Triple PCR (Polymerase Chain Reaction) detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus

Abstract

The invention belongs to the technical field of biological detection, and provides a bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus triple PCR detection method which comprises the following steps: comparing nucleotide sequences of BVDV, BEV and BPV to obtain a gene conserved region, and constructing three pairs of specific primers; rNA of BVDV, BEV and BPV cell viruses is extracted respectively; taking the cDNA of the BVDV and the BEV and the DNA of the BPV as templates, and respectively carrying out single PCR (Polymerase Chain Reaction) amplification by utilizing the constructed three pairs of specific primers; carrying out plasmid construction and identification on the BVDV, the BEV and the BPV; establishing and optimizing a triple PCR reaction system; according to the invention, a triple PCR detection method is constructed through 5 'UTR gene sequences of BVDV and BEV and a VP2 gene sequence of BPV, and infection conditions of three cattle pathogens can be mastered only through one-time detection, so that the detection efficiency is greatly improved, the sample treatment time is shortened, and the detection cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, in particular to a triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus. Background Art

[0002] Bovine viral diarrhea virus (BVDV for short) belongs to the Flaviviridae family and the Pestivirus genus. The clinical symptoms caused by this virus are diverse and severe, mainly including fever, erosion and ulcers of the digestive system and nasal mucosa, diarrhea, and abortion and fetal abnormalities in cows. These symptoms not only seriously affect the health of cattle, but also greatly reduce their production performance. At present, bovine viral diarrhea virus has been widely distributed around the world, especially in countries with a high level of animal husbandry development. The prevalence of this disease has caused a huge impact on the cattle industry, resulting in serious economic losses and affecting the market supply and price stability of beef and dairy products;

[0003] Bovine enterovirus (BEV) is a non-enveloped single-stranded positive-sense RNA virus belonging to the Picornaviridae family and the Enterovirus genus. Clinically, BEV is mainly pathogenic to the respiratory and digestive systems of cattle. In severe cases, it may also invade the reproductive system and cause infertility. BEV is extremely common in clinical practice. It can infect cattle alone, or co-infect with other pathogens or appear as a secondary infection, thereby causing a series of clinical symptoms, posing a serious threat to the healthy development of the global cattle industry.

[0004] Bovine parvovirus (BPV for short) belongs to the Parvoviridae family. Currently known viruses of the same genus include human bocavirus, canine parvovirus, porcine bocavirus, gorilla bocavirus, California sea lion bocavirus, feline bocavirus, and canine bocavirus, all of which can produce similar symptoms. BPV can be transmitted vertically, that is, it can cross the placental barrier and be transmitted directly from the pregnant cow to the fetus. This process often leads to miscarriage and stillbirth in the cow. In addition, for newborn calves, once infected with BPV, their digestive system may be significantly affected, showing symptoms of digestive dysfunction, which poses a serious threat to the healthy growth of calves.

[0005] PCR technology is an efficient detection technology. With its fast detection speed, high sensitivity and significant specificity, it plays a pivotal role in gene cloning, medical identification, DNA sequence analysis and pathogenic gene screening. At the same time, this technology also provides a powerful platform for the rapid detection of microorganisms. Through PCR technology, researchers can quickly amplify specific DNA fragments, thereby achieving accurate recognition and identification of microorganisms.

[0006] BVDV, BEV, and BPV can all cause symptoms such as diarrhea and stunted growth in cattle and often spread rapidly among cattle, causing serious economic losses to the animal husbandry industry. These viruses not only affect the health of cattle, but also reduce their production performance, significantly increasing breeding costs. At present, scholars have reported PCR detection methods for BVDV, BEV, and BPV, but a method for simultaneously detecting and distinguishing BVDV, BEV, and BPV triple PCR has not been reported. When faced with mixed infections of multiple viruses, traditional single virus detection methods need to be tested separately, which is time-consuming, labor-intensive, and costly.

[0007] To this end, technicians in this field have proposed a triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus, aiming to simultaneously detect BVDV, BEV and BPV, improve detection efficiency, and save time and cost. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus to solve the problems raised in the background technology.

