CN117305519A - Primer probe combination for Niu Duo tumor virus triple qPCR detection and detection method thereof - Google Patents
Primer probe combination for Niu Duo tumor virus triple qPCR detection and detection method thereof Download PDFInfo
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Abstract
The invention discloses a primer probe combination for triple qPCR detection of bovine polyoma virus and a detection method thereof, and relates to the technical field of biological detection. The primer probe combination comprises a primer probe combination for detecting bovine polyoma virus BPyV1, BPyV2a, BPyV2b and BPyV3, wherein the nucleotide sequences of a forward primer, a reverse primer and a probe are respectively shown as SEQ ID NO. 1-11, and the 5' ends of the probes of the BPyV1 and the BPyV3 are respectively coupled with a first fluorescent group and a third fluorescent group; the 5 'ends of the probes of BPyV2a and BPyV2b are coupled with a second fluorescent group, and the 3' ends of the probes of BPyV1, BPyV2a, BPyV2b and BPyV3 are coupled with a quenching group; the first fluorescent group, the second fluorescent group and the third fluorescent group are three different types of fluorescent groups. The primer probe combination can cover 3 genotypes, has wider coverage range, can rapidly judge whether BPyV pollution exists in a sample according to a Ct value and an amplification curve and determine the specific genotype of the BPyV, can detect a plurality of target genes at one time, and has high detection efficiency.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer probe combination for triple qPCR detection of bovine polyomavirus and a detection method thereof.
Background
Niu Duo the tumor virus (Bovine polyomavirus, BPyV) belongs to the genus polyomavirus (Polyomaviridae) in the family of Papovaviridae (Papovaviridae) and is a non-enveloped, closed-loop, double-stranded DNA virus with viral particles of 40-45nm. BPyV is classified into 3 genotypes, BPyV1, BPyV2, BPyV3, respectively, according to nucleotide sequences, wherein two subtypes of BPyV2 are BPyV2a, BPyV2b, respectively.
BPyV is a refractory pollutant, widely exists in bovine serum and beef products, and has strong inactivation resistance. Commercial calf/fetal bovine serum was tested for BPyV contamination multiple times, as well as adult bovine serum. The detection of BPyV DNA in culture supernatants of MDBK, vero, etc. has been shown to be the cause of the presence of BPyV contamination in bovine serum. It has been found that serum from humans in intimate contact with bovine and bovine-derived products often detects BPyV positivity and that BPyV has been reported in the literature to cause carcinogenesis in immunocompromised neonatal mice. The use of bovine serum in cell culture and biological product production processes is very common, and the preparation and quality control of animal cell matrix for biological product production detection in three parts of the current edition of Chinese pharmacopoeia in China clearly indicate that the established MCB/WCB/production terminal cells should carry out bovine-derived virus detection when the bovine serum is used in the establishment or passage of the cell matrix. The United states Pharmacopeia also indicates the use of a suitable method to detect BPyV in bovine serum for the production of biological products for human use.
The real-time fluorescent quantitative PCR (TaqMan probe method) has the advantages of strong specificity, high sensitivity, good repeatability and the like, and is widely used for virus detection. Therefore, the development of a PCR system capable of specifically detecting BPyV3 genotypes is of great significance to the fields of food safety and biological product safety.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a primer probe combination for bovine polyoma virus triple qPCR detection and a detection method thereof, which can detect 3 genotypes of BPyV simultaneously so as to meet the safety quality control detection requirements of serum and related biological products using the serum. The method is realized by the following specific techniques:
in a first aspect of the invention, a primer probe combination for triple qPCR detection of bovine polyomavirus is provided, which comprises a primer probe combination for detecting bovine polyomavirus BPyV1, BPyV2a, BPyV2b and BPyV3, wherein the nucleotide sequences of a forward primer, a reverse primer and a probe of the BPyV1 are respectively shown as SEQ ID NO. 1-3; the nucleotide sequences of the forward primer and the probe of the BPyV2a are respectively shown as SEQ ID NO. 4-5; the nucleotide sequences of the forward primer and the probe of the BPyV2b are respectively shown in SEQ ID NO. 6-7; the nucleotide sequences of the reverse primers of the BPyV2a and the BPyV2b are shown in SEQ ID NO: shown as 8; the nucleotide sequences of the forward primer, the reverse primer and the probe of the BPyV3 are respectively shown as SEQ ID NO. 9-11;
the 5' ends of probes for detecting bovine polyoma viruses BPyV1 and BPyV3 are respectively coupled with a first fluorescent group and a third fluorescent group; the 5 'end of the probe for detecting the bovine polyomaviruses BPyV2a and BPyV2b is coupled with a second fluorescent group, and the 3' end of the probe for detecting the bovine polyomaviruses BPyV1, BPyV2a, BPyV2b and BPyV3 is coupled with a quenching group; the first fluorescent group, the second fluorescent group and the third fluorescent group are three different types of fluorescent groups.
