CN107653348A - For detecting the primer and probe of the type of pig circular ring virus 3, PCR kit for fluorescence quantitative and method, application - Google Patents
For detecting the primer and probe of the type of pig circular ring virus 3, PCR kit for fluorescence quantitative and method, application Download PDFInfo
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Abstract
It is used to detect the primer and probe of the type of pig circular ring virus 3, PCR kit for fluorescence quantitative and method, application the invention discloses a kind of, wherein, the nucleotide sequence of primer is as follows：Sense primer：5'ACGTCGGGAAATCTGACTGAA 3', anti-sense primer：5' CCGCCCATAGCGTATAAATACTG 3'；The sequence of probe is as follows：5' FAM‑TTGCGGAGAAGATG‑MGB 3'.The beneficial effects of the invention are as follows：The fluorescent quantitative PCR detection method built using primer and probe of the present invention, with the advantage such as high specificity, high sensitivity, reproducible, quick, easy and reliable technical tool is provided for PCV3 early stage quick detection, and its epidemiology survey and prevention and control China PCV3 prevalence.
Description
Technical Field
The invention relates to the technical field of virus PCR detection, and particularly relates to a primer and a probe for detecting porcine circovirus type 3, a fluorescent quantitative PCR kit, a fluorescent quantitative PCR method and application.
Background
Porcine Circovirus (PCV) belongs to the family circoviridae, the genus circovirus, is a single stranded circular DNA virus without a membrane, and is the smallest animal virus found today. PCV has two serotypes, PCV-1 and PCV-2. PCV-1 is a conventional virus that infects pigs and is not pathogenic to pigs. PCV2 was first reported in 1998 to be pathogenic to pigs, causing clinically mainly Postweaning Multisystemic Wasting Syndrome (PMWS), dermatitis nephrotic syndrome (PDNS), reproductive disorders and respiratory and intestinal diseases, causing significant economic losses to the swine industry worldwide.
2016, a new circovirus serotype, PCV3, was reported to emerge in the United states and cause dermatitis nephrotic syndrome, reproductive disorders in swine, and cardiac and multisystemic inflammatory responses. At present, PCV3 virus is not successfully isolated, and pathogenicity is not studied deeply. Molecular epidemiological investigations have shown that PCV3 is widely prevalent in the us herd. In 2016 to 2017, the presence of PCV3 was also detected successively in Anhui, Fujian, Hebei, Henan, Shandong, Chongqing, etc. of China. At present, the morbidity and prevalence of PCV3 in swine herds in China are unclear, so that any detection reagent capable of quickly and accurately detecting PCV3 does not appear. The establishment of a rapid and accurate PCV3 laboratory diagnosis method is of great significance for further understanding the prevalence of PCV3 in swinery of China.
Disclosure of Invention
In order to provide a detection reagent for rapidly and accurately detecting PCV3, and provide a rapid, simple and reliable technical tool for rapid detection of PCV3, epidemiological investigation and prevention and control of the prevalence of PCV3 in China, the invention provides a specific primer and probe for detecting porcine circovirus type 3, a fluorescent quantitative PCR kit, a fluorescent quantitative PCR method and application.
In order to achieve the above object, the present invention provides a primer and a probe for detecting porcine circovirus type 3 specificity, wherein:
the nucleotide sequence of the primer is as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'。
in order to better achieve the above object, the present invention also provides a fluorescent quantitative PCR detection reagent for detecting porcine circovirus type 3, which comprises the following components: reaction mixed liquid, water and primers and probes for detecting the specificity of the porcine circovirus type 3;
the nucleotide sequence of the primer is as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'。
the reaction mixture was Premix Ex Taq (Probe qPCR) manufactured by TaKaRa.
The Water is RNase-free Water produced by TaKaRa company.
A fluorescent quantitative PCR kit for preparing a reagent kit for detecting porcine circovirus type 3, which contains the fluorescent quantitative PCR detection reagent of any one of claims 2 to 4.
The primer and the probe or the reagent or the kit are applied to the detection of the porcine circovirus type 3 or the preparation of a detection product for detecting the porcine circovirus type 3.
