, centrifuging at 12000rpm for 1min, discarding the filtrate, adding rinsing solution PW, and centrifuging. The CP4 preparative column was returned to the collection tube and centrifuged at 12000rpm for 2min to remove the rinsing solution PW. The CP4 preparative column was placed in a clean centrifuge tube, and ethanol was evaporated for 5min with the lid open. 100 μ L of RNA-free ddH2O was added dropwise to the center of the prepared inner membrane, and left to stand for 2 min. The solution was centrifuged at 12000rpm for 2 min. Collecting the filtrate, taking 1 mu L of the filtrate to deliver to the worker organism Limited company for sequencing, and obtaining the GETV-NSP1-pBM16A positive control standard after comparing the obtained product.
9. Reagent kit reaction system and conditions
The copy number of the GETV-NSP1-PBM16A positive control standard is measured by an enzyme-labeling instrument, GETV NSP1-PBM16A is used as a template, and a nested PCR reaction system and conditions are as follows:
first round PCR reaction (10. mu.L): 2 XTaq PCR MasterMix 5. mu.L, first pair of primers prepared in example 1 each 0.4. mu. L, ddH2O3.2. mu.L, template cDNA 1. mu.L.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 50s at 95 ℃, 30s at 50 ℃, 1min at 72 ℃, 30 cycles, and finally 10min at 72 ℃.
Second round PCR reaction (10. mu.L): the PCR product obtained in the first round was used as a template for nested PCR (1. mu.L), 2 XTaq PCR MasterMix (5. mu.L), and the second pair of primers prepared in example 1 was 0.4. mu. L, ddH each2O 3.2μL。
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 50s at 95 ℃, 30s at 52 ℃, 1min at 72 ℃, 30 cycles, and finally 10min at 72 ℃.
The PCR product was detected by electrophoresis on a 1.1% agarose gel. The negative control was obtained by replacing the template in the original reaction system with ddH 2O. And (4) sending the PCR product to Shanghai worker for sequencing, and detecting to determine the PCR product as a GETV positive product.
10. Liaoning pig GETV-NSP1 gene nest type PCR specificity and sensitivity test
The nucleic acid extraction kit is used for extracting nucleic acid from PRRSV, PCV, PRV, PTOV, PKV, SVV and ASFV genes, and GETV, PRRSV, PCV, PRV, PTOV, PKV, SVV and ASFV genes are subjected to PCR operation according to the method, the result is detected by 1.1% agarose gel electrophoresis, and the negative control is to replace a template in the original reaction system with ddH2O, so that the specificity of the invention on the GETV genes is verified.
The prepared GETV-NSP1-pBM16A standard recombinant plasmid is expressed by 8 multiplied by 106～8×10-1The copies/. mu.L are diluted 10-fold in serial and used as templates, from 8X 106copies/. mu.L of2 mu L of each dilution is taken, the kit established by the invention is utilized to carry out nested PCR reaction, and the obtained PCR product is analyzed and detected by 1.2 percent agarose gel through electrophoresis technology so as to evaluate and compare the sensitivity of the kit and the conventional commercial RT-PCR detection kit. The negative control was obtained by replacing the template in the original reaction system with ddH 2O.
Second, experimental results
1. Conversion of GETV-NSP1 standard substance concentration
The concentration of the positive standard plasmid was 117.8 ng/. mu.L as determined by the microplate reader according to the formula (6.02X 10)23)×(ng/μL×10-9) /(DNA length. times.660) ═ copies/. mu.L. The copy number after conversion is 8 × 106copies/μL。
2. Liaoning pig GETV-NSP1 gene nested PCR amplified target fragment
As shown in FIG. 1, specific bands with fragments of about 544bp and 326bp are amplified from a positive sample by using the GETV NSP1 gene nested PCR detection kit established by the invention. The resulting size of the target band fragment corresponds to the expected result. The obtained PCR product is sent to Beijing Bomaide company for sequencing, and the obtained part gene sequence of the Gatasavir NSP1 is determined by NCBI online sequence alignment software BLAST.
