CN106148568B - Immunomagnetic bead kit and application thereof in detection of peste des petits ruminants virus antigen - Google Patents
Immunomagnetic bead kit and application thereof in detection of peste des petits ruminants virus antigen Download PDFInfo
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Abstract
The invention discloses an immunomagnetic bead kit and application thereof in detection of peste des petits ruminants virus antigen, belonging to the technical field of biology. The kit comprises rabbit anti-PPRV N protein IgG coupled immunomagnetic beads, a primer pair for detecting peste des petits ruminants virus antigen by taking PPRVM gene segments as targets, RT-PCR reaction mixed liquor and RNA polymerase. The sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. The invention overcomes the problems that PPRV pathogen is easy to degrade in the detection process of the peste des petits ruminants virus antigen, and false negative results occur because the extracted RNA cannot meet the minimum requirement of RT-PCR detection due to the limited volume of the venom to be detected in the extraction process of the pathogenic RNA, and provides an effective technical means for the detection of the peste des petits ruminants virus antigen.
Description
Technical Field
The invention relates to an immunomagnetic bead kit and application thereof, in particular to an immunomagnetic bead kit for detecting peste des petits ruminants virus antigen and application thereof, and belongs to the technical field of biology.
Background
Peste des petits ruminants (PPR) is also called as plague, mainly infects Peste des petits ruminants, particularly goats and sheep are highly susceptible, occasionally occur in wild animals, and is a serious acute, virulent and highly contagious disease caused by Peste des petits ruminants virus (PPRV), no infected people report at present, the morbidity and mortality of the infected people can reach 100 percent at most, the animal plague reported by the world animal health group (OIE) is listed as the animal plague reported by law, and the animal plague is also listed as a type of plague in China. The scheme for preventing and treating medium and long-term animal epidemic diseases in China (2012 and 2020) lists it as one of the 13 kinds of foreign animal epidemic diseases for important prevention. At present, the Peste des petits ruminants occur occasionally in most areas of China, seriously threaten the animal health safety of China and the development of the animal husbandry of China, and are one of animal epidemic diseases which need to be strictly prevented, controlled and extinguished.
The Immunomagnetic beads (IMB) technology is a new diagnostic technology based on antigen-antibody reaction and mediated by magnetic carrier technology. Due to the characteristics of rapidness, high efficiency, low toxicity, high specificity, simple operation and the like, the method is widely applied to the field of biomedicine. Jacobsen uses immunomagnetic bead technology to detect Listeria monocytogenes, and 3x10 in culture solution can be detected by sensitivity of Jacobsen4Bacteria with a recovery rate of95%, can well remove non-target bacteria in the mixed culture solution, and has good specificity. Yangwen and the like establish a method for rapidly and quantitatively detecting rotavirus in water by combining an immunomagnetic bead separation technology with real-time quantitative PCR, and successfully apply to the detection of the rotavirus in water by preparing specific immunomagnetic beads capable of separating the rotavirus in water. The method for combining immunomagnetic bead separation technology and impedance measurement technology is adopted in the small flies and the like, so that the specificity rapid detection of the avian influenza H5 subtype virus is realized. The research results show that the immunomagnetic bead separation technology can effectively separate and purify the Tazewski pathogen and provides a new idea for detecting recessive infection by serological antigen.
There are many conventional detection and diagnostic methods for PPRV infection, and the standard detection methods prescribed by OIE are competitive ELISA and RT-PCR methods. The competitive ELISA is mainly used as a serological detection method of the PPRV antibody, and because the vaccine immune antibody and the natural infection antibody cannot be distinguished and identified, the final diagnosis in the PPRV infection detection still needs to be matched with the antigen diagnosis of PCR to judge whether the PPRV infection occurs. PPRV is a single-stranded negative strand RNA virus, is sensitive to the external environment, and can be inactivated and degraded quickly at 50 ℃ for 30min or under the irradiation of sunlight. Because PCR detection cannot be carried out in the field, the virus content in the tissue pathological material is easily reduced in the process of collecting and storing the pathological material, so that a false negative detection result is caused.
The M protein is one of the main structural proteins of PPRV, plays a key role in the process of releasing mature virus particles from cells, viruses lose the capacity of infecting the cells under the condition of lacking the M protein, and the existing detection method aiming at the PPRV M gene is few.
