CN112662819A - Detection kit and detection method for porcine gata virus NSP1 gene - Google Patents
Detection kit and detection method for porcine gata virus NSP1 gene Download PDFInfo
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Abstract
The invention belongs to the field of biomedical specialty, and particularly relates to a detection kit for a swine Gata virus NSP1 gene and a detection method thereof. The diagnostic kit comprises a specific primer and a fluorescent probe, wherein the sequence of an upstream primer in the specific primer is 5 '-CCATCCTGGACGTGGGTAGT-3, and the sequence of a downstream primer in the specific primer is 5' -TTTCGCCAGTTTTCGAGCGT-3; the fluorescent probe sequence is 5 '-FAM-ACCACACCTACCACTGCATCTGCCCCA-TAMRA-3'. The kit can be used for early detection of the Getavirus (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 detection kit for a swine Gata virus NSP1 gene and a detection method thereof.
Background
Gattavirus (GETV) belongs to a new infectious disease, mainly outbreaks between horses and pigs, can cause newborn piglets to have local tremor, listlessness and higher mortality, and is very important for the early rapid diagnosis of the disease. 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 a Gata virus and a detection method thereof. Through earlier researches, the invention designs a fluorescent probe with high specificity and good repeatability based on a probe (TAMRA) with TaqMan minor groove combination as a base, and the fluorescent probe can be used as a rapid, simple and specific GETV infection diagnosis kit. The specific primer and the fluorescent probe of the kit are designed based on the conserved region of NSP1 gene of 6 GETV genome sequences in GenBank, and have good conservation, thereby completing the invention.
The invention provides the following technical scheme:
a detection kit for a swine gata virus NSP1 gene comprises a specific primer and a fluorescent probe, wherein the sequence of an upstream primer in the specific primer is 5 '-CCATCCTGGACGTGGGTAGT-3, and the sequence of a downstream primer in the specific primer is 5' -TTTCGCCAGTTTTCGAGCGT-3; the fluorescent probe sequence is 5 '-FAM-ACCACACCTACCACTGCATCTGCCCCA-TAMRA-3'. Wherein FAM marked by the fluorescent probe is a fluorescent reporter group, and TAMRA is a fluorescent quenching group.
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 or reverse transcription of the sample GETV-NSP1 to obtain cDNA, and constructing a sample GETV-NSP1-pBM16 cloning plasmid as a template;
2) preparing a PCR reaction system by using the template, the Probe qPCR MIX, the specific primer, the fluorescent Probe and water, and carrying out PCR amplification reaction;
3) and (3) carrying out PCR amplification reaction by taking a positive standard GETV-PBM16A as a template under the same condition, and drawing a standard curve by taking the logarithmic value of the copy concentration of the standard solution as an abscissa and the Ct value of the standard solution as an ordinate.
4) When the Ct value of the sample is less than or equal to 28.0 and a typical amplification curve appears, the detection is positive;
5) and (4) according to the Ct value measured by the sample, contrasting with the standard curve to obtain the copy concentration of the sample.
Further, the concentration of the specific primer in the PCR system of the step 2) or 3) is 4umol/L, the concentration of the fluorescent probe is 6umol/L, and the PCR reaction conditions are as follows: denaturation at 95 ℃ for 5min, amplification at 95 ℃ for 30sec and 50 ℃ for 20 sec were carried out for 40 cycles.
The minimum nucleic acid detection amount of this method was 8.8369X 101copies/μL。
The fluorescent probe kit for detecting the GETV-NSP1 gene has the advantages of high specificity and low background value. The specific primers and fluorescent probes in this kit are designed based on the non-structural protein gene sequences, as they are highly conserved in the genome, but contain enough genetic variation to distinguish them from other alphaviruses. When the positive control CT value is less than or equal to 28.0 and a typical amplification curve appears, and the negative control has no CT value and no amplification curve, the detection result is positive.
The detection kit established in the research is beneficial to quickly, sensitively and specifically detecting and quantitatively detecting the porcine gatherina virus (GETV). The fluorescent probe kit can be used for rapid diagnosis and virus monitoring of GETV infection, and can also be used for research and application. Compared with the conventional RT-PCR detection method, the GETV-NSP1 kit has the advantages of good specificity, convenience and rapidness, and the key to success lies in the design of specific primers and fluorescent probes, the specific combination of the primers and the probes with virus target genes, and no cross property with different pathogens.
In addition, the detection results using the specific primers and probes show good sensitivity and do not cross-react with other closely related insect-borne viruses (PRRSV, PRV, PCV, SVV, PTOV) sharing mosquito-borne with GETV. Therefore, the method has important significance for monitoring the mosquito virus, and is particularly suitable for rapid detection and quantitative analysis of low-grade GETV infection in mosquito and bovine serum samples.
Drawings
FIG. 1 is a standard curve generated by 10-fold serial dilutions using GETV-NSP1 standard.
FIG. 2 is a graph showing the fluorescent probe amplification curves of GETV-NSP1 standard, which are 8.8369X 10 from left to right10-8.8369×100copies/μ L, a. first amplification curve; B. a second amplification curve; C. third amplification curve.
