CN115478118A - Taqman multiplex fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus - Google Patents
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Abstract
The application provides a porcine circovirus Taqman multiplex fluorescence quantitative PCR detection method and a corresponding detection kit thereof, wherein primers and probes which are designed aiming at PCV1, PCV-2, PCV-3 and PCV-4 and have sequences of SEQ ID NO.1-12 are used in the method and the kit. The method and the kit have comprehensive detection objects, complete detection in one step in a 4-fold Taqman multiplex fluorescence quantitative PCR, and have good sensitivity and specificity.
Description
The invention belongs to the field of the invention:
the application belongs to the technical field of molecular biology detection, and particularly provides a porcine circovirus Taqman multiplex fluorescence quantitative PCR detection method and a corresponding detection kit thereof.
The background art comprises the following steps:
porcine Circovirus (PCV) is an unencapsulated, single-stranded, closed circular DNA virus of the genus circovirus of the family circoviridae, with virions in a regular icosahedral symmetric structure, about 17-22nm in diameter, and about 1.7-2.0kb in whole genome size, and is one of the smallest animal viruses currently known. The porcine circovirus is subdivided into four genotypes according to the difference of nucleotide homology, namely porcine circovirus type 1 (PCV 1), porcine circovirus type 2 (PCV 2), porcine circovirus type 3 (PCV 3) and porcine circovirus type 4 (PCV 4). PCV1 was first discovered in 1974 and was originally identified as a PK15 porcine kidney cell line contaminant, but was not pathogenic; PCV2 and PCV3 were isolated and identified in 1991 and 2016, respectively, and were both highly susceptible to pigs, and clinically caused Postweaning multi-system wasting Syndrome (PMWS), porcine Dermatitis Nephrotic Syndrome (PDNS), porcine respiratory disease Syndrome (PRDC), and Porcine Proliferative and Necrotizing Pneumonia (PNP). These diseases are collectively referred to as Porcine circovirus disease (PCVD) or Porcine circovirus associated disease (PCVAD); PCV4 is first discovered in Hunan province in 2019 in China, and the pathogenicity of PCV4 is not clear. Porcine circovirus usually causes severe immunosuppression of organisms, presents multiple pathogen mixed infection clinically, and has great harm to the pig industry, so that the porcine circovirus is particularly important for differential diagnosis of the porcine circovirus in the prevention and treatment process.
The porcine circovirus infected with different genotypes has similar clinical symptoms and pathological changes, is usually mixed infection of various pathogens, and is difficult to make accurate judgment simply by means of clinical diagnosis and pathological section. The results of the molecular level diagnosis in the laboratory are therefore usually further confirmed as gold standards, i.e.by detection of pathogens by molecular biological methods and by detection of serum-neutralizing antibodies by serological methods. The molecular biology method mainly comprises the following steps: polymerase Chain Reaction (PCR) and in situ nucleic acid hybridization assay (ISH); the serological method mainly comprises the following steps: enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), immunohistochemical assay (IHC), immunoperoxidase monolayer assay (IPMA), agar test, colloidal gold immunochromatography assay, and the like. Although the methods described above are numerous, the most widely used and most studied methods are currently the PCR and ELISA methods. In the prior art, various PCR detection methods for PCV1-3 exist.
The in vitro diagnostic technique of PCV1-3, especially PCV2 and PCV3, has been studied relatively well, but there are few reports on all members of porcine circovirus, i.e. in vitro diagnostic methods for detecting PCV1-4 simultaneously. The method for establishing the rapid and effective TaqMan multiplex real-time fluorescence quantitative PCR method for PCV1-4 type can efficiently and rapidly detect early infectors and recessive carriers in swinery, provides help for mastering PCV epidemic situation and subsequent PCV prevention and control purification, and has great significance.