Further, the first fluorescent group, the second fluorescent group and the third fluorescent group are FAM, TET, NED, ROX, CY3, CY5, VIC, JOE, HEX or Texas Red fluorescent groups; the quenching group is TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1 or BHQ2 quenching group.
Further, the first fluorescent group is a HEX fluorescent group, the second fluorescent group is a Texas Red fluorescent group, and the third fluorescent group is a FAM fluorescent group; the quenching group is a BHQ1 quenching group.
According to the invention, three different types of fluorescent groups are designed at the 3' end of the probe of the BPyV1, the BPyV2 (comprising two subtypes of the BPyV2a and the BPyV2 b) and the BPyV3, and the fluorescent groups are combined with primers to be used in a triple qPCR reaction system, so that specific genotypes of bovine polyoma viruses can be specifically amplified and distinguished, whether the BPyV pollution exists in a sample or not can be judged, and the specific genotypes of the BPyV can be determined.
In a second aspect of the invention, a product of a bovine polyomavirus triple qPCR detection is provided, comprising the primer probe combination.
In a third aspect of the invention, the application of the primer probe combination is provided, and a product for detecting bovine polyoma virus is prepared.
In a fourth aspect of the present invention, there is provided a method for detecting bovine polyoma virus which is not aimed at diagnosis and treatment of diseases, using the above-mentioned product.
Further, the detection method includes the steps of: extracting nucleic acid in a sample to be detected; creating a first fluorescent group detection channel, a second fluorescent group detection channel and a third fluorescent quantitative group detection channel on a qPCR instrument, and performing triple qPCR reaction on nucleic acid of a sample to be detected; and judging the detection result according to the amplification curve and the Ct value generated by the triple qPCR reaction.
Further, the reaction system of the triple qPCR reaction is as follows: 10 mu L of Probe qPCR MIX, 0.2uL of each of forward primer and reverse primer for detecting bovine polyoma virus BPyV1 and BPyV3, 0.1uL of each of forward primer and reverse primer for detecting bovine polyoma virus BPyV2a and BPyV2b, 0.4uL of each of probes for detecting bovine polyoma virus BPyV1, BPyV2a, BPyV2b and BPyV3 and 7.2uL of template to be detected. The amplification procedure for the triple qPCR reaction described above was: firstly, pre-denaturing for one time at 25 ℃ for 10min and 95 ℃ for 30 s; then, the mixture was circulated 42 times at 95℃for 5 seconds and 60℃for 30 seconds.
Further, the determination of the detection result is specifically: when the Ct value of the first fluorescent group detection channel is less than or equal to 38 and an obvious amplification curve exists, judging that the bovine polyoma virus BPyV1 is positive; when the Ct value of the second fluorescent group detection channel is less than or equal to 38 and an obvious amplification curve exists, judging that the bovine polyoma virus BPyV2a or BPyV2b is positive; when the Ct value of the third fluorescent group detection channel is less than or equal to 38 and an obvious amplification curve exists, judging that the bovine polyoma virus BPyV3 is positive;
when the Ct value of the first fluorescent group, the first fluorescent group or the third fluorescent group detection channel is more than 38 and an obvious amplification curve exists, the judgment result is invalid;
when the first fluorescent group detection channel has no Ct value and no obvious amplification curve, judging that the bovine polyoma virus BPyV1 is negative; when the second fluorescent group detection channel has no Ct value and no obvious amplification curve, judging that the bovine polyoma viruses BPyV2a and BPyV2b are negative; and when the third fluorescent group detection channel has no Ct value and no obvious amplification curve, judging that the bovine polyoma virus BPyV3 is negative.
Compared with the prior art, the invention has the following advantages:
1. the existing BPyV qPCR detection method only can detect BPyV1 through comparison, and the primer probe combination for bovine polyoma virus triple qPCR detection provided by the invention can cover 3 genotypes, and compared with the prior art, the coverage area is wider.