Disclosure of Invention
Aiming at the problems that PPRV pathogen is easy to degrade in the peste des petits ruminants virus antigen detection process in the prior art, and the extracted RNA cannot meet the minimum requirement of RT-PCR detection due to the limited volume of the venom to be detected used in the pathogen RNA extraction process, and a false negative result occurs, on one hand, the invention designs a pair of specific primers which use PRRVM as a target point and can be used for PPRV detection, on the other hand, prepares a specific immunomagnetic bead which can be used for enriching PPRV pathogen, and finally establishes an immunomagnetic bead kit which can be used for detecting peste des petits ruminants virus antigen.
Specifically, in order to achieve the above purpose, the invention adopts the following technical means:
the invention relates to an immunomagnetic bead kit for detecting peste des petits ruminants virus antigen, which comprises immunomagnetic beads coupled with rabbit anti-PPRVN protein IgG, a primer pair for detecting peste des petits ruminants virus antigen by taking PPRV M gene fragments as targets, RT-PCR reaction mixed liquid and RNA polymerase, wherein the sequences of the primer pair are respectively shown as SEQ ID No.1 and SEQ ID No. 2.
In the invention, the concentration of the rabbit anti-PPRV N protein IgG coupled immunomagnetic beads is preferably 10 mg/mL.
In the present invention, preferably, the immunomagnetic bead kit further comprises a PBS buffer, and more preferably, the PBS buffer is 10mM and the pH value is 7.4.
In the present invention, preferably, the rabbit anti-PPRV N protein IgG-coupled immunomagnetic beads are prepared by the following method:
1) washing of magnetic beads: taking a magnetic bead stock solution in an EP tube, washing for 3 times by using a PBS buffer solution by utilizing the adsorption effect of a magnetic frame, and finally resuspending by using the PBS buffer solution;
2) coupling of the antibody: adding excessive rabbit anti-PPRV N protein IgG antibody into the magnetic beads after the heavy suspension in the step 1), uniformly mixing, and oscillating for 15-60min at 37 ℃ to obtain a suspension containing rabbit anti-PPRV N protein IgG coupled immunomagnetic beads;
3) separation of rabbit anti-PPRV N protein IgG-coupled immunomagnetic beads: adsorbing the suspension obtained in the step 2) by using a magnetic frame, removing the supernatant, washing the suspension for 3 times by using PBS (phosphate buffer solution), and finally resuspending the suspension by using the PBS buffer solution to obtain the rabbit anti-PPRV N protein IgG coupled immunomagnetic beads.
Wherein, preferably, the volume of the stock solution of the magnetic beads in the step 1) is 200 μ L, wherein the weight of the contained magnetic beads is 6mg, and the stock solution is resuspended by using 600 μ L of LPBS buffer solution; the dosage of the rabbit anti-PPRV N protein IgG antibody in the step 2) is more than 100 mu g.
Among them, the PBS buffer solution in step 1) and step 3) is preferably 10mM PBS buffer solution with pH value of 7.4.
Wherein, the shaking time in the step 2) is preferably 20 min.
Preferably, the final concentration of the rabbit anti-PPRV N protein IgG coupled immunomagnetic beads is 10 mg/mL.
Furthermore, the invention also discloses application of the immunomagnetic bead kit in detection of peste des petits ruminants virus antigen.
When the immunomagnetic bead kit is used for detecting peste des petits ruminants virus antigen, the method comprises the following steps:
1) and (3) enrichment of viruses: putting immunomagnetic beads into a 1.5mL EP tube, adding a sample to be detected, and carrying out oscillation reaction at 37 ℃ for 20-60 min;
2) washing of magnetic beads: after the completion of virus enrichment, placing an EP tube on a magnetic frame, discarding the supernatant, washing for 3 times by using PBS buffer solution, and then adding 2-5 times of PBS buffer solution to re-suspend the virus-enriched immunomagnetic beads;
3) extraction of viral RNA: destroying the virus structure by thermal cracking method, and releasing virus RNA;
4) and (3) PCR amplification: the kit is used for carrying out amplification of one-step RT-PCR;
5) the amplification results were analyzed by electrophoresis in a 1% agarose gel.
Preferably, the quantity of the immunomagnetic beads used for detecting each 400 μ L sample to be detected in the step 1) is 20 μ L, and the oscillation reaction time at 37 ℃ is 30 min; obtaining viral RN by thermal cracking adopted in step 2)The procedure for A was: cracking at 95 deg.C for 10min, and rapidly ice-cooling for 3 min; the amplification reaction system of the one-step RT-PCR in the step 3) is as follows: PrimeScript 1Step enzymeMix 2. mu.L; 2X 1Step Buffer 25. mu.L; PPRV RNA template 6 uL; RNase Free dH2O15 mu L; 1 mu L of upstream primer; the downstream primer was 1. mu.L, and the total volume was 50. mu.L.