[0009] A triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus comprises the following steps:

[0010] S1. Download the sequences of BVDV, BEV, and BPV from GenBank, use Megalign to compare the nucleotide sequences, obtain the conserved regions of the genes, and construct three pairs of specific primers using Primer premier6.0;

[0011] S2, extracting RNA of BVDV, BEV, and BPV cell viruses respectively;

[0012] S3, using the cDNA of BVDV, BEV and DNA of BPV as templates, single PCR amplification was performed using the three pairs of specific primers constructed;

[0013] S4. Take 4 μL of the single PCR amplification product obtained in S3 and perform electrophoresis on a 1.5% agarose gel at a constant voltage of 120 V and a current of 100 mA for 35 minutes. Cut the correct target fragment from the agarose gel and recover it using a gel recovery kit. If a single band of the correct size appears on the gel recovery product after 1.5% agarose gel electrophoresis, it is considered to be recovered successfully.

[0014] S5. Construct recombinant cloning plasmids with the target genes successfully recovered from the gel. The ligation system is 10 μL, 4 μL of gel recovery product, 5 μL of Solution Buffer, and 1 μL of pMTM 19-T Vector. The reaction system is fully pipetted and mixed in a PCR reaction tube and ligated overnight at 4°C.

[0015] S6. Transform and identify the recombinant clone plasmid, and obtain a positive bacterial solution with a correct sequencing result through amplification;

[0016] S7. Take 4 mL of the amplified positive bacterial solution respectively, extract the BVDV, BEV, and BPV recombinant plasmids, name them pMD19-T-BVDV, pMD19-T-BEV, and pMD19-T-BPV, respectively, and dilute the plasmids to 5 ng / μL, and store them in a -20°C refrigerator for later use;

[0017] S8. Mix equal volumes of plasmid diluted to 5 ng / μL as templates, use analysis software Primerpremier6.0 to determine the theoretical Tm values ​​of three pairs of primers, set the annealing temperature to 58.9°C as the optimal annealing temperature for BVDV, BEV, and BPV, and use the chessboard method to optimize the volumes of upstream and downstream primers of the three viruses to determine the optimal primer volume; determine the optimal number of cycles by comparing the effects of different numbers of cycles on amplification efficiency.

[0018] Preferably, the extraction step of step S2 is:

[0019] S201, take a 1.5ml centrifuge tube and add 20μL Proteinase K;

[0020] S202, inject 200 μL of serum or plasma sample into the centrifuge tube, then add an equal amount of 200 μL of Buffer GL solution, and use a vortex oscillator to mix thoroughly for 15 seconds to evenly blend the sample and the buffer solution;

[0021] S203, placing the centrifuge tube in a 56° C. water bath and heating it for 15 minutes to release the nucleic acid in the sample, and then briefly centrifuging it to allow the solution on the tube wall to completely settle to the bottom of the tube;

[0022] S204, adding 250 μL of anhydrous ethanol to the centrifuge tube, mixing again using a vortex oscillator for 15 seconds, leaving the centrifuge tube at room temperature for 5 minutes to fully precipitate impurities, and then briefly centrifuging to allow the solution to completely settle to the bottom of the tube;

[0023] S205, transferring the solution obtained in S204 to the adsorption column loaded into the collection tube, centrifuging at a speed of 12000 rpm for 1 minute, pouring out the waste liquid, and putting the adsorption column back into the collection tube;

[0024] S206, adding 500 μL of Buffer GW1 solution to the adsorption column, adding anhydrous ethanol before use, centrifuging again at a speed of 12000 rpm for 1 minute, pouring out the waste liquid, and placing the adsorption column back into the collection tube;

[0025] S207, repeat the operation of S206, use Buffer GW2 solution for washing, centrifuge at a speed of 12000 rpm for 1 minute, pour out the waste liquid, and put the adsorption column back into the collection tube;

[0026] S208, adding 500 μL of anhydrous ethanol to the adsorption column again, centrifuging at 12000 rpm for 1 minute, discarding the waste liquid, and placing the adsorption column back into the collection tube for the final washing;

[0027] S209. Centrifuge the adsorption column at 12,000 rpm for 3 minutes to completely remove the residual waste liquid, place the adsorption column at room temperature for several minutes to completely dry it, then transfer the adsorption column to a new collection tube, add 20-150 μL of Buffer RE or sterile water to the middle part of the adsorption column membrane, let it stand at room temperature for 2 to 5 minutes to fully dissolve the nucleic acid, and centrifuge it at 12,000 rpm for 1 minute to collect the resulting nucleic acid solution.

[0028] Preferably, the reaction conditions for the single PCR amplification in step S3 are: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s; annealing temperature at 50°C to 60°C for 30 s; extension at 72°C for 20 s; final extension at 72°C for 2 min; 35 cycles in total, stored at 4°C, and the amplified product was detected by 1.5% agarose gel electrophoresis.