In order to better achieve the above object, the present invention further provides a fluorescent quantitative PCR detection method for detecting porcine circovirus type 3, the detection method comprising:
(1) designing and synthesizing primers and probes; wherein the nucleotide sequence of the primer is as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'；
(2) preparing a positive plasmid standard substance;
(3) optimizing the reaction conditions of the fluorescent quantitative PCR, and establishing a fluorescent quantitative PCR detection system;
(4) and detecting the virus by adopting a fluorescent quantitative PCR detection system.
Wherein the step (2) specifically comprises:
extracting DNA of the internal organ homogenate, the virus culture and the serum respectively by using a DNA/RNA nucleic acid co-extraction kit, dissolving the DNA in 20 mu L of RNase-free water, and directly using the obtained DNA for fluorescent quantitative PCR or storing the DNA at the temperature of-20 ℃ for later use;
PCV3 target gene is amplified by conventional PCR with Primer F/R as Primer, and the product is purified with recovery kit and connected to pMD18-T vector and converted into PCV3 target geneE.coliDH5 α colibacillus, obtaining positive clone plasmid as plasmid standard substance through sequence determination, converting into copy number after DNA concentration determination, and diluting to 1010copies•μL-1And stored at-20 ℃ and diluted before use as a plasmid standard.
In the step (3), the optimized reaction conditions are as follows: 5min at 95 ℃; 94 ℃ for 15sec, 60 ℃ for 45sec, 40 cycles.
In the step (3), the total volume of the fluorescent quantitative PCR reaction system is 25. mu.L, and the fluorescent quantitative PCR reaction system contains 12.5. mu.L of 2 XPrefmix ExTaq (Probe qPCR), 10. mu.L of RNase-free water, 0.5. mu.L of Probe (10. mu.M), 0.5. mu.L of Primer F (10. mu.M), 0.5. mu.L of Primer R (10. mu.M), and 1. mu.L of plasmid standard.
The invention has the beneficial effects that: the primers and the probes of the invention are a pair of specific primers and a specific TaqMan-MGB probe designed according to the sequence of Porcine circovirus type 3 (PCV 3) in GenBank, and a specific TaqMan-MGB fluorescent quantitative PCR method for rapidly and accurately detecting PCV3 viral load is established, so that the method is performed at 4.78x101-4.78x109copies•μL-1Has good linear relation in the range and the sensitivity reaches 4.78x101copies•μL-1Is 100 times of the conventional PCR method. Compared with the conventional PCR method, the method can monitor the result in real time without further gel electrophoresis analysis. After DNA is extracted from a sample to be detected, fluorescent quantitative PCR reaction is carried out, PCV3 in the sample can be rapidly and accurately detected within 2h, qualitative detection can be carried out, and accurate determination can be carried outAmount of the compound (A). Furthermore, tests show that the fluorescent quantitative PCR detection method constructed by using the primers and the probes has the advantages of strong specificity, high sensitivity, good repeatability and the like, and provides a quick, simple and reliable technical tool for early rapid detection of PCV3, epidemiological investigation and prevention and control of the prevalence of PCV3 in China.
Drawings
FIG. 1 is a schematic diagram showing the PCR amplification result of the target gene of PCV3 in example 5 of the present invention; wherein, M: DL2000 Marker; 1-2: PCV 3; 3: and (5) negative control.
FIG. 2 is a graph showing the fluorescent quantitative PCR kinetics of PCV3 TaqMan-MGB in example 5 of the present invention; wherein, 1 to 9 are respectively 4.78x109-4.78x101copies•μL-1The standard substance of (1).
FIG. 3 is a standard curve diagram of PCV3 TaqMan-MGB fluorescent quantitative PCR in example 5 of the present invention.
FIG. 4 is a schematic diagram showing the result of PCV3 TaqMan-MGB fluorescent quantitative PCR specificity assay in example 5 of the present invention; wherein, 1: a standard substance; 2-10 are CSFV, PRRSV, PRV, PEDV, TGEV, RV, PPV, PCV2 and negative control, respectively.