The amplification procedure was: 30min at 50 ℃; 3min at 94 ℃; denaturation at 94 ℃ for 30s, renaturation at 57 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; after extension at 72 ℃ for 10min, the cells were stored at 4 ℃.
The invention overcomes the problems that PPRV pathogen is easy to degrade in the detection process of the peste des petits ruminants virus antigen, and false negative results occur because the extracted RNA cannot meet the minimum requirement of RT-PCR detection due to the limited volume of the venom to be detected in the extraction process of the pathogenic RNA, and provides an effective technical means for the detection of the peste des petits ruminants virus antigen.
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FIG. 1 shows the purification of rabbit anti-PPRV N protein serum IgG;
m: pre-dyeing a protein Marker; 1: eluent 1; 2: eluent 2; 3: eluent 3;
FIG. 2 shows the results of detection of the specificity of rabbit anti-PPRV N protein IgG coupled immunomagnetic beads by SDS-PAGE;
1: products after binding PPRV-N protein; 2: PPRV-N protein as such; 3: product in combination with PPRV vaccine dilutions: 4: PPRV vaccine diluent;
FIG. 3 shows the result of RT-PCR amplification;
m: DNA Marker DL 2000; 1-5: PPRV RNA amplification result; 6: negative control
FIG. 4 shows the result of RT-PCR amplification (sensitivity detection) performed after enrichment of rabbit anti-PPRV N protein IgG-coupled immunomagnetic beads.
M is DNA Marker DL 2000; 1, negative control; 2: 3mL of vaccine diluent; 3:2mL of vaccine diluent; 4-9: 1mL of the diluted vaccine is sequentially diluted by ten times.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. However, the examples are only for illustrating the present invention and do not set any limit to the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 design and Synthesis of primer pairs for detecting Peste des petits ruminants Virus antigens targeting PPRV M Gene fragments
The inventor designs a specific primer pair aiming at the M gene according to the whole genome sequence (accession number: HQ197753.1) of the PPRV Nigeria 75/1 strain in GenBank, wherein the sequences of the primer pair are as follows:
upstream primer 1-up: ATGACCGAGATCTACGATT (shown in SEQ ID NO. 1);
downstream primer 1-low: ACAGGATCTTGAACAGGCC (shown in SEQ ID NO. 2).
The primer pair was synthesized by bio-corporation.
Example 2 preparation of rabbit anti-PPRV N protein hyperimmune serum
Specific primers for the H gene were designed based on the whole genome sequence of the PPRV Nigeria 75/1 strain in GenBank (accession number: HQ197753.1), and the primers were synthesized by Biometrics. The primer sequences are as follows:
PPRV-N up: 5'-CCGGAATTCATGGCTACTCTCCTTAAA-3' (shown in SEQ ID NO. 3)
5'-ATTTGCGGCCGCTCAGCTGAGGAGATCCTT-3' (shown in SEQ ID NO. 4) is PPRV-N down
The N gene is obtained by amplification with a peste des petits ruminants virus vaccine (the vaccine is purchased from Xinjiang Tiankang animal biotechnology, Inc.) as a template, and the N protein is obtained by prokaryotic expression and purification.
10 healthy adult male rabbits (New Zealand white rabbits) with the weight of about 2kg are selected, and the subcutaneous immunogen nucleus at the neck, the back and the inner side of the thigh of the hind limb expresses the purified PPRV N protein.
The specific immunization program is as follows: collecting blood from ear marginal vein of rabbit before first immunization, mixing with Freund's complete adjuvant and target protein in equal amount, and emulsifying with emulsifier to obtain immune dose of about 5mg for each rabbit; 2 weeks after the first immunization, performing blood sampling on the ear marginal vein to detect the antibody level, and performing boosting immunization, wherein the immunization dose is increased to about 10mg for each rabbit; collecting blood respectively at 2 weeks and 3 weeks after the second immunization for antibody detection, and performing third immunization when the titer of the antibody reaches the highest value and begins to decrease, wherein the immune amount is the same as that of the second immunization; and (3) blood is collected one week after the three-immunization to detect the antibody titer, and if the ELISA method detection titer reaches 1:6400, the rabbit anti-PPRV N protein hyperimmune serum is obtained by heart blood collection.