Disclosure of Invention
In view of the above, in one aspect, the present application provides a Taqman multiplex fluorescence quantitative PCR detection method for porcine circovirus for non-diagnostic use, the method comprising performing Taqman multiplex fluorescence quantitative PCR using the following primers: PCV1-F with a sequence of SEQ ID NO.1, PCV2-F with a sequence of SEQ ID NO.4, PCV3-F with a sequence of SEQ ID NO.7, PCV4-F with a sequence of SEQ ID NO.10, PCV1-R with a sequence of SEQ ID NO.2, PCV2-R with a sequence of SEQ ID NO.5, PCV3-R with a sequence of SEQ ID NO.8 and PCV4-R with a sequence of SEQ ID NO. 11.
Further, the following probes are also used in the Taqman multiplex fluorescence quantitative PCR: PCV1-Probe with the sequence of SEQ ID NO.3, PCV2-Probe with the sequence of SEQ ID NO.6, PCV3-Probe with the sequence of SEQ ID NO.9 and PCV4-Probe with the sequence of SEQ ID NO. 12.
Further, the 5 'end and the 3' end of the probe are respectively marked with the following fluorescent groups: PCV1-Probe: CY5 and BHQ-2, PCV2-Probe: FAM and BHQ-2, PCV3-Probe: HEX-BHQ1, PCV4-Probe: ROX and BHQ-2.
Further, the Taqman multiplex fluorescence quantitative PCR reaction system is 20 mu: 2X T5 Fast qPCR Mix 10. Mu.L, 10. Mu.M PCV1, 3, 4 upstream primer 0.1. Mu.L each, 10. Mu.M PCV1, 3, 4 downstream primer 0.1. Mu.L each, 10. Mu.M PCV2 upstream primer 0.3. Mu.L, 10. Mu.M PCV2 downstream primer 0.3. Mu.L, 10. Mu.M probe 0.02. Mu.L each, template DNA 0.5. Mu.L, ddH 2 O 8.22μL。
Further, the Taqman multiplex fluorescence quantitative PCR reaction program is as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 10s and 61 ℃ for 50 s.
On the other hand, the application provides a porcine circovirus Taqman multiplex fluorescence quantitative PCR kit, which comprises the following primers: PCV1-F with a sequence of SEQ ID NO.1, PCV2-F with a sequence of SEQ ID NO.4, PCV3-F with a sequence of SEQ ID NO.7, PCV4-F with a sequence of SEQ ID NO.10, PCV1-R with a sequence of SEQ ID NO.2, PCV2-R with a sequence of SEQ ID NO.5, PCV3-R with a sequence of SEQ ID NO.8 and PCV4-R with a sequence of SEQ ID NO. 11.
Further, the kit further comprises the following probes: PCV1-Probe with the sequence of SEQ ID NO.3, PCV2-Probe with the sequence of SEQ ID NO.6, PCV3-Probe with the sequence of SEQ ID NO.9 and PCV4-Probe with the sequence of SEQ ID NO. 12.
Further, the 5 'end and the 3' end of the probe are respectively marked with the following fluorescent groups: PCV1-Probe: CY5 and BHQ-2, PCV2-Probe: FAM and BHQ-2, PCV3-Probe: HEX-BHQ1, PCV4-Probe: ROX and BHQ-2.
Further, the kit also comprises T5 Fast qPCR Mix.
Further, the application also provides application of the following primers and probes in preparing a Taqman multiplex fluorescence quantitative PCR kit for detecting porcine circovirus, wherein the primers and the probes are as follows: PCV1-F with a sequence of SEQ ID NO.1, PCV2-F with a sequence of SEQ ID NO.4, PCV3-F with a sequence of SEQ ID NO.7, PCV4-F with a sequence of SEQ ID NO.10, PCV1-R with a sequence of SEQ ID NO.2, PCV2-R with a sequence of SEQ ID NO.5, PCV3-R with a sequence of SEQ ID NO.8 and PCV4-R with a sequence of SEQ ID NO. 11; PCV1-Probe with the sequence of SEQ ID NO.3, PCV2-Probe with the sequence of SEQ ID NO.6, PCV3-Probe with the sequence of SEQ ID NO.9 and PCV4-Probe with the sequence of SEQ ID NO. 12; the 5 'end and the 3' end of the probe are respectively marked with the following fluorescent groups: PCV1-Probe: CY5 and BHQ-2, PCV2-Probe: FAM and BHQ-2, PCV3-Probe: HEX-BHQ1, PCV4-Probe: ROX and BHQ-2.