[0029] Preferably, the step of glue recovery in step S4 is:

[0030] S401. Cut out the agarose gel containing the target DNA under ultraviolet light, chop the gel block, and calculate the volume of the gel block as 1 mg = 1 μL;

[0031] S402, adding Buffer GM to the gel block, the amount added is based on the gel concentration and volume: 1.0% plus 3 gel volumes; 1.0% to 1.5% plus 4 gel volumes; 1.5% to 2.0% plus 5 gel volumes;

[0032] S403, dissolve the rubber block at room temperature of 15-25°C after uniform mixing, and use intermittent oscillation mixing to fully dissolve the rubber block;

[0033] S404, placing the Spin Column in the reagent kit on the Collection Tube;

[0034] S405, transferring the solution obtained in S403 to a Spin Column, centrifuging at 12,000 rpm for 1 minute, and discarding the filtrate;

[0035] S406, add 700 μL of Buffer WB to the Spin Column, centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the filtrate;

[0036] S407, place the Spin Column on the Collection Tube, and centrifuge at 12,000 rpm for 1 minute at room temperature;

[0037] S408. Place the Spin Column in a new 1.5 ml centrifuge tube, add 30 μL Elution Buffer to the center of the Spin Column membrane, and let stand at room temperature for 1 minute;

[0038] S409, centrifuge at 12,000 rpm for 1 minute at room temperature to elute the DNA.

[0039] Preferably, the step S6 of transforming and identifying the recombinant clone plasmid is:

[0040] S601. Take out 100 μL of DH5α competent cells from the -80°C refrigerator, place on ice to melt, add 10 μL of ligation product, and place on ice for 30 minutes;

[0041] S602, place in a water bath preheated to 42°C for 45 seconds and immediately cool on ice for 2 minutes;

[0042] S603. Under sterile conditions, add 890 μL of SOC liquid culture medium preheated to 37°C, mix well, and culture on a shaker at 37°C for 1 h.

[0043] S604, spread on LB solid medium containing ampicillin, spread evenly, and after all the bacterial solution is absorbed, incubate inverted at 37°C for 12 to 14 hours;

[0044] S605. Randomly select a single colony on the solid culture medium and inoculate it into 4 ml of LB liquid culture medium, culture it at 37°C with shaking overnight, perform PCR verification on the bacterial solution using BVDV-F / R, BEV-F / R, and BPV-F / R as primers, and sequence the positive bacterial solution.

[0045] Preferably, step S7 further uses a full-wavelength multifunctional microplate reader to measure the plasmid OD260 / OD280 value, and calculates the standard plasmid copy number according to the copy number formula (copies / μL)=(6.02×1023)×(ng / μL×1 0-9) / (DNA length×660).

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. The present invention constructs a triple PCR detection method through the 5'UTR gene sequence of BVDV and BEV, and the VP2 gene sequence of BPV, and optimizes the reaction system and reaction conditions of the method so that it can diagnose and detect BVDV, BEV and BPV at the same time. The technology shows a high degree of specificity. During the detection process of Huni virus, MRV, Escherichia coli, Bacillus cereus and Clostridium difficile, no cross reaction was observed.

[0048] 2. The triple PCR method established in the present invention can simultaneously detect BVDV, BEV and BPV, and the infection status of the three cattle pathogens can be grasped with only one test, which greatly improves the detection efficiency, shortens the sample processing time, reduces the detection cost, and provides valuable epidemic monitoring information for the development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a diagram of the positive sample cDNA result of the present invention, wherein M: DL2000 DNA marker; 1: negative control 2: BPV; 3: BEV; 4: BVDV.

[0050] Figure 2 This is the triple PCR annealing temperature optimization diagram of the present invention, wherein M: DL2000 DNA marker; 1: 50°C; 2: 52.2°C; 3: 54.4°C; 4: 55.6°C; 5: 56.8°C; 6: 57.9°C; 7: 58.9°C; 8: 60°C; 9: Nuclease-Free Water.

[0051] Figure 3 This is a triple PCR primer volume optimization diagram of the present invention, wherein M: DL2000 DNA marker; 1: Nuclease-Free Water; 2-10: primer final volume 0.2-1.0 μL increased by 0.1 μL.

[0052] Figure 4This is the optimization diagram of the triple PCR cycle number of the present invention, wherein M: DL2000 DNA marker; 1-7: 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles; 8: Nuclease-Free Water.