Example 3 preparation of rabbit anti-PPRV N protein IgG coupled immunomagnetic beads
Purification of rabbit anti-PPRV N protein hyperimmune serum IgG:
the method specifically comprises the following operation steps of an AKTA FPLC protein purification system of Swedish FamaXia company:
1) respectively putting infusion inlets (A, B) of the instrument into corresponding required buffer solutions, starting a computer, selecting UNICON software, selecting a manual pump wash FPLC to select an inlet A used in a system control interface, and executing functions to wash and purify pipelines and pumps of the system;
2) connecting a protein G affinity chromatography protein purification column;
3) system setting: in a system control interface, selecting a 'manual pump Flow' input Flow rate, and inputting the pressure of a pump in 'inert alarm & monalarm pressure';
4) and (3) balancing and purifying the column: equilibrating the pH of the purification column with 1M Tris-HCl buffer, pH 9.0;
5) loading: rabbit sera immunized with PPRV N protein were made with distilled water as 1: 5 after dilution, adding 10mL of Binding Buffer into a sample adding tube, controlling the conductance of the Binding Buffer at 50-60mS/cm, pH at 7.0-7.4, controlling the flow rate at 1.0mL/min, flowing through for about 0.5h, collecting the flow-through liquid, and collecting 1 tube per 2 mL.
6) And (3) elution: after the absorption peak is stable, adding an Elution Buffer, controlling the pH value of the Elution Buffer to be about 2.5-3.0, and controlling the flow rate to be 0.75mL/min, observing the absorption peak, controlling a sample receiver to receive eluent when the absorption peak rises rapidly, collecting one tube per 2mL of eluent when collecting the eluent, sequentially naming the tube as eluent 1, immediately adding a Tris-HCl Buffer solution with the pH value of 9.0 to neutralize the pH value of the eluent after the eluent 2 … … is collected, and keeping the pH value of the eluent at neutral or alkalescent.
7) After the elution is finished, the pump and the pipeline are washed by distilled water, and then the pump and the pipeline are continuously washed by 20 percent ethanol.
8) The recovered purified IgG concentration was determined by NanoDrop2000A280 and the purified IgG purity was analyzed by SDS-PAGE, and the purification results are shown in FIG. 1.
Preparation of (II) rabbit anti-PPRV N protein IgG coupled immunomagnetic beads
1) Washing of magnetic beads: mu.L (about 6mg) of stock solution of magnetic beads (purchased from life Bio Inc.) was taken in an EP tube, washed 3 times with PBS buffer by magnetic frame adsorption, and finally resuspended in 600. mu.L of PBS buffer.
2) Coupling of the antibody: adding excessive anti-PPRV N protein IgG antibody (more than 100 mu g) into the magnetic bead suspension obtained in the step 1), uniformly mixing, and oscillating for 20min at 37 ℃ to obtain a suspension containing rabbit anti-PPRV N protein IgG coupled immunomagnetic beads;
3) separation of rabbit anti-PPRV N protein IgG-coupled immunomagnetic beads: adsorbing the suspension obtained in the step 2) by using a magnetic frame, removing the supernatant, washing the suspension for 3 times by using PBS (phosphate buffer solution), and finally resuspending the suspension by using the PBS buffer solution to obtain the rabbit anti-PPRV N protein IgG coupled immunomagnetic beads with the concentration of 10 mg/mL.
Wherein the PBS buffer used is 10mM PBS buffer with pH value of 7.4.
(III) specific detection of rabbit anti-PPRV N protein IgG coupled immunomagnetic beads:
1) 50 μ L of each prepared immunomagnetic bead was placed in 3 1.5mL EP tubes, and 200 μ L of the unpurified PPRV N protein and 1:10 times diluted Peste des petits ruminants live vaccine (the vaccine is purchased from Xinjiang Tiankang animal biotechnology, Inc., and the virus content is about 10)4TCID50/mL). Sucking and uniformly mixing by using a liquid transfer gun, reacting at 37 ℃ for 2h or reacting overnight at 4 ℃ (uniformly mixing by using the liquid transfer gun irregularly in the reaction process);
2) after full reaction, taking out the EP tube, placing the EP tube on a magnetic frame for adsorption for 2min, and completely sucking out the supernatant by using a pipette;
3) taking off the EP tube from the magnetic frame, adding 1mL of PBS with pH 7.4 for washing, after fully mixing, putting the tube on the magnetic frame again for adsorbing for 2min, sucking out all the supernatant by a pipette gun, repeating the washing for 3-5 times, finally resuspending the magnetic beads by 200 muL of PBS with pH 7.4, sucking part of the suspension, adding 4 x protein electrophoresis sample loading buffer solution, directly carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis without boiling, and the result is shown in figure 2, which shows that the immunomagnetic beads can well combine PPRV N protein and PPRV whole virus.