FIG. 5 is a graph showing the results of conventional PCR sensitivity test of PCV3 in example 5 of the present invention; wherein, M: DL2000 Marker; 1-7: the dilution was 4.21X107-4.21×101copies•μL-1The template DNA of (1).
FIG. 6 is a schematic diagram showing the result of repeated PCR fluorescent quantitative PCR assay with PCV3 TaqMan-MGB in example 5 of the present invention.
Detailed Description
Conventional methods for detecting viruses include virus isolation and identification, indirect immunofluorescence antibody assay (IFA), serum neutralization assay (SN), Polymerase Chain Reaction (PCR), sequencing, and the like. When the conventional methods are used for detecting PCV3, the defects exist in the aspects of specificity, sensitivity, timeliness and the like, and particularly the method is obvious in early diagnosis of virus infection. The real-time fluorescent quantitative PCR combines the conventional PCR and the fluorescent probe detection technology, has the advantages of strong specificity, high sensitivity, good repeatability and the like, is simple and convenient to operate, short in time consumption and less in pollution, can be automatically and quantitatively analyzed through analysis software, is more accurate and visual in result, is suitable for qualitative and quantitative detection of a large batch of viruses, and gradually becomes an important method for pathogen detection.
The invention designs a pair of specific primers and a specific TaqMan-MGB probe according to the gene sequence of Porcine circovirus type 3 (PCV 3) in GenBank, and establishes a specific TaqMan-MGB fluorescent quantitative PCR method for rapidly detecting PCV3 virus load. The method is optimized to 4.78x10 by optimizing the reaction conditions and the reaction system1～4.78x109copies•μL-1Has good linear relation in the range and the sensitivity reaches 4.78x101copies•μL-1Is 100 times of the conventional PCR method. Compared with the conventional PCR method, the method can monitor the result in real time without further gel electrophoresis analysis.
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1 primers and probes
The embodiment of the invention provides a primer and a probe for detecting porcine circovirus type 3 specificity, wherein the primer comprises the following components in parts by weight:
the nucleotide sequence of the primer is as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'。
example 2 fluorescent quantitative PCR detection reagent
The embodiment of the invention provides a fluorescent quantitative PCR detection reagent for detecting porcine circovirus type 3, which comprises the following components: reaction mixed liquid, water and primers and probes for detecting the specificity of the porcine circovirus type 3; wherein,
the nucleotide sequences of the primers are as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'。
the reaction mixture was Premix Ex Taq (Probe qPCR) manufactured by TaKaRa Co., Ltd; the Water was RNase-free Water produced by TaKaRa.
Example 3 fluorescent quantitative PCR kit
The embodiment of the invention provides a fluorescent quantitative PCR kit for detecting porcine circovirus type 3, and particularly provides a kit containing the fluorescent quantitative PCR detection reagent in the embodiment 2.
Example 4 fluorescent quantitative PCR detection method
The embodiment of the invention provides a fluorescent quantitative PCR detection method for detecting porcine circovirus type 3, which comprises the following steps:
(1) designing and synthesizing primers and probes; wherein the nucleotide sequence of the primer is as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'；
(2) preparing a positive plasmid standard substance;
(3) optimizing the reaction conditions of the fluorescent quantitative PCR, and establishing a fluorescent quantitative PCR detection system;
(4) and detecting the virus by adopting a fluorescent quantitative PCR detection system.
Wherein the step (2) specifically comprises:
extracting DNA of the internal organ homogenate, the virus culture and the serum respectively by using a DNA/RNA nucleic acid co-extraction kit, dissolving the DNA in 20 mu L of RNase-free water, and directly using the obtained DNA for fluorescent quantitative PCR or storing the DNA at the temperature of-20 ℃ for later use;
PCV3 target gene is amplified by conventional PCR with Primer F/R as Primer, and the product is purified with recovery kit and connected to pMD18-T vector and converted into PCV3 target geneE.coliDH5 α colibacillus, obtaining positive clone plasmid as plasmid standard substance through sequence determination, converting into copy number after DNA concentration determination, and diluting to 1010copies•μL-1And stored at-20 ℃ and diluted before use as a plasmid standard.
In the step (3), the optimized reaction conditions are as follows: 5min at 95 ℃; 94 ℃ for 15sec, 60 ℃ for 45sec, 40 cycles.