[0053] Figure 5 This is a triple PCR specificity test diagram of the present invention, wherein M: DL2000 DNA marker; 1: Nuclease-Free Water; 2: Positive control; 3: BVDV; 4: BEV; 5: BPV; 6: Escherichia coli; 7: BufHuV; 8: MRV; 9: Bacillus cereus; 10: Bacillus fusoniformis.

[0054] Figure 6 This is a triple PCR sensitivity test diagram of the present invention, wherein M: DL2000 DNA marker; 1-11: 10 0 ~10 10 Dilute recombinant plasmid mixed template; 12: Nuclease-Free Water

[0055] Figure 7 This is a graph of the triple PCR repeatability test of the present invention, wherein M: DL2000 DNA marker; 1, 6, 11: Nuclease-Free Water; 2, 7, 12: positive control; 3-5: first amplification result; 7-10: second amplification result; 13-15: third amplification result. DETAILED DESCRIPTION

[0056] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0057] Embodiment: The present invention provides a triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus, comprising the following steps:

[0058] S1. Download the sequences of BVDV, BEV, and BPV from GenBank, use Megalign to compare the nucleotide sequences, obtain the conserved regions of the genes, and construct three pairs of specific primers using Primer premier6.0; the sequence information of the triple PCR primers is shown in Table 1 below:

[0059] Table 1 Triple PCR primer sequence information

[0060]

[0061] 2. Extract RNA of BVDV, BEV, and BPV cell viruses respectively; the extraction steps are as follows:

[0063] S201, take a 1.5ml centrifuge tube and add 20μL Proteinase K;

[0064] S202, inject 200 μL of serum or plasma sample into the centrifuge tube, then add an equal amount of 200 μL of Buffer GL solution, and use a vortex oscillator to mix thoroughly for 15 seconds to evenly blend the sample and the buffer solution;

[0065] S203, placing the centrifuge tube in a 56° C. water bath and heating it for 15 minutes to release the nucleic acid in the sample, and then briefly centrifuging it to allow the solution on the tube wall to completely settle to the bottom of the tube;

[0066] S204, adding 250 μL of anhydrous ethanol to the centrifuge tube, mixing again using a vortex oscillator for 15 seconds, leaving the centrifuge tube at room temperature for 5 minutes to fully precipitate impurities, and then briefly centrifuging to allow the solution to completely settle to the bottom of the tube;

[0067] S205, transferring the solution obtained in S204 to the adsorption column loaded into the collection tube, centrifuging at a speed of 12000 rpm for 1 minute, pouring out the waste liquid, and putting the adsorption column back into the collection tube;

[0068] S206, adding 500 μL of Buffer GW1 solution to the adsorption column, adding anhydrous ethanol before use, centrifuging again at a speed of 12000 rpm for 1 minute, pouring out the waste liquid, and placing the adsorption column back into the collection tube;

[0069] S207, repeat the operation of S206, use Buffer GW2 solution for washing, centrifuge at a speed of 12000 rpm for 1 minute, pour out the waste liquid, and put the adsorption column back into the collection tube;

[0070] S208, adding 500 μL of anhydrous ethanol to the adsorption column again, centrifuging at 12000 rpm for 1 minute, discarding the waste liquid, and placing the adsorption column back into the collection tube for the final washing;

[0071] S209. Centrifuge the adsorption column at 12,000 rpm for 3 minutes to completely remove the residual waste liquid, place the adsorption column at room temperature for several minutes to completely dry it, then transfer the adsorption column to a new collection tube, add 20-150 μL of Buffer RE or sterile water to the middle part of the adsorption column membrane, let it stand at room temperature for 2 to 5 minutes to fully dissolve the nucleic acid, and centrifuge it at 12,000 rpm for 1 minute to collect the resulting nucleic acid solution.

[0072] S3. Using the cDNA of BVDV, BEV and DNA of BPV as templates, single PCR amplification was performed respectively using the three pairs of specific primers constructed according to the reaction system in Table 2; the reaction conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s; annealing temperature at 50°C-60°C for 30 s; extension at 72°C for 20 s; final extension at 72°C for 2 min; 35 cycles in total, stored at 4°C, and the amplified products were detected by 1.5% agarose gel electrophoresis.

[0073] Table 2 PCR reaction system

[0074]

[0075] like Figure 1 As shown, a pair of primers for BVDV, BEV and BPV were designed and screened according to the conserved gene sequence, and the cDNA / DNA of BVDV, BEV and BPV were extracted as reaction templates to amplify specific bands consistent with the size of the target fragment, with corresponding lengths of 467bp, 350bp and 257bp, respectively. The amplified products were sequenced and compared using the alignment tool of NCBI. The results showed that the amplified specific genes of BVDV, BEV and BPV were sequenced correctly.