3. Nest-type PCR (polymerase chain reaction) specificity test result of GETV-NSP1 gene of Liaoning pig
As shown in FIG. 2, only GETV positive samples can amplify specific target bands of 544bp and 326bp by using the nested PCR kit established by the invention, and no bands are amplified by PRRSV, PCV, PRV, PTOV, PKV, SVV and ASFV, which indicates that the nested PCR kit has good specificity (FIG. 2).
4. Nest's PCR sensitivity test result of Liaoning pig GETV-NSP1 gene
As shown in FIG. 3, the detection limit of the nested PCR kit established by the invention is 8 copies/. mu.L, which is higher than the sensitivity (90 copies/. mu.L) of the commercial Sudoku virus RT-PCR detection kit (BTN 13-46800, Beijing Baiolai Pacobi technology Co., Ltd.), and the sensitivity of the nested PCR kit is higher than that of the commercial Sudoku virus RT-PCR detection kit.
While the invention has been described in connection with a preferred embodiment, it will be understood that various changes and modifications may be effected therein by one skilled in the art after reading the foregoing description, and equivalents may be resorted to, falling within the scope of the invention as defined by the appended claims.
Sequence listing
<110> Shenyang agriculture university
<120> kit for detecting porcine Galtavirus and detection method thereof
<130> 2021
<160> 4
<170> PatentIn version 3.3
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<213> Artificial sequence
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Claims (7)
1. A kit for detecting porcine gata virus comprises two pairs of nested PCR primers and is characterized in that: the first pair of primers is 5 '-ATGGCGGACGTGTGACATCAC-3 and 5' -GTAACCTTCGCATGACACCACC-3, and the second pair of primers is 5 '-GCTACTCCACATAGTGAGAG-3 and 5' -AACATGAATGGAGTGGTATC-3.
2. The kit for detecting porcine gatifloxacin according to claim 1, further comprising Probe qPCR MIX.
3. The kit for detecting porcine adefovir according to claim 1, wherein the NSP1 gene of porcine adefovir is detected by the kit.
4. A method for using the kit for detecting porcine gata virus according to claim 1, characterized in that the method comprises the steps of:
1) extracting DNA or reverse transcription of a sample GETV-NSP1 gene to obtain cDNA, and constructing a sample GETV-NSP1-pBM16 cloning plasmid;
2) performing a first round of PCR amplification by using a first pair of primers by taking GETV-NSP1-pBM16 clone plasmid as a template;
3) after the first round of PCR amplification is finished, taking a PCR product as a template, performing second round of PCR amplification by using a second pair of primers, and performing gel electrophoresis;
4) if a 326bp band appears in electrophoresis, the test proves to be positive.
5. The method according to claim 4, wherein the PCR reaction conditions of step 2) are as follows: pre-denaturation at 95 ℃ for 5 min; 50s at 95 ℃, 30s at 50 ℃, 1min at 72 ℃, 30 cycles, and finally 10min at 72 ℃.
6. The method according to claim 4, wherein the PCR reaction conditions in 3) are as follows: pre-denaturation at 95 ℃ for 5 min; 50s at 95 ℃, 30s at 52 ℃, 1min at 72 ℃, 30 cycles, and finally 10min at 72 ℃.
7. The use according to claim 4, wherein the minimum nucleic acid detection level is 8 copies/. mu.L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117192113A (en) * | 2023-09-18 | 2023-12-08 | 南京农业大学三亚研究院 | Colloidal gold test strip for detecting antibody of getta virus, preparation method and application thereof |
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| CN108950083A (en) * | 2018-08-24 | 2018-12-07 | 河南农业大学 | The multiplex RT-PCR method of GETV, PEDV, TGEV, PDCoV and PoRV are detected simultaneously |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108950083A (en) * | 2018-08-24 | 2018-12-07 | 河南农业大学 | The multiplex RT-PCR method of GETV, PEDV, TGEV, PDCoV and PoRV are detected simultaneously |
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| LI YUAN YUAN等: "Identification of a Newly Isolated Getah Virus in the China-LaoS Border,China", 《BIOMEDICAL AND ENVIRONMENTAL SCIENCES》 * |
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| CN117192113A (en) * | 2023-09-18 | 2023-12-08 | 南京农业大学三亚研究院 | Colloidal gold test strip for detecting antibody of getta virus, preparation method and application thereof |
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