In step (3), the total volume of the fluorescent quantitative PCR reaction system is 25. mu.L, and the system contains 12.5. mu.L of 2 XPromix Ex Taq (Probe qPCR), 10. mu.L of RNase-free water, 0.5. mu.L of Probe (10. mu.M), 0.5. mu.L of Primer F (10. mu.M), 0.5. mu.L of Primer R (10. mu.M), and 1. mu.L of plasmid standard.
Example 5 application
The embodiment provides a primer and a probe, or a fluorescent quantitative PCR detection reagent, or a fluorescent quantitative PCR kit, which are used for the application of specific detection of porcine circovirus type 3, and specifically comprises the following steps:
1 materials and methods
1.1 instruments and reagents
The CFX96 fluorescent quantitative PCR instrument is purchased from Bio-Rad company, and the ultraviolet spectrophotometer is purchased from Thermo company; the plasmid extraction kit is purchased from AxyGen company;E.coliDH5 α was purchased from TaKaRa, DNA/RNA nucleic acid Co-extraction kit was purchased from Invitrogen, Premix Ex Taq (Probe qPCR) was purchased from TaKaRa, and DNA gel recovery kit was purchased from AxyGen.
1.2 primers and probes
The specific primer pair and the TaqMan-MGB fluorescent probe are as follows:
the sequences of the primer pairs are as follows:
the sequence of the upstream Primer is Primer F: 5 'ACGTCGGGAAATCTGACTGAA 3',
the sequence of the downstream Primer is Primer R: 5 'CCGCCCATAGCGTATAAATACTG 3';
the Probe sequence is Probe 5'FAM-TTGCGGAGAAGATG-MGB 3'.
The fluorescent reporter group marked at the 5 'end of the probe is FAM, and the fluorescent quencher group marked at the 3' end is Non-fluorescent transducer and Minor Groove Binder (MGB).
1.3 extraction of viral DNA and preparation of plasmid standards
DNA of the visceral homogenate, the virus culture and the serum are respectively extracted by using a DNA/RNA nucleic acid co-extraction kit according to the operation instruction, and dissolved in 20 mu L of RNase-free water, and the obtained DNA can be directly used for fluorescence quantitative PCR or stored at the temperature of-20 ℃ for later use.
PCV3 target gene is amplified by conventional PCR with Primer F/R as Primer, and the product is purified with recovery kit and connected to pMD18-T vector and converted into PCV3 target geneE.coliDH5 α colibacillus, obtaining positive clone plasmid as plasmid standard substance through sequence determination, converting into copy number after determining DNA concentration, and diluting to 1010copies•μL-1And stored at-20 ℃ and diluted before use as a plasmid standard.
1.4 optimization of fluorescent quantitative PCR reaction conditions
Taking a plasmid standard product as a template, carrying out conventional PCR reaction at different annealing temperatures, analyzing the amplified product by agarose gel electrophoresis, and determining the optimal primer annealing temperature. The primer and probe concentration of the fluorescent quantitative PCR is optimized by using a matrix method so as to obtain the optimal reaction system and reaction conditions.
1.5 establishment of fluorescent quantitative PCR Standard Curve
Plasmid standards were diluted 10-fold to a concentration range of 4.78 × 101-4.78x109copies•μL-1. And setting 3 times for each dilution, performing fluorescent quantitative PCR detection, and drawing a standard curve.
1.6 specificity test
Nucleic acids of viruses such as Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine pseudorabies virus (PRV), Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), Rotavirus (RV), Porcine Parvovirus (PPV), porcine circovirus type 2 (PCV 2) and the like are respectively extracted, and the established fluorescent quantitative PCR method is utilized to detect the nucleic acids to verify the specificity of the method.
1.7 sensitivity test
Positive DNA was diluted 10-fold to a minimum concentration of 4.21X101copies•μL-1And performing fluorescent quantitative PCR reaction. Meanwhile, the DNA is used as a template to carry out conventional PCR reaction, and the upstream primer and the downstream primer are respectively 5 'CGGGAAATCTGACTGAAGTT 3' and 5 'ACTCCTCCGGTACAACATTA 3'. 10. mu.L of the amplified product was analyzed by 1.5% agarose gel electrophoresis to compare the differences in sensitivity.