[0076] S4. Take 4 μL of the single PCR amplification product obtained in S3 and perform electrophoresis on a 1.5% agarose gel at a constant voltage of 120 V and a current of 100 mA for 35 minutes. Cut the correct target fragment from the agarose gel and recover it using a gel recovery kit. If a single band of the correct size appears after 1.5% agarose gel electrophoresis, the recovery is considered successful. The steps of gel recovery are as follows:

[0077] S401. Cut out the agarose gel containing the target DNA under ultraviolet light, chop the gel block, and calculate the volume of the gel block as 1 mg = 1 μL;

[0078] S402, adding Buffer GM to the gel block, the amount added is based on the gel concentration and volume: 1.0% plus 3 gel volumes; 1.0% to 1.5% plus 4 gel volumes; 1.5% to 2.0% plus 5 gel volumes;

[0079] S403, dissolve the rubber block at room temperature of 15-25°C after uniform mixing, and use intermittent oscillation mixing to fully dissolve the rubber block;

[0080] S404, placing the Spin Column in the reagent kit on the Collection Tube;

[0081] S405, transferring the solution obtained in S403 to a Spin Column, centrifuging at 12,000 rpm for 1 minute, and discarding the filtrate;

[0082] S406, add 700 μL of Buffer WB to the Spin Column, centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the filtrate;

[0083] S407, place the Spin Column on the Collection Tube, and centrifuge at 12,000 rpm for 1 minute at room temperature;

[0084] S408. Place the Spin Column in a new 1.5 ml centrifuge tube, add 30 μL Elution Buffer to the center of the Spin Column membrane, and let stand at room temperature for 1 minute;

[0085] S409, centrifuge at 12,000 rpm for 1 minute at room temperature to elute the DNA.

[0086] S5. Construct recombinant cloning plasmids with the target genes successfully recovered from the gel. The ligation system is 10 μL, 4 μL of gel recovery product, 5 μL of Solution Buffer, and 1 μL of pMTM 19-T Vector. The reaction system is fully pipetted and mixed in a PCR reaction tube and ligated overnight at 4°C.

[0087] S6. Transform and identify the recombinant clone plasmid, the steps are:

[0088] S601. Take out 100 μL of DH5α competent cells from the -80°C refrigerator, place on ice to melt, add 10 μL of ligation product, and place on ice for 30 minutes;

[0089] S602, place in a water bath preheated to 42°C for 45 seconds and immediately cool on ice for 2 minutes;

[0090] S603. Under sterile conditions, add 890 μL of SOC liquid culture medium preheated to 37°C, mix well, and culture on a shaker at 37°C for 1 h.

[0091] S604, spread on LB solid medium containing ampicillin, spread evenly, and after all the bacterial solution is absorbed, incubate inverted at 37°C for 12 to 14 hours;

[0092] S605. Randomly select a single colony on the solid culture medium and inoculate it into 4 ml of LB liquid culture medium, shake and culture it at 37°C overnight, perform PCR verification on the bacterial solution using BVDV-F / R, BEV-F / R, and BPV-F / R as primers, and sequence the positive bacterial solution. The positive bacterial solution with correct sequencing results is obtained by amplification.

[0093] S7. Take 4 mL of the amplified positive bacterial solution, extract the BVDV, BEV, and BPV recombinant plasmids, and name them pMD19-T-BVDV, pMD19-T-BEV, and pMD19-T-BPV, respectively. Use a full-wavelength multifunctional microplate reader to measure the plasmid OD260 / OD280 value, and calculate the standard plasmid copy number according to the copy number formula (copies / μL) = (6.02×1023)×(ng / μL×10-9) / (DNA length×660). Dilute the plasmid to 5 ng / μL and store it in a -20℃ refrigerator for later use;

[0094] S8. Mix equal volumes of plasmid diluted to 5 ng / μL as templates, use analysis software Primerpremier6.0 to determine the theoretical Tm values ​​of three pairs of primers, set the annealing temperature to 58.9°C as the optimal annealing temperature for BVDV, BEV, and BPV, and use the chessboard method to optimize the volumes of upstream and downstream primers of the three viruses to determine the optimal primer volume; determine the optimal number of cycles by comparing the effects of different numbers of cycles on amplification efficiency.