2. By utilizing the primer probe combination provided by the invention to perform triple qPCR, whether BPyV pollution exists in a sample or not can be rapidly judged according to the Ct value and the amplification curve, the specific genotype of the BPyV can be determined, a plurality of target genes can be detected at one time, the detection process is accelerated, and the detection efficiency is high.
3. The BPyV triple qPCR reaction system has strong specificity, and the primer probe combination has no cross reaction with human and animal cells and non-BPyV bovine-derived virus genome.
4. The BPyV triple qPCR reaction system has high sensitivity, and the detection limit can reach 100copies/reaction.
5. The BPyV triple qPCR reaction system has strong durability and matrix interference resistance, and the BPyV with detection limit can be normally detected by introducing the BPyV into 293/CHO/Vero cell genome.
Drawings
FIG. 1 is a graph of the sensitivity verification results of the single qPCR described in example 4;
FIG. 2 is a graph showing the results of sensitivity verification of triple qPCR described in example 4;
FIG. 3 is a graph showing the results of the cross-reaction between the BPyV qPCR detection system and BADV or BPV described in example 5;
FIG. 4 is a graph of the results of the specificity verification described in example 5;
fig. 5 is a diagram of the durability verification result described in example 6.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Compared with the single qPCR, the triple qPCR provided by the invention has the advantages that a plurality of nucleic acid fragments are amplified simultaneously in the same reaction tube, so that base pairing interference and non-specific binding of different primer pairs can occur in the reaction, the condition in the triple qPCR reaction is more difficult to search, and the primer probe sequence, concentration, annealing temperature and other factors in the reaction system can be interfered. The primer probe combination, the reaction system of the triple qPCR and the amplification program provided in the following embodiments are determined through a great deal of researches and experiments, and specific genotypes of the bovine polyomaviruses can be specifically amplified and distinguished, so that whether the sample is polluted by the genotypes of the bovine polyomaviruses or not can be judged.
In the primer probe combination for detecting bovine polyomaviruses BPyV1, BPyV2a, BPyV2b and BPyV3, the 5' end of the probe is respectively coupled with different fluorescent markers so as to distinguish different genotypes of the bovine polyomaviruses; the 3' ends are coupled with quenching groups. Thus, based on the commonly used fluorescent label and the type of quencher, the fluorescent label may be selected from FAM, TET, NED, ROX, CY, CY5, VIC, JOE, HEX or Texas Red fluorescent labels; the quenching group may be selected from TAMRA, NFQ, ECLIPSE, DABCYL, BHQ or BHQ2.
For convenience of verification, in the following example 1, when designing primers and probes, 5' ends of the probes of BPyV1 and BPyV3 were coupled with a HEX fluorescent group and a FAM fluorescent group, respectively; the 5 'ends of the probes of BPyV2a and BPyV2b are coupled with Texas Red fluorescent groups, and the 3' ends of the probes of BPyV1, BPyV2a, BPyV2b and BPyV3 are coupled with BHQ1 quenching groups.
Example 1 design and Synthesis of BPyV primer probes
The applicant carried out sequence alignment of genomes of BPyV1, BPyV2 (BPyV 2a, BPyV2 b) and BPyV3 downloaded from NCBI, and designed primer probes capable of specifically detecting each genotype of BPyV, respectively, with the sequences shown in Table 1 below. Wherein each primer is custom made by Tian Yi Yuan Biotechnology Co., ltd (PAGE purification), and the probe is custom made by biological organisms (HPLC purification).
TABLE 1BPyV qPCR primer and probe sequences
Remarks: in the case of single qPCR, the detection probes of BPyV1, BPyV2a, BPyV2b and BPyV are FAM fluorescent groups.
Example 2 establishment of BPyV Single qPCR reaction System and procedure
The linearity and sensitivity of the BPyV qPCR reactions were optimized using the reaction systems and procedures in tables 2.1-2.4.
TABLE 2.1BPyV1 reaction System
| Component (A) | Usage amount (μl) |
| AceQ Universal U+Probe Master Mix V2 | 10 |
| Upstream primer (10. Mu.M) | 1 |
| Downstream primer (10. Mu.M) | 1 |
| Probe (10 mu M) | 0.8 |
| RNase-free water+ template to be detected | 7.2 |
| Total volume of | 20 |
TABLE 2.2BPyV2 reaction System
TABLE 2.3BPyV3 reaction System
| Component (A) | Usage amount (μl) |
| AceQ Universal U+Probe Master Mix V2 | 10 |
| Upstream primer (10. Mu.M) | 1 |
| Downstream primer (10. Mu.M) | 1 |
| Probe (10 mu M) | 0.8 |
| RNase-free water+ template to be detected | 7.2 |
| Total volume of | 20 |
TABLE 2.4BPyV reaction procedure
Example 3 establishment of BPyV triple qPCR reaction System and program
The linearity and sensitivity of the BPyV triple qPCR reactions were optimized using the reaction systems and procedures in tables 3.1-3.2.