1.8 repeatability test
The reproducibility test was carried out for 4 batches and between batches using 5 concentrations of the standard plasmid and the coefficient of variation was calculated from the Ct values.
1.9 testing of clinical samples
47 suspected PCV3 clinical samples were collected by Xinhope six and animal protection centers, and subjected to fluorescent quantitative PCR detection and conventional PCR detection, respectively, and the results were compared and analyzed.
2 results
2.1 preparation of plasmid Standard
PCV3 target gene is amplified by conventional PCR with Primer F/R as Primer, the amplification result is shown in figure 1, the product is purified by a recovery kit and then connected with pMD18-T vector, and the product is transformed toE.coliDH5 α colibacillus, obtaining positive clone plasmid as plasmid standard substance through sequence determination, converting into copy number after determining DNA concentration, and diluting to 1010copies•μL-1And stored at-20 ℃ and diluted before use as a plasmid standard.
2.2 optimization of the fluorescent quantitative PCR reaction conditions
The optimal reaction conditions of the fluorescent quantitative PCR method are finally determined by optimizing the annealing temperature and the concentrations of the primers and the probes. The total volume of the fluorescent quantitative PCR reaction was 25. mu.L, containing 12.5. mu.L of 2 XPrimx Ex Taq (Probe qPCR), 10. mu.L of LRNase-free water, 0.5. mu.L of Probe (10. mu.M), 0.5. mu.L of Primer F (10. mu.M), 0.5. mu.L of Primer R (10. mu.M), and 1. mu.L of plasmid standard. The reaction conditions are as follows: 5min at 95 ℃; 94 ℃ for 15sec, 60 ℃ for 45sec, 40 cycles.
2.3 establishment of fluorescent quantitative PCR Standard Curve
Taking the concentration of 4.78x101-4.78x109copies•μL-1The standard plasmid is used as a template to carry out fluorescence quantitative PCR amplification and establish a standard curve. As can be seen from FIGS. 2 and 3, the linear relationship is good, the standard curve is successfully established, and the method can be used for clinical detection.
2.4 specificity test
The established fluorescent quantitative PCR method is used for detecting CSFV, PRRSV, PRV, PEDV, TGEV, RV, PPV and PCV2, the detection results are negative, the amplification curves of the viruses are all horizontal lines and do not reach the detection threshold (figure 4), and the test results show that the method has good specificity.
2.5 sensitivity test
The lowest concentration of the DNA template detected by the fluorescent quantitative PCR method is 4.78x101copies•μL-1(FIG. 2), whereas the lowest template concentration detectable by conventional PCR is 4.21X103copies•μL-1(FIG. 5), the test results show that the sensitivity of the established fluorescent quantitative PCR method is about 100 times that of the conventional PCR method.
2.6 repeatability test
Within-batch and between-batch reproducibility tests were performed with 5 different concentrations of standards, respectively. The test results show that the variation coefficient of the repeatability tests in batches and between batches is less than 2.3 percent (Table 1 and figure 6), which indicates that the method has good repeatability and stability.
TABLE 1 PCV3 TaqMan-MGB fluorescent quantitative PCR repeatability test results
2.7 clinical sample test results
47 collected clinical samples of suspected PCV3 were subjected to fluorescent quantitative PCR detection and routine PCR detection, respectively. The detection result shows that the positive detection rate (21.3%) of the fluorescent quantitative PCR method is higher than that of the conventional PCR method (14.9%) (Table 2). The detection result of the clinical sample shows that the fluorescence quantitative method has higher sensitivity and is more suitable for clinical detection of PCV3, especially when the virus content in the early infection is lower.