Non-diagnostic uses described herein include, but are not limited to, environmental testing, feed testing, food testing, epidemiological investigations, and the like. Samples that may be used include, but are not limited to, water, environmental sampling swabs, feces, feed products, foods of varying degrees of processing, and the like.
The types of reagents, reaction systems, and reaction parameters in the methods and kits of the present application are not limited to those shown in the summary of the invention and in the examples section, and those skilled in the art can obtain suitable types of reagents, reaction systems, and reaction parameters according to the performance and use of commercially available products and conventional knowledge in the art.
Has the beneficial effects that:
the method and the kit have the advantages that all porcine circovirus from PCV-1 to PCV-4 is detected by the detection object, the detection is completed in one 4-fold Taqman multiplex fluorescence quantitative PCR at one time, and the sensitivity and the specificity are good.
Drawings
FIG. 1 is 10 7 –10 3 Multiplex real-time fluorescent quantitative PCR amplification curve of copies/muL concentration gradient positive standard.
FIG. 2 is 10 7 –10 3 Multiplex real-time fluorescence quantitative PCR standard curve of copies/muL concentration gradient positive standard.
FIG. 3 is a multiplex real-time fluorescent quantitative PCR sensitivity experiment.
FIG. 4 shows a general PCR sensitivity experiment (1-5 4 –10 0 A positive standard substance with a copies/mu L concentration gradient; n: negative control).
FIG. 5 shows multiplex real-time fluorescent quantitative PCR specificity assays (1-7: amplification curves for PrV, ASFV, PPV, PRRSV, RV, PEDV and PK-15 cell lines, respectively; NC: negative control).
Figure 6 data of repeated experiments.
Detailed Description
The present invention will be described in detail with reference to specific examples, but the scope of the present invention is not limited to the following examples.
Example 1 primer and Probe design
And 4-8 PCV1-4 ORF2 Gene sequences separated from different countries published in NCBI GeneBank are selected, snap Gene is used for sequence comparison, a high-conservative region in ORF2 Gene is selected, oligo7 is used for primer probe design, and the designed primers are used for performing Blast comparison verification specificity on NCBI. Primer probe sequence is synthesized by Opticalceae Biotechnology Limited company, and dd H for synthesized primer probe 2 O is diluted to 10 mu mol/L and stored at 4 ℃ for later use. Through the practical verification of multiplex PCR, the following primers and probes are finally selected from 13 pairs of designed primers and 8 probes:
table 1 selected primer and probe sequences
Example 2 sample treatment and nucleic acid extraction
Adding 1mL of PBS into a sterile 2mL lysine Matrix tissue grinding tube, adding a proper amount of PCV2, PCV3, prV, ASFV, PPV, PRRSV, PRV and PEDV positive tissue samples, placing the sample in a FastPrep-24TM5G grinder, grinding for 30s, taking down the grinding tube, placing the tube in a centrifuge at 8000rpm, centrifuging for 1min, taking the supernatant, and storing at-20 ℃ for later use. The PK15 cell line, pUC57-PCV1-ORF2 and pUC57-PCV4-ORF2 were subjected to the following DNA extraction procedure without this step.
And (3) performing DNA extraction by using a Tian _ KingFisher _ Duo.bdz program by referring to the instruction of the magnetic bead method virus DNA/RNA extraction kit and matching with a KingFisher DUO automatic extraction instrument. After the procedure was completed, the eluted DNA was transferred to a 1.5mL centrifuge tube and stored at-20 ℃ for further use.