Table 31BPyV triple qPCR reaction system
| Component (A) | Usage amount (μl) |
| Probe qPCR MIX，with UNG(2×) | 10 |
| RP1+RP2+RP3(50μM) | 0.2+0.2+0.2 |
| FP1+FP3(50μM) | 0.2+0.2 |
| FP2a+FP2b(50μM) | 0.1+0.1 |
| probe1+probe2a+Probe2b+probe3(20μM) | 0.4+0.4+0.4+0.4 |
| RNase-free water+ template to be detected | 7.2 |
| Total volume of | 20 |
TABLE 3.2BPyV triple qPCR program
Example 4 plasmid sensitivity and Massa Medicata Fermentata of BPyV qPCR detection System
Artificially synthesizing genome sequences of 4 BPyVs (BPyV 1, BPyV2a, BPyV2b and BPyV 3) and constructing recombinant plasmid preparation standard substances, and establishing a standard curve: artificial construction of pUC57-BPyV1, pUC57-BPyV2a, pUC57-BPyV2b, pUC57-BPyV3 recombinant plasmids using Plasmid Mini KitI (OMEGA, D6943-02) was subjected to plasmid standard extraction, plasmid concentration was measured, and plasmid copy number was calculated according to the following formulas 1 and 2, diluted to 2X 10 6 copies/μl-2×10 2 The copies/. Mu.l plasmid standard solution was used as the points of the standard curve. 2X 10 1 The copies/. Mu.l plasmid standard solution served as a sensitivity control. qPCR was performed using the reaction systems and amplification procedures of tables 2.1-2.4, and 3 independent replicates were performed on different 3 days, each run sensitivity control replicated 8 wells. The results show that: the amplification efficiency of the 4BPyV plasmid markers is more than 90%, and the correlation coefficient R 2 The sensitivity of the plasmid reaches 100copies/reaction, which is more than or equal to 0.99. The sensitivity detection results of the single qPCR reaction system are shown in Table 4 and FIG. 1.
Equation 1:
equation 2: assume that the total volume after dilution is 100uL
TABLE 4 sensitivity detection of a singleplex qPCR reaction System
pUC57-BPyV1, pUC57-BPyV2a, pUC57-BPyV2b, pUC57-BPyV3 recombinant plasmids were diluted to 2X 10, respectively 9 cobies/. Mu.l; 10ul 2X 10 each 9 The copies/. Mu.l of pUC57-BPyV1, pUC57-BPyV2a or pUC57-BPyV2b, pUC57-BPyV3 were added with 70ul RNase-free water to prepare a BPyV mixture containing 3 genotypes; 10-fold gradient dilution to 2X 10 1 COPIES/. Mu.l. Taking 2X 10 6 COPIES/. Mu.l to 2X 10 2 The copies/. Mu.l plasmid standard solution was used as the standard curve point, 2X 10 1 The copies/. Mu.l plasmid standard solution served as a sensitivity control. qPCR was performed using the reaction systems and amplification procedures of tables 3.1-3.2, at different 3 daysThe experiment was repeated 3 times independently, 8 wells being repeated for each experimental sensitivity control. The results show that: the mixed plasmid standard of BPyV1, BPyV2a and BPyV3 is used as a template or the mixed plasmid standard of BPyV1, BPyV2b and BPyV3 is used as a template, the amplification efficiency of each standard curve is more than 90 percent, and the correlation coefficient R 2 The sensitivity of the plasmid reaches 100copies/reaction, which is more than or equal to 0.99. The sensitivity detection results of the triple qPCR reaction system are shown in Table 5 and FIG. 2.
TABLE 5 sensitivity detection of triple qPCR reaction systems
In conclusion, the sensitivity of the single qPCR reaction system and the sensitivity of the triple qPCR reaction system can reach 100copies/reaction, which indicates that the sensitivity of the triple qPCR reaction system provided by the invention is not affected.