TABLE 2 clinical sample test results
In conclusion, by applying the primers and the probes of the invention to the specificity detection of porcine circovirus type 3 and the sample results, it can be seen that the fluorescent quantitative PCR detection method constructed by the primers and the probes of the invention has no cross reaction with other porcine circovirus, has good specificity, and has sensitivity 100 times that of the conventional PCR method, and the method directly performs the fluorescent quantitative PCR reaction after extracting DNA from the sample to be detected, and can rapidly and accurately detect PCV3 in the sample within 2h by using the established fluorescent quantitative PCR method, thereby not only performing qualitative detection, but also performing accurate quantification. The kit has the advantages of high sensitivity, strong specificity, good repeatability and the like, and provides a rapid and accurate detection means for laboratory diagnosis of the porcine circovirus type 3 and investigation of the prevalence of the porcine circovirus type 3 in swine herds in China.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The primer and the probe for detecting the porcine circovirus type 3 specificity are characterized in that,
the nucleotide sequence of the primer is as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'。
2. the fluorescent quantitative PCR detection reagent for detecting porcine circovirus type 3 is characterized by comprising the following components: reaction mixed liquid, water and primers and probes for detecting the specificity of the porcine circovirus type 3;
the nucleotide sequence of the primer is as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'。
3. the reagent of claim 2, wherein the reaction mixture is Premix Ex Taq (Probe qPCR) manufactured by TaKaRa.
4. The PCR detection reagent for detecting porcine circovirus type 3 of claim 2 or 3, wherein the Water is RNase-free Water manufactured by TaKaRa.
5. A fluorescent quantitative PCR kit for preparing a reagent kit for detecting porcine circovirus type 3, which contains the fluorescent quantitative PCR detection reagent of any one of claims 2 to 4.
6. The primer and the probe as claimed in claim 1, or the detection reagent as claimed in any one of claims 2 to 4, or the kit as claimed in claim 5, for use in detecting porcine circovirus type 3 or in preparing a detection product for detecting porcine circovirus type 3.
7. The fluorescent quantitative PCR detection method for detecting porcine circovirus type 3 is characterized by comprising the following steps:
(1) designing and synthesizing primers and probes; wherein the nucleotide sequence of the primer is as follows:
an upstream primer: 5 'ACGTCGGGAAATCTGACTGAA 3' of the formula I,
a downstream primer: 5 'CCGCCCATAGCGTATAAATACTG 3';
the sequence of the probe is as follows:
5' FAM-TTGCGGAGAAGATG-MGB 3'；
(2) preparing a positive plasmid standard substance;
(3) optimizing the reaction conditions of the fluorescent quantitative PCR, and establishing a fluorescent quantitative PCR detection system;
(4) and detecting the virus by adopting a fluorescent quantitative PCR detection system.
8. The fluorescent quantitative PCR detection method for detecting porcine circovirus type 3 according to claim 7, wherein the step (2) is specifically:
extracting DNA of the internal organ homogenate, the virus culture and the serum respectively by using a DNA/RNA nucleic acid co-extraction kit, dissolving the DNA in 20 mu L of RNase-free water, and directly using the obtained DNA for fluorescent quantitative PCR or storing the DNA at the temperature of-20 ℃ for later use;
PCV3 target gene is amplified by conventional PCR with Primer F/R as Primer, and the product is purified with recovery kit and connected to pMD18-T vector and converted into PCV3 target geneE.coliDH5 α colibacillus, obtaining positive clone plasmid as plasmid standard substance through sequence determination, converting into copy number after DNA concentration determination, and diluting to 1010copies•μL-1And stored at-20 ℃ and diluted before use as a plasmid standard.
9. The fluorescent quantitative PCR detection method for detecting porcine circovirus type 3 according to claim 7 or 8, wherein in the step (3), the optimized reaction conditions are as follows: 5min at 95 ℃; 94 ℃ for 15sec, 60 ℃ for 45sec, 40 cycles.
10. The fluorescent quantitative PCR detection method for detecting porcine circovirus type 3 of any one of claims 7-9, wherein in the step (3), the fluorescent quantitative PCR reaction system is 25 μ L in total, and contains 12.5 μ L of 2 XPPremixEx Taq (Probe qPCR), 10 μ L of RNase-free water, 0.5 μ L of Probe (10 μ M), 0.5 μ L of Primer F (10 μ M), 0.5 μ L of Primer R (10 μ M), and 1 μ L of plasmid standard.
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