EXAMPLE 3 preparation of recombinant plasmid Standard
The TaqMan real-time fluorescent quantitative PCR primer pair designed by the experiment is used for extracting PCV1-4 DNA for common PCR amplification. The reaction conditions are as follows: 2min at 98 ℃; 35 cycles of 98 ℃ for 10s,56 ℃ for 15s, and 72 ℃ for 20 s; 5min at 72 ℃. (25. Mu.L reaction system: 1.1X gold medal Mix Ver.2. Mu.L, 10. Mu.M upstream primer 1. Mu.L, 10. Mu.M downstream primer 1. Mu.L, template DNA 1. Mu.L).
The PCR product was subjected to electrophoresis on a 3% agarose gel, set at a voltage of 120V, and run for 30min. After electrophoresis, cutting a target band, recovering and purifying according to the instruction of a DNA gel recovery kit, and immediately performing ligation transformation on a recovered product.
The recovered gel products were T-A ligated using pClone007 Simple Vector Kit to prepare 10. Mu.L of a reaction system (template 4. Mu.L), pClone007 Simple Vector 1. Mu.L), 10XTopo Mix 1. Mu.L), and ddH 2 O make up 10. Mu.L)).
The above-described ligation system was placed in a metal bath at 25 ℃ for 5min and immediately transformed after completion using Trelife TM 5. Alpha. Competent cells according to the instructions. The transformed competent cells were plated on LB medium containing Amp antibiotics and cultured at 37 ℃ for 12 hours. After the culture, a single clone was selected and inoculated in LB liquid medium, and shaken overnight at 37 ℃ and 250 rpm. The overnight cultured bacterial liquid is subjected to PCV2 and PCV3 recombinant plasmid extraction by using a high-purity plasmid DNA small-amount extraction kit according to instructions and sent to the Protechinics Biotech limited company for sequencing. The successfully ligated, sequence-free recombinant plasmids were assayed for concentration using a nucleic acid protein instrument, the copy number was calculated and 10-fold gradient dilutions were performed. The diluted plasmid is used as a positive standard substance and stored at-20 ℃ for later use. Copy number calculation formula:
copy number (copies/. Mu.L) = C.times.N A /MW
Wherein C is positive plasmid concentration with unit ng/. Mu.L, N A Take 6.02X 10 23 copies/mol, MW is the average molecular weight, in units of Dolton.
The copy numbers of PCV1-4 type plasmids are respectively as follows: PCV1 5.86X 10 11 copies/μL、PCV2 9.69×10 11 copies/μL、PCV3 6.54×10 11 copies/μL、PCV4 5.11×10 11 copies/μL。
Example 3 optimization of multiplex PCR reactions
The specific primers and the probes determined in the example 1 are used, the prepared positive standard substance is used as a template, a conventional reaction system and reaction conditions are recommended according to a reagent specification to carry out TaqMan multiplex real-time fluorescent quantitative PCR amplification, the specificity of the primers is preliminarily verified, and a basis is provided for subsequent optimization. The reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 10s and 60 ℃ for 50 s. 20 μ L reaction system 2X T5 Fast qPCR Mix 10 μ L, 10 μ M upstream primer 0.8 μ L each, 10 μ M downstream primer 0.8 μ L each, 10 μ M probe 0.4 μ L each, template DNA 0.5 μ L, ddH 2 O 1.5μL。
Adding the extracted four positive templates into the same reaction system to carry out TaqMan multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) primary amplification. The result shows that the negative control is established, the four positive templates have typical amplification curves, the specificity of the primer probe is good, and the reaction system can be further optimized.