Example 5 specificity verification of BPyV detection method
(1) Human and animal cell genomic DNA
Bovine kidney cells MDBK, african green monkey kidney cells Vero, chinese hamster ovary cells CHO-K1, and human embryonic kidney cells 293 were all maintained by the present company, and genomic DNA was extracted using QIAamp O R Mini kit (50) (QIAGEN, 51304).
(2) Viral genomic DNA
Bovine adenovirus BADV and bovine parvovirus BPV were all maintained by the company, and genomic DNA was extracted using Viral Nucleic Acid purification kit (simgen, 4002050).
After the nucleic acid extraction is completed, the preparation of the working concentration genome DNA is carried out: measuring the concentration of the above extracted cell genome DNA by using a micro-spectrophotometer, diluting the human and animal cell genome to 20 ng/. Mu.l by using RNase-free Water according to the measured concentration, and taking the physical titer of virus BADV and BPV to be 10 8 DNA was extracted from the copies/ml virus solution, and 5ul of the extracted DNA was used as a template to participate in qPCR. 3 pUC57-BPyV plasmid markers 100copies/reaction were used as qPCR sensitivity controls, and the reaction system and amplification procedure were as described in tables 3.1-3.2.BPyV qPCR detection system and BADV and BPV with or without cross-reactionThe results of the verification are shown in FIG. 3, which shows that the BPyV qPCR detection system has no cross reaction with common engineering cell genome and common bovine-derived virus BADV and BPV genome, and the amplification condition of non-bovine polyoma virus genomic DNA is summarized in Table 6.
TABLE 6 non-bovine polyoma viral genomic DNA amplification
The qPCR reaction systems and amplification procedures of tables 3.1-3.2 were used to detect BPyV1, BPyV2a, BPyV2b, and BPyV3, respectively, using 4 pUC57-BPyV plasmid markers, respectively, as templates. As shown in FIG. 4, the FAM channel amplified only BPyV3, the HEX channel amplified only BPyV1, and the Texas Red channel amplified only BPyV2a and BPyV2b. The invention can specifically amplify and distinguish BPyV1, BPyV2a/2b and BPyV3.
Experimental example 6 durability verification of BPyV-qPCR detection method
Bovine serum is widely used in the cell culture and biological product production processes, a common engineering cell line 293/CHO/Vero is selected for durability verification, and whether the normal detection of BPyV is affected or not is detected by the common engineering cell genome DNA on BPyV target amplification inhibition.
The 293/CHO/Vero cell genome was extracted, measured and diluted to 80 ng/. Mu.l for use. With 100copies/reaction pUC57-BPyV1, pUC57-BPyV2a, pUC57-BPyV2b and pUC57-BPyV3 plasmid standards as controls, 400ng/reaction of 293/CHO/Vero cell genome was introduced into each BPyV plasmid standard of 100copies/reaction, and the qPCR reaction system and the procedure were the same as in tables 2.1-2.4. As shown in FIG. 5 and Table 7, the introduction of 293T/CHO/Vero cell genome did not affect BPyV detection and did not result in false negative of the samples.
TABLE 7 durability verification results for 293/CHO/Vero
Application example of application example BPyV-qPCR detection method
The cultured Vero cell samples (medium dmem+10% fbs) were subjected to BPyV examination using the triple fluorescence qPCR method in this protocol to determine whether there was BPyV contamination. The specific method comprises the following steps:
negative control: 200ul PBS was used as a negative control;
sample to be detected: take 10 6 Adding 200uLPBS into Vero cells;
positive control: respectively diluting 100uL with PBS to 10 4 PFU/mL of BPyV1, BPyV2a and BPyV3 pseudoviruses are added with 700uLPBS to prepare virus liquid, and 200uL is taken as positive control;
suitability control: adding the same virus liquid as the positive control into the detection sample;
all the above samples were subjected to qPCR detection using Viral Nucleic Acid purification kit (simmen, 4002050) to extract DNA, and 5ul was used as a working solution.
Template-free control: rnase-free water;
sensitivity control: and (3) taking BPyV1, BPyV2b and BPyV3 mixed plasmid solution to carry out gradient dilution by 1000 times, namely respectively carrying out sensitivity control on the BPyV1, the BPyV2b and the BPyV3 containing 100copies/reaction.
The reaction system and the amplification procedure are as shown in tables 3.1-3.2. The test results are shown in Table 8: and judging that the Vero cells have no BPyV pollution according to the BPyV qPCR detection method judgment standard.