Example 4 preparation of immunomagnetic bead kit for detecting Peste des petits ruminants Virus antigen
The kit comprises rabbit anti-PPRV N protein IgG coupled immunomagnetic beads (10mg/mL, prepared in example 2), a primer pair (50pM) for detecting peste des petits ruminants virus antigen by taking a PPRVM gene fragment as a target spot (shown in SEQ ID NO.1 and SEQ ID NO.2 respectively), PBS Buffer solution (10mM, pH value of 7.4), 2 x 1Step Buffer and PrimeScript 1Step Enzyme Mix.
Example 5 use of the kit of the invention for detecting Peste des petits ruminants virus antigen
1. Test sample
1:10 times diluted peste des petits ruminants virus vaccine. (the vaccine is purchased from Xinjiang Tiankang animal biotechnology GmbH, and the virus content is about 10)4TCID50/mL)
2. Method of producing a composite material
1) And (3) enrichment of viruses: 20 mu L of rabbit anti-PPRV N protein IgG coupled immunomagnetic beads (10mg/mL) are put into a 1.5mL EP tube, 400 mu L of diluted peste des petits ruminants virus vaccine is added, and the concussion reaction is carried out for 30min at 37 ℃;
2) washing of magnetic beads: after the completion of virus enrichment, the EP tube is placed on a magnetic frame, the supernatant is discarded, the EP tube is washed for 3 times by PBS buffer solution, and then 1.2ml of PBS buffer solution (10mM, pH value of 7.4) is added to resuspend the virus-enriched immunomagnetic beads;
3) extraction of viral RNA: destroying the virus structure by thermal cracking method, and releasing virus RNA; the procedure for obtaining viral RNA by thermal cracking is as follows: cracking at 95 deg.C for 10min, and rapidly ice-cooling for 3 min;
4) and (3) PCR amplification: amplification of one-step RT-PCR was performed using the kit described in example 4;
the amplification reaction system of the one-step RT-PCR is as follows:
the amplification procedure was as follows:
5) the amplification results were analyzed by electrophoresis in a 1% agarose gel.
The results are shown in figure 3, which shows that the kit of the invention can well amplify peste des petits ruminants virus and has good repeatability.
Example 6: sensitivity test of the kit of the present invention
The peste des petits ruminants virus vaccine is diluted by 1:10 times (the virus content is about 10)3TCID50/mL), 1mL of virus diluent was diluted ten-fold, 10 each0、10-1、10-2、10-3、10-4、10-5Adding 3mL of vaccine dilution stock solution, 2mL of vaccine dilution stock solution and 1mL of 10 vaccine dilution stock solution respectively0、10-1、10-2、10-3、10-4、10-5The ten fold ratio of the diluent of the vaccine is enriched by rabbit anti-PPRV N protein IgG coupled immunomagnetic beads, viral genome RNA is extracted, RT-PCR is carried out, the method is the same as example 5, and the result is shown in figure 4. The result shows that the lowest detectable sample virus concentration by adopting the kit of the invention is 0.1TCID50/mL。
Claims (11)
1. An immunomagnetic bead kit for detecting peste des petits ruminants virus antigen is characterized by comprising rabbit anti-PPRV N protein IgG coupled immunomagnetic beads, a primer pair for detecting peste des petits ruminants virus antigen by taking PPRVM gene fragments as targets, RT-PCR reaction mixed liquid and RNA polymerase, wherein the sequences of the primer pair are respectively shown as SEQ ID No.1 and SEQ ID No. 2.
2. The immunomagnetic bead kit of claim 1, wherein the kit further comprises a PBS buffer.
3. The immunomagnetic bead kit of claim 2, wherein the PBS buffer is 10mM PBS buffer with pH 7.4.
4. The immunomagnetic bead kit of claim 1, wherein the concentration of the rabbit anti-PPRV N protein IgG-conjugated immunomagnetic beads is 10 mg/mL.