Based on the reaction system and the reaction conditions, a gradient is set to optimize the annealing temperature and the cycle number, and a matrix method is adopted to optimize the primer concentration and the probe concentration. Primer final concentrations were set at 0.05. Mu.M, 0.1. Mu.M, 0.15. Mu.M, 0.2. Mu.M, 0.25. Mu.M, 0.3. Mu.M, 0.35. Mu.M, 0.4. Mu.M, 0.45. Mu.M, 0.5. Mu.M; the final concentration of the probe was set to 0.01. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.15. Mu.M, 0.2. Mu.M, 0.25. Mu.M, 0.3. Mu.M, 0.35. Mu.M, and the annealing temperature was optimized at 65 ℃, 64 ℃, 63 ℃, 62 ℃,61 ℃,60 ℃, 59 ℃, 58 ℃ and 30, 35, and 40 cycles. Under the same template concentration, the reaction conditions with smaller Ct value are preferably selected, and if the Ct values are the same, the annealing temperature is selected to be higher.
The annealing temperature is controlled to be 59-61 ℃ under the same template concentration, the amplification efficiency and the fluorescence intensity are consistent, and the Ct value change is small. The final annealing temperature of 61 ℃ was chosen because higher annealing temperatures reduced the chance of non-specific amplification. Under the condition that other conditions are not changed, the PCV2 and PCV3 amplification reactions show different degrees of non-specific signals after the 35 th cycle, and the final selection cycle number is 35 times. And optimally screening two groups of primers and probe concentrations with high fluorescence intensity and consistent amplification efficiency by using a matrix method, and finally selecting a group of primers and probe concentrations with lower concentrations. And (3) optimizing the final concentration of the primer: PCV1, PCV3 and PCV4 are all 0.05 μ M, PCV2 is 0.15 μ M; and (3) optimizing the final concentration of the probe: PCV1-4 is 0.01. Mu.M.
The optimized 20-microliter reaction system is as follows: 2X T5 Fast qPCR Mix 10. Mu.L, 10. Mu.M PCV1, 3, 4 upstream primer 0.1. Mu.L each, 10. Mu.M PCV1, 3, 4 downstream primer 0.1. Mu.L each, 10. Mu.M PCV2 upstream primer 0.3. Mu.L, 10. Mu.M PCV2 downstream primer 0.3. Mu.L 10. Mu.M probe 0.02. Mu.L each, template DNA 0.5. Mu.L, ddH 2 O 8.22μL。
EXAMPLE 4 creation of Standard Curve
Selecting 0.5 mu L of positive standard substance with the copy number of 107-103 copies/mu L after 10 times of gradient dilution as a template, adding the template into the optimized reaction system, replacing a negative control template with ddH2O, and carrying out multiple fluorescent quantitative PCR amplification according to the optimized reaction conditions. The amplification results were analyzed using Bio-Rad CFX Maestro to establish a standard curve.
The TaqMan multiplex real-time fluorescent quantitative PCR amplification curve (figure 1) and the standard curve (figure 2) show that the Ct value (y) and the copy number (x) of the positive standard substance show good linear relation in a gradient concentration interval of 107-103 copies/mu L, and the standard curves corresponding to four target genes are respectively as follows: PCV1: y = -3.293x +36.838, R 2 =1.000, amplification efficiency (E) =101.2%; PCV2: y = -3.515x + (38.358) 2 =0.998, amplification efficiency (E) =92.5%; PCV3: y = -3.025x +36.828 2 =0.999, amplification efficiency (E) =114.1%; PCV4: y = -3.342x+38.392, R 2 =0.999, amplification efficiency (E) =99.2%.
Example 5 sensitivity, specificity, reproducibility experiments
TaqMan multiplex real-time fluorescent quantitative PCR sensitivity experiment
The copy number after 10 times of gradient dilution is selected to be 10 4 -10 0 0.5. Mu.L of positive standard substance of copies/. Mu.L is used as template, and ddH is used as negative control template 2 And O, respectively carrying out TaqMan multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) amplification and common PCR amplification, and comparing the sensitivity degrees of the two methods. And respectively detecting and simultaneously detecting the positive standard substance by adopting a TaqMan multiplex real-time fluorescent quantitative PCR method, repeating the detection for 3 times, and further evaluating the influence of the simultaneous existence of various pathogens on the sensitivity.