[0095] Eight annealing temperatures with different gradients were set, namely 50°C, 52.2°C, 54.4°C, 55.6°C, 56.8°C, 57.9°C, 58.9°C, and 60°C, and the triple PCR detection method was used to determine the optimal annealing temperature for BVDV, BEV, and BPV. Figure 2 As shown, the results showed that the three viruses could amplify specific bands under 8 annealing temperature conditions. In order to ensure the specificity of the detection results, the optimal annealing temperature of triple PCR was determined to be 58.9℃.

[0096] The volumes of upstream and downstream primers of the three viruses were optimized by chessboard test and set to 0.2μL, 0.3μL, 0.4μL, 0.5μL, 0.6μL, 0.7μL, 0.8μL, 0.9μL, and 1.0μL, respectively, to determine the optimal primer volume for triple PCR detection method. Figure 3 As shown in the experimental results, the final reaction system was determined as follows: 25μL: Premix TaqTM 12.5μL, template 2μL, BVDV F / R 0.7μL each, BEV F / R 0.8μL each, BPV F / R 0.8μL each, Nuclease-Free Water 5.9μL.

[0097] like Figure 4As shown in the figure, under the above conditions of optimal primer volume and annealing temperature, the number of cycles was set to 15, 20, 25, 30, 35, 40, and 45 cycles, respectively, and the number of triple PCR cycles was optimized for plasmid sample cDNA / DNA. The results showed that specific bands existed in all 7 cycles, and the target bands were clear in 15, 20, 25, and 30 cycles. Non-specific bands appeared in the range of 45 cycles, and the bands were clearest in 35 and 40 cycles. 35 cycles were selected as the optimal reaction cycle number under the premise of saving time.

[0098] Experimental example:

[0099] Specificity test: The optimized triple PCR detection method was used to detect BVDV, BEV, BPV and BufHuV, MRV, Escherichia coli, Bacillus cereus, and Bacillus fusoniformis with an equal volume mixture of BVDV, BEV, and BPV plasmids as a positive control and Nuclease-Free Water as a negative control to evaluate the specificity of the established triple PCR detection method.

[0100] like Figure 5 The results showed that only BVDV, BEV, and BPV could amplify a single target band, while no bands were amplified for other viruses, indicating that the triple PCR detection method established in this experiment had good specificity.

[0101] Sensitivity test: The standard plasmid was calculated according to the copy number formula, and the corresponding copy number was: 3.28×10 9 copies / μLBVDV, 9.35×10 9 copies / μLBEV, 1.28×10 10 copies / μL BPV, dilute 10 times with sterile water, i.e. 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 There were 11 dilution gradients in total. Equal volumes of plasmids with the same dilution multiples were mixed and used as templates. Amplification was performed according to the optimized reaction system and conditions to determine the sensitivity of the triple PCR detection method.

[0102] like Figure 6 The results showed that the minimum sensitivity of BVDV was 3.28×10 5copies / μL, the minimum sensitivity of BEV is 9.35×10 5 copies / μL, and the minimum sensitivity of BPV was 1.28×10 5 copies / μL.

[0103] Repeatability test: Using the optimized triple PCR detection method, take an equal volume of 5ng / μL plasmid standard as a positive control, and Nuclease-Free Water as a negative control. The cDNA / DNA of BVDV, BEV, and BPV prepared in the same batch was amplified three times to determine the stability of the triple PCR detection method.

[0104] like Figure 7 The experimental results showed that the three amplifications all produced clear and single target bands, and the sizes of these bands were consistent with expectations. This result proved that the established triple PCR detection method has good repeatability.

[0105] Three pairs of primers were designed with the 5'UTR gene of BVDV and BEV and the VP2 gene of BPV as the target genes. The optimal reaction conditions were obtained by optimizing factors such as annealing temperature, primer volume, and cycle number. Only specific bands were amplified for BVDV, BEV, and BPV, and the target fragment sizes were 467bp, 350bp, and 257bp, respectively. No non-specific bands were amplified for other virus detection, indicating that the triple PCR detection method has good specificity. At the same time, in the sensitivity test, the minimum detection limit of BVDV was 3.28×10 5 copies / μL, BEV 9.35×10 5 copies / μL, BPV 1.28×10 5 copies / μL, and the surface triple PCR detection method had good sensitivity.