5. The immunomagnetic bead kit of claim 1, wherein the rabbit anti-PPRV N protein IgG-coupled immunomagnetic beads are prepared by the following method:
1) washing of magnetic beads: taking a magnetic bead stock solution in an EP tube, washing for 3 times by using a PBS buffer solution by utilizing the adsorption effect of a magnetic frame, and finally resuspending by using the PBS buffer solution;
2) coupling of the antibody: adding excessive rabbit anti-PPRV N protein IgG antibody into the magnetic beads after the heavy suspension in the step 1), uniformly mixing, and oscillating for 15-60min at 37 ℃ to obtain a suspension containing rabbit anti-PPRV N protein IgG coupled immunomagnetic beads;
3) separation of rabbit anti-PPRV N protein IgG-coupled immunomagnetic beads: adsorbing the suspension obtained in the step 2) by using a magnetic frame, removing the supernatant, washing the suspension for 3 times by using PBS (phosphate buffer solution), and finally resuspending the suspension by using the PBS buffer solution to obtain the rabbit anti-PPRV N protein IgG coupled immunomagnetic beads.
6. The immunomagnetic bead kit of claim 5, wherein the volume of the stock solution of the magnetic beads in step 1) is 200 μ L, wherein the weight of the contained magnetic beads is 6mg, and 600 μ L PBS buffer solution is used for resuspension; the dosage of the rabbit anti-PPRV N protein IgG antibody in the step 2) is more than 100 mu g.
7. The immunomagnetic bead kit of claim 5, wherein the PBS buffer in step 1) and step 3) is 10mM PBS buffer with pH 7.4.
8. The immunomagnetic bead kit of claim 5, wherein the shaking time in step 2) is 20 min.
9. The immunomagnetic bead kit of claim 5, wherein the final concentration of the rabbit anti-PPRV N protein IgG-conjugated immunomagnetic beads is 10 mg/mL.
10. An immunomagnetic bead kit according to any of claims 1-9, characterized in that for the detection of peste des petits ruminants virus antigens, the following steps are performed:
1) and (3) enrichment of viruses: putting immunomagnetic beads into a 1.5mL EP tube, adding a sample to be detected, and carrying out oscillation reaction at 37 ℃ for 20-60 min;
2) washing of magnetic beads: after the completion of virus enrichment, placing an EP tube on a magnetic frame, discarding the supernatant, washing for 3 times by using PBS buffer solution, and then adding 2-5 times of PBS buffer solution to re-suspend the virus-enriched immunomagnetic beads;
3) extraction of viral RNA: destroying the virus structure by thermal cracking method, and releasing virus RNA;
4) and (3) PCR amplification: performing one-step RT-PCR amplification by using the kit as described in claim 1;
5) the amplification results were analyzed by electrophoresis in a 1% agarose gel.
11. The immunomagnetic bead kit according to claim 10, wherein the amount of immunomagnetic beads used in the step 1) per 400 μ L of the sample to be detected is 20 μ L, and the reaction time is 30min at 37 ℃ with shaking; the thermal cracking method adopted in the step 2) is as follows: crack at 95 ℃Performing rapid ice-bath for 3min after 10 min; the amplification reaction system of the one-step RT-PCR in the step 3) is as follows: PrimeScript 1Step Enzyme Mix 2. mu.L; 2X 1Step Buffer 25. mu.L; PPRV RNA template 6 uL; RNase Free dH2O15 mu L; 1 mu L of upstream primer; 1 mu L of downstream primer and 50 mu L of total volume;
the amplification procedure was: 30min at 50 ℃; 3min at 94 ℃; denaturation at 94 ℃ for 30s, renaturation at 57 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; after extension at 72 ℃ for 10min, the cells were stored at 4 ℃.
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| CN102230938A (en) * | 2011-06-22 | 2011-11-02 | 中国科学院武汉病毒研究所 | Kit and method for detecting influenza A virus based on immune magnetic bead enrichment |
| CN103224914A (en) * | 2013-05-17 | 2013-07-31 | 中国农业科学院哈尔滨兽医研究所 | Construction and application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein |
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| CN102230938A (en) * | 2011-06-22 | 2011-11-02 | 中国科学院武汉病毒研究所 | Kit and method for detecting influenza A virus based on immune magnetic bead enrichment |
| CN103224914A (en) * | 2013-05-17 | 2013-07-31 | 中国农业科学院哈尔滨兽医研究所 | Construction and application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein |
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