FIG. 3 is a graph showing the results of electrophoresis. M is 2000bp DNA Marker, 1-11 is 8.8369X 1010～8.8369×100copies/. mu.L, 12 is negative control (negative control is the replacement of template cDNA in the original reaction system with ddH)2O)。
Detailed Description
EXAMPLE 1 design and Synthesis of specific primers and fluorescent probes
6 strains of GETV sequences (KY363863.1, LC223132.1, EU015063.1, KY399029.1, MK693225.1 and AY702913.1) are downloaded from GenBank, and after alignment of the gene sequences of the non-structural proteins NSP1 is carried out by using SnapGene6.1, a conserved region is used as an identification region, and primer and probe sequences are calculated in the region.
The sequence of the upstream primer is 5' -CCATCCTGGACGTGGGTAGT-3;
the sequence of the downstream primer is 5' -TTTCGCCAGTTTTCGAGCGT-3;
the fluorescent probe sequence is 5 '-FAM-ACCACACCTACCACTGCATCTGCCCCA-TAMRA-3'.
Wherein FAM marked by the fluorescent probe is a fluorescent reporter group, and TAMRA is a fluorescent quenching group. Both primers and probes were synthesized by Bomaide GmbH. The amplified fragment was approximately 128 bp.
Example 2
Firstly, experimental steps
Drawing a standard curve
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 specific primers designed in example 1 were added, specifically, the reaction system was 2uL of template cDNA, 0.8uL of each of the specific 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 taking 5 mu L of the amplification product, carrying out electrophoresis on 1.2% agarose gel, detecting a GETV band by using a gel imaging system, and carrying out up-to-the-sea biological sequencing on the RT-PCR positive product of the GETV NSP 1.
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 10uL of recombinant vector GETV NSP1-pBM16A into 50 uL of DH5 alpha chemically competent cells, carrying out ice bath for 30min, carrying out water bath heat shock at 42 ℃ for 90sec, carrying out ice bath again for 5min, adding 500 uL of LB culture medium, and putting into a shaker at 37 ℃ 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. Optimization of kit reaction system and conditions
The copy number of the GETV-NSP1-PBM16A positive control standard is measured by a microplate reader, the GETV NSP1-PBM16A gradient concentration is used as a template, the total system is determined to be 10 mu L, the fluorescent probe and the specific primer are subjected to different concentration ratios (the concentration is controlled to be 0.1-2umol/L) to select an optimal concentration, the optimal annealing temperature is used for adjusting the reaction temperature and the reaction time (the temperature is 50-60 ℃, the time is 20-40s), the cycle parameters are respectively 35, 40 or 45, and optimization is carried out according to the conditions.
10. Establishment of a Standard Curve
The recombinant plasmid 8.8369X 10 of the prepared GETV-NSP1-pBM16A standard product0-8.8369×1010The copies/. mu.L was diluted 10-fold and used as a template. And (3) performing GETV-NSP1 fluorescent probe detection according to the optimized reaction system and reaction conditions, and establishing a standard curve by taking the logarithm of the standard substance concentration as an x axis and the CT value as a y axis.
(II) specificity, sensitivity and repeatability test of GETV-NSP1 fluorescent probe of Liaoning pig
Extracting DNA or RNA of GETV, PRRSV, PRV, PCV, SVV, PTOV and other genes. The method of the fluorescent probe kit established by the invention is used for detection by taking cDNA reverse transcribed by DNA or RNA as a template, and when the positive control CT value is less than or equal to 28.0 and a typical amplification curve appears, and the negative control has no CT value and no amplification curve, the test is established. Therefore, the specificity of the Liaoning pig GETV NSP1 fluorescent probe method is verified.
The prepared GETV-NSP1-pBM16A standard recombinant plasmid is expressed according to 8.8369 x 100-8.8369×1010The copies/μ L was diluted 10-fold in serial doubleAs a template, after being detected by a commercial RT-PCR kit (BTN 13-46800, Beijing Baiolai Pacobo technology Co., Ltd.), the sensitivity of the fluorescent probe kit established by the invention and the sensitivity of the conventional commercial RT-PCR kit are evaluated and compared. The obtained PCR product is analyzed and detected by electrophoresis technology through 1.2 percent agarose gel, and the negative control is that the template cDNA in the original reaction system is replaced by ddH2O。
Carrying out 10-fold gradient dilution on the GETV-NSP1-pBM16A standard recombinant plasmid, selecting samples with different concentrations for detection, repeating each sample for 3 times, repeating the test for 3 times in different time periods, calculating the standard deviation of the average value by using a biological statistical method, and verifying the repeatability of the method.
Second, experimental results
1. The optimal detection kit reaction system and conditions are determined as follows:
the optimal concentration of the specific primer is 4umol/L, and the optimal concentration of the fluorescent probe is 6 umol/L. The reaction system was 10. mu.L, and contained 5. mu.L of Probe Qpcr MIX, 3.5. mu.L of RNase free ddH20, 0.2. mu.L of each of the upstream and downstream primers, 0.1. mu.L of fluorescent Probe, and 1. mu.L of template. The optimum annealing temperature was 50 ℃ and the cycle parameters were 40. The details are shown in Table 4.