The results in fig. 3 and 4 show that the lowest copy number detection limit of the TaqMan multiplex real-time fluorescent quantitative PCR method is: PCV 1.5.86X 10 1 copies/μL、PCV2 9.69×10 2 copies/μL、PCV36.54×10 1 copies/μL、PCV4 5.11×10 1 copies/μL。
The lowest copy number detection limits of the common PCR method are respectively: PCV1 5.86X 10 2 copies/μL、PCV2 9.69×10 3 copies/μL、PCV3 6.54×10 3 copies/μL、PCV4 5.11×10 2 copies/. Mu.L. The self-constructed TaqMan multiplex real-time fluorescent quantitative PCR method is about 10 to 100 times better than the common PCR method in the aspect of sensitivity.
TaqMan multiplex real-time fluorescent quantitative PCR specificity experiment
The method comprises the steps of taking extracted positive standard substance, porcine pseudorabies virus (PRV), african Swine Fever Virus (ASFV), porcine Parvovirus (PPV) DNA, porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine rotavirus (PoRV) and Porcine Epidemic Diarrhea Virus (PEDV) to extract RNA, then carrying out reverse transcription to obtain cDNA and PK-15 cell line nucleic acid as templates, carrying out TaqMan multiplex real-time fluorescent quantitative PCR amplification according to an optimized reaction system and conditions, and verifying the specificity of the established TaqMan multiplex real-time fluorescent quantitative PCR method. The result of FIG. 5 shows that only PCV positive standard has a typical amplification curve, and no amplification curve appears in nucleic acids of other pathogens and cells, which indicates that the established TaqMan multiplex real-time fluorescence quantitative PCR method has good specificity.
TaqMan multiplex real-time fluorescent quantitative PCR repeatability experiment
The copy number after 10 times of gradient dilution is 10 6 -10 4 Taking a positive standard substance of copies/mu L as a template, respectively carrying out 3 times of repeated detection by using an established TaqMan multiplex real-time fluorescent quantitative PCR method, and carrying out an intra-group repeated test; taking the positive standard substance, and carrying out repeated detection on the positive standard substance for 3 times at intervals of one week under the same reaction condition to obtain a repeated test among groups. And calculating the variation coefficient between groups to verify whether the repeatability of the established TaqMan multiplex real-time fluorescence quantitative PCR method is good or not.
The results of fig. 6 show that the coefficient of variation within the group of the method is between 0.33% and 2.48%, and the coefficient of variation between the groups is between 0.53% and 3.59% (tables 2 to 7), both of which are less than 5%, indicating that the established method has better repeatability and reproducibility.
In addition, the applicant uses the kit of the present application and the existing commercially available six porcine circovirus single/multiple TaqMan real-time fluorescence quantitative detection kit for real-time detection and comparison (30 mixed samples of spleen and lymph node collected for inspection by a innocent treatment plant of Chengdu City of 2021 year 6), and the results show that the 4-fold PCR method of the present application is not inferior or even superior to the existing commercially available kit in terms of sensitivity and specificity.
Claims (10)
1. A Taqman multiplex fluorescence quantitative PCR detection method for porcine circovirus with non-diagnostic use is characterized by comprising the following primers:
PCV1-F with a sequence of SEQ ID NO.1, PCV2-F with a sequence of SEQ ID NO.4, PCV3-F with a sequence of SEQ ID NO.7, PCV4-F with a sequence of SEQ ID NO.10, PCV1-R with a sequence of SEQ ID NO.2, PCV2-R with a sequence of SEQ ID NO.5, PCV3-R with a sequence of SEQ ID NO.8 and PCV4-R with a sequence of SEQ ID NO. 11.