[0106] The established triple PCR method can detect BVDV, BEV and BPV at the same time, and only one test is needed to grasp the infection status of the three cattle pathogens, which greatly improves the detection efficiency, shortens the sample processing time, reduces the detection cost, and provides valuable epidemic monitoring information for the development of animal husbandry. At the same time, this method also has good sensitivity and specificity, and can accurately detect even when the pathogen load is low, effectively avoiding the occurrence of false positives or false negatives. It helps to take targeted prevention and control measures in a timely manner, effectively control the spread of pathogens, and reduce the risk of epidemic outbreaks, thereby protecting the health of the cattle herd and maintaining the economic benefits of the cattle farm, providing an efficient, accurate and economical monitoring method for disease prevention and control in cattle farms.

[0107] Importantly, it should be noted that the construction and arrangement of the present application shown in a plurality of different exemplary embodiments are only exemplary. Although only a few embodiments are described in detail in this disclosure, it should be readily understood by those who refer to this disclosure that many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described in the application. Without departing from the scope of the present invention, other replacements, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the exemplary embodiments. Therefore, the present invention is not limited to specific embodiments, but extends to a variety of modifications still falling within the scope of the appended claims.

[0108] Additionally, in order to provide a concise description of exemplary embodiments, all features of an actual embodiment (ie, those features that are not relevant to the best mode presently contemplated for carrying out the invention or those that are not relevant to implementing the invention) may not be described.

[0109] It should be understood that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but for those of ordinary skill having the benefit of this disclosure, without undue experimentation, the development effort will be a routine task of design, fabrication, and production.

[0110] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus, characterized in that: The following steps are involved: S1. Download the sequences of BVDV, BEV, and BPV from GenBank, use Megallign to compare the nucleotide sequences, obtain the conserved regions of the genes, and construct three pairs of specific primers using Primer premier6.0; S2, extracting RNA of BVDV, BEV, and BPV cell viruses respectively; S3, using the cDNA of BVDV, BEV and DNA of BPV as templates, single PCR amplification was performed using the three pairs of specific primers constructed; S4. Take 4 μL of the single PCR amplification product obtained in S3 and perform electrophoresis on a 1.5% agarose gel at a constant voltage of 120 V and a current of 100 mA for 35 minutes. Cut the correct target fragment from the agarose gel and recover it using a gel recovery kit. If a single band of the correct size appears on the gel recovery product after 1.5% agarose gel electrophoresis, it is considered to be recovered successfully. S5. Construct recombinant cloning plasmids with the target genes successfully recovered from the gel. The ligation system is 10 μL, 4 μL of gel recovery product, 5 μL of Solution Buffer, and 1 μL of pMTM 19-T Vector. The reaction system is fully pipetted and mixed in a PCR reaction tube and ligated overnight at 4°C. S6. Transform and identify the recombinant clone plasmid, and obtain a positive bacterial solution with a correct sequencing result through amplification; S7. Take 4 mL of the amplified positive bacterial solution respectively, extract the BVDV, BEV, and BPV recombinant plasmids, name them pMD19-T-BVDV, pMD19-T-BEV, and pMD19-T-BPV, respectively, and dilute the plasmids to 5 ng / μL, and store them in a -20°C refrigerator for later use; S8. Mix equal volumes of plasmid diluted to 5 ng / μL as templates, use analysis software Primer premier6.0 to determine the theoretical Tm values ​​of three pairs of primers, set the annealing temperature to 58.9°C as the optimal annealing temperature for BVDV, BEV, and BPV, and use the chessboard method to optimize the volumes of upstream and downstream primers of the three viruses to determine the optimal primer volume; determine the optimal number of cycles by comparing the effects of different cycle numbers on amplification efficiency.