TABLE 4 optimal conditions for PCR
2. Conversion of GETV-NSP1 standard substance concentration
The concentration of the GETV NSP1-pBM16A standard substance detected by a microplate reader is 180.6 ng/. mu.L. According to the formula (6.02X 10)23)×(ng/uL×10-9) /(DNA length × 660) ═ copies/uL. The copy number after conversion is about 8 × 1010copies/μL。
3. Drawing of standard curve
The linear relationship between the Ct value and the logarithm of the target gene concentration is analyzed to obtain a standard curve as shown in FIG. 1, the slope of the curve is-2.6316, the intercept is 34.119, and the standard curve equation is y-2.6316 x +34.119 (R)2＝0.9856)。
4. GETV-NSP1 fluorescent probe sensitivity test results
The GETV-NSP1 fluorescent probe amplification curve is generated by detecting positive standard plasmid of GETV NSP1-pBM16A diluted in gradient, and the copy number range is 8.8369 × 100-8.8369×1010copies, Ct value ≦ 28.0, and a typical amplification curve appeared. As can be seen from FIG. 2, the lowest nucleic acid detection amount of the GETV-NSP1 fluorescent probe was 8.8369X 101copies/. mu.L, and as shown in the electrophoresis result of FIG. 3, the lowest nucleic acid copy number of the known commercial porcine gata virus RT-PCR detection kit is 8.8369X 103copies/mu L, which shows that the sensitivity of the GETV-NSP1 fluorescent probe is far higher than that of the commercial swine Gata virus RT-PCR detection kit.
5. GETV-NSP1 fluorescent probe specificity test result
The specific result shows that the GETV fluorescent probe of the Liaoning pig can specifically amplify GETV cDNA to generate a specific amplification curve, the Ct value of a positive control is less than or equal to 28.0, a typical amplification curve is generated, and the negative control has no Ct value and no amplification curve. And the detection results of the known common pig pathogens are negative, which indicates that the Liaoning pig GETV NSP1 fluorescent probe method has good specificity.
6. GETV-NSP1 fluorescent probe repeatability test result
The intra-batch repeatability and the inter-batch repeatability of the GETV-NSP1 detection kit in detecting the samples are respectively evaluated through the calculation of the coefficient of variation values. The CV values in the measurement batches and among the measurement batches are 0.208-0.611% and 0.045-0.908% respectively (shown in Table 3), which indicates that the GETV fluorescent probe of Liaoning pigs has good repeatability.
TABLE 3 intra-and inter-batch variation as determined by Liaoning pig GETV NSP1 fluorescent Probe
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> detection kit for swine cottavirus NSP1 gene and detection method thereof
<130> 2021
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<170> PatentIn version 3.3
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<213> Artificial sequence
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<212> DNA
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Claims (7)
1. A detection kit for an NSP1 gene of a porcine Gaulta virus is characterized by comprising a specific primer and a fluorescent probe, wherein the sequence of an upstream primer in the specific primer is 5 '-CCATCCTGGACGTGGGTAGT-3, and the sequence of a downstream primer in the specific primer is 5' -TTTCGCCAGTTTTCGAGCGT-3; the fluorescent probe sequence is 5 '-FAM-ACCACACCTACCACTGCATCTGCCCCA-TAMRA-3'.
2. The kit for detecting the NSP1 gene of the porcine Gatas virus according to claim 1, wherein FAM labeled by the fluorescent probe is a fluorescent reporter group and TAMRA is a fluorescent quencher group.
3. The kit for detecting NSP1 gene of Sus domestica Galavirus according to claim 1, further comprising Probe Qpcr MIX.
4. A method for using the detection kit for the NSP1 gene of the porcine podavirus according to claim 1, characterized in that the method comprises the following steps:
1) extracting DNA or reverse transcription of a sample GETV-NSP1 gene to obtain cDNA, and constructing a sample GETV-NSP1-pBM16 clone plasmid as a template;
2) preparing a PCR reaction system by using the template, the Probe qPCR MIX, the specific primer, the fluorescent Probe and water, and carrying out PCR amplification reaction;
3) carrying out PCR amplification reaction under the same condition by taking a positive standard GETV-PBM16A as a template, and drawing a standard curve by taking a logarithmic value of the copy concentration of a standard solution as an abscissa and a Ct value of the standard solution as an ordinate;
4) when the Ct value of the sample is less than or equal to 28.0 and a typical amplification curve appears, the detection is positive;
5) and (4) according to the Ct value measured by the sample, contrasting with the standard curve to obtain the copy concentration of the sample.
5. The use method according to claim 4, wherein the PCR reaction conditions of step 2) or 3) are as follows: denaturation at 95 ℃ for 5min, amplification at 95 ℃ for 30sec and 50 ℃ for 20 sec were carried out for 40 cycles.
6. The use method of claim 4, wherein the concentration of the specific primer and the concentration of the fluorescent probe in the PCR system in step 2) or 3) are respectively 4umol/L and 6umol/L, respectively.
7. The use according to claim 4, wherein the minimum nucleic acid detection amount is 8.8369X 101copies/μL。
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