2. The method of claim 1, wherein the following probes are also used in the Taqman multiplex quantitative PCR: PCV1-Probe with the sequence of SEQ ID NO.3, PCV2-Probe with the sequence of SEQ ID NO.6, PCV3-Probe with the sequence of SEQ ID NO.9 and PCV4-Probe with the sequence of SEQ ID NO. 12.
3. The method of claim 2, wherein the probes are labeled at the 5 'and 3' ends with the following fluorophores, respectively: PCV1-Probe: CY5 and BHQ-2, PCV2-Probe: FAM and BHQ-2, PCV3-Probe: HEX-BHQ1, PCV4-Probe: ROX and BHQ-2.
4. The method according to any one of claims 1 to 3, wherein the Taqman multiplex quantitative PCR reaction system is 20 μ:2X T5 Fast qPCR Mix 10. Mu.L, 10. Mu.M PCV1, 3, 4 upstream primer 0.1. Mu.L each, 10. Mu.M PCV1, 3, 4 downstream primer 0.1. Mu.L each, 10. Mu.M PCV2 upstream primer 0.3. Mu.L, 10. Mu.M PCV2 downstream primer 0.3. Mu.L, 10. Mu.M probe 0.02. Mu.L each, template DNA 0.5. Mu.L, ddH 2 O 8.22μL。
5. The method of claim 4, wherein the Taqman multiplex quantitative PCR reaction program is: 5min at 95 ℃; 30 cycles of 95 ℃ for 10s and 61 ℃ for 50 s.
6. The porcine circovirus Taqman multiplex fluorescence quantitative PCR kit is characterized by comprising the following primers: PCV1-F with a sequence of SEQ ID NO.1, PCV2-F with a sequence of SEQ ID NO.4, PCV3-F with a sequence of SEQ ID NO.7, PCV4-F with a sequence of SEQ ID NO.10, PCV1-R with a sequence of SEQ ID NO.2, PCV2-R with a sequence of SEQ ID NO.5, PCV3-R with a sequence of SEQ ID NO.8 and PCV4-R with a sequence of SEQ ID NO. 11.
7. The kit of claim 6, wherein the kit further comprises the following probes: PCV1-Probe with the sequence of SEQ ID NO.3, PCV2-Probe with the sequence of SEQ ID NO.6, PCV3-Probe with the sequence of SEQ ID NO.9 and PCV4-Probe with the sequence of SEQ ID NO. 12.
8. The kit according to claim 7, wherein the probes are labeled at the 5 'and 3' ends with the following fluorophores, respectively: PCV1-Probe: CY5 and BHQ-2, PCV2-Probe: FAM and BHQ-2, PCV3-Probe: HEX-BHQ1, PCV4-Probe: ROX and BHQ-2.
9. The kit of claim 7 or 8, wherein the kit further comprises a T5 Fast qPCR Mix.
10. The application of the following primers and probes in preparing a Taqman multiplex fluorescence quantitative PCR kit for detecting porcine circovirus is characterized in that the primers and the probes are as follows: PCV1-F with a sequence of SEQ ID NO.1, PCV2-F with a sequence of SEQ ID NO.4, PCV3-F with a sequence of SEQ ID NO.7, PCV4-F with a sequence of SEQ ID NO.10, PCV1-R with a sequence of SEQ ID NO.2, PCV2-R with a sequence of SEQ ID NO.5, PCV3-R with a sequence of SEQ ID NO.8 and PCV4-R with a sequence of SEQ ID NO. 11; PCV1-Probe with the sequence of SEQ ID NO.3, PCV2-Probe with the sequence of SEQ ID NO.6, PCV3-Probe with the sequence of SEQ ID NO.9 and PCV4-Probe with the sequence of SEQ ID NO. 12; the 5 'end and the 3' end of the probe are respectively marked with the following fluorescent groups: PCV1-Probe: CY5 and BHQ-2, PCV2-Probe: FAM and BHQ-2, PCV3-Probe: HEX-BHQ1, PCV4-Probe: ROX and BHQ-2.
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