2. A triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus as claimed in claim 1, characterized in that: The extraction steps of step S2 are: S201, take a 1.5ml centrifuge tube and add 20μL Proteinase K; S202, inject 200 μL of serum or plasma sample into the centrifuge tube, then add an equal amount of 200 μL of Buffer GL solution, and use a vortex oscillator to mix thoroughly for 15 seconds to evenly blend the sample and the buffer solution; S203, placing the centrifuge tube in a 56° C. water bath and heating it for 15 minutes to release the nucleic acid in the sample, and then briefly centrifuging it to allow the solution on the tube wall to completely settle to the bottom of the tube; S204, adding 250 μL of anhydrous ethanol to the centrifuge tube, mixing again using a vortex oscillator for 15 seconds, leaving the centrifuge tube at room temperature for 5 minutes to fully precipitate impurities, and then briefly centrifuging to allow the solution to completely settle to the bottom of the tube; S205, transferring the solution obtained in S204 to the adsorption column loaded into the collection tube, centrifuging at a speed of 12000 rpm for 1 minute, pouring out the waste liquid, and putting the adsorption column back into the collection tube; S206, adding 500 μL of Buffer GW1 solution to the adsorption column, adding anhydrous ethanol before use, centrifuging again at a speed of 12000 rpm for 1 minute, pouring out the waste liquid, and placing the adsorption column back into the collection tube; S207, repeat the operation of S206, use Buffer GW2 solution for washing, centrifuge at a speed of 12000 rpm for 1 minute, pour out the waste liquid, and put the adsorption column back into the collection tube; S208, adding 500 μL of anhydrous ethanol to the adsorption column again, centrifuging at 12000 rpm for 1 minute, discarding the waste liquid, and placing the adsorption column back into the collection tube for the final washing; S209. Centrifuge the adsorption column at 12,000 rpm for 3 minutes to completely remove the residual waste liquid, place the adsorption column at room temperature for several minutes to completely dry it, then transfer the adsorption column to a new collection tube, add 20-150 μL of Buffer RE or sterile water to the middle part of the adsorption column membrane, let it stand at room temperature for 2 to 5 minutes to fully dissolve the nucleic acid, and centrifuge it at 12,000 rpm for 1 minute to collect the resulting nucleic acid solution.

3. A triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus according to claim 1, characterized in that: The reaction conditions of the single PCR amplification in step S3 are: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds; annealing temperature at 50°C-60°C for 30 seconds; extension at 72°C for 20 seconds; final extension at 72°C for 2 minutes; 35 cycles in total, stored at 4°C, and the amplified product was detected by 1.5% agarose gel electrophoresis.

4. A triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus as claimed in claim 1, characterized in that: The steps of glue recovery in step S4 are as follows: S401. Cut out the agarose gel containing the target DNA under ultraviolet light, chop the gel block, and calculate the volume of the gel block as 1 mg = 1 μL; S402, adding Buffer GM to the gel block, the amount added is based on the gel concentration and volume: 1.0% plus 3 gel volumes; 1.0% to 1.5% plus 4 gel volumes; 1.5% to 2.0% plus 5 gel volumes; S403, dissolve the rubber block at room temperature of 15-25°C after uniform mixing, and use intermittent oscillation mixing to fully dissolve the rubber block; S404, placing the Spin Column in the reagent kit on the Collection Tube; S405, transferring the solution obtained in S403 to a Spin Column, centrifuging at 12,000 rpm for 1 minute, and discarding the filtrate; S406, add 700 μL of Buffer WB to the Spin Column, centrifuge at 12000 rpm for 30 seconds at room temperature, and discard the filtrate; S407, place the Spin Column on the Collection Tube, and centrifuge at 12000 rpm for 1 minute at room temperature; S408. Place the Spin Column in a new 1.5 ml centrifuge tube, add 30 μL Elution Buffer to the center of the Spin Column membrane, and let stand at room temperature for 1 minute; S409, centrifuge at 12000 rpm for 1 minute at room temperature to elute the DNA.

5. A triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus as claimed in claim 1, characterized in that: The step S6 of transforming and identifying the recombinant clone plasmid is as follows: S601. Take out 100 μL of DH5α competent cells from the -80°C refrigerator, place on ice to melt, add 10 μL of ligation product, and place on ice for 30 minutes; S602, place in a water bath preheated to 42°C for 45 seconds and immediately cool on ice for 2 minutes; S603. Under sterile conditions, add 890 μL of SOC liquid culture medium preheated to 37°C, mix well, and culture on a shaker at 37°C for 1 h. S604, spread on LB solid medium containing ampicillin, spread evenly, and after all the bacterial solution is absorbed, incubate inverted at 37°C for 12 to 14 hours; S605. Randomly select a single colony on the solid culture medium and inoculate it into 4 ml of LB liquid culture medium, culture it at 37°C with shaking overnight, perform PCR verification on the bacterial solution using BVDV-F / R, BEV-F / R, and BPV-F / R as primers, and sequence the positive bacterial solution.

6. A triple PCR detection method for bovine viral diarrhea virus, bovine enterovirus and bovine parvovirus as claimed in claim 1, characterized in that: In step S7, the plasmid OD260 / OD280 value is measured using a full-wavelength multifunctional microplate reader, and the standard plasmid copy number is calculated according to the copy number formula (copies / μL)=(6.02×1023)×(ng / μL×10-9) / (DNA length×660).

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