TABLE 8 detection results
The result judgment standard of the BPyV-qPCR detection method is as follows:
1. test effectiveness
The following requirements are satisfied simultaneously in the same experiment, otherwise, the experiment is ineffective.
1) Template-free control: no Ct value, no obvious amplification curve.
2) Sensitivity control: has obvious amplification curve and Ct value less than or equal to 38.
3) Negative control: no Ct value, no obvious amplification curve.
4) Positive control: has obvious amplification curve, and Ct value is less than or equal to 35.
5) Suitability control: there is an obvious amplification curve, if the Ct value becomes larger, the difference from the positive control Ct value is within 3Ct value.
2. Sample detection result determination
The criteria for determining the results of the sample detection are shown in the following table 9:
TABLE 9 sample test result criterion
The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.
Claims (10)
1. The primer probe combination for detecting the Niu Duo tumor virus triple qPCR is characterized by comprising primer probe combinations for detecting bovine polyomavirus BPyV1, BPyV2a, BPyV2b and BPyV3, wherein the nucleotide sequences of a forward primer, a reverse primer and a probe of the BPyV1 are respectively shown as SEQ ID NO. 1-3; the nucleotide sequences of the forward primer and the probe of the BPyV2a are respectively shown as SEQ ID NO. 4-5; the nucleotide sequences of the forward primer and the probe of the BPyV2b are respectively shown in SEQ ID NO. 6-7; the nucleotide sequences of the reverse primers of the BPyV2a and the BPyV2b are shown in SEQ ID NO. 8; the nucleotide sequences of the forward primer, the reverse primer and the probe of the BPyV3 are respectively shown as SEQ ID NO. 9-11;
the 5' ends of probes for detecting bovine polyoma viruses BPyV1 and BPyV3 are respectively coupled with a first fluorescent group and a third fluorescent group; the 5 'end of the probe for detecting the bovine polyomaviruses BPyV2a and BPyV2b is coupled with a second fluorescent group, and the 3' end of the probe for detecting the bovine polyomaviruses BPyV1, BPyV2a, BPyV2b and BPyV3 is coupled with a quenching group; the first fluorescent group, the second fluorescent group and the third fluorescent group are three different types of fluorescent groups.
2. The primer probe combination for bovine polyomavirus triple qPCR detection of claim 1, wherein the first, second, third fluorescent groups are FAM, TET, NED, ROX, CY3, CY5, VIC, JOE, HEX or Texas Red fluorescent groups; the quenching group is TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1 or BHQ2 quenching group.
3. The primer probe combination for bovine polyomavirus triple qPCR detection of claim 2, wherein the first fluorescent group is a HEX fluorescent group, the second fluorescent group is a Texas Red fluorescent group, and the third fluorescent group is a FAM fluorescent group; the quenching group is a BHQ1 quenching group.
4. A product of triple qPCR detection of bovine polyomavirus comprising the primer probe combination of any one of claims 1-3.
5. Use of a primer probe combination according to any one of claims 1-3, characterized in that a product for detection of bovine polyoma virus is prepared.
6. A method for detecting bovine polyoma virus not for disease diagnosis or treatment, characterized in that the product of claim 4 is used for detection.
7. The method of detecting according to claim 6, comprising the steps of: extracting nucleic acid in a sample to be detected; creating a first fluorescent group detection channel, a second fluorescent group detection channel and a third fluorescent quantitative group detection channel on a qPCR instrument, and performing triple qPCR reaction on nucleic acid of a sample to be detected; and judging the detection result according to the amplification curve and the Ct value generated by the triple qPCR reaction.
8. The method according to claim 7, wherein the reaction system of the triple qPCR reaction is: 10 mu L of Probe qPCR MIX, 0.2uL of each of forward primer and reverse primer for detecting bovine polyoma virus BPyV1 and BPyV3, 0.1uL of each of forward primer and reverse primer for detecting bovine polyoma virus BPyV2a and BPyV2b, 0.4uL of each of probes for detecting bovine polyoma virus BPyV1, BPyV2a, BPyV2b and BPyV3 and 7.2uL of template to be detected.
9. The method of claim 7, wherein the amplification procedure of the triple qPCR reaction is: firstly, pre-denaturing for one time at 25 ℃ for 10min and 95 ℃ for 30 s; then, the mixture was circulated 42 times at 95℃for 5 seconds and 60℃for 30 seconds.
10. The method according to claim 7, wherein the determination of the detection result is specifically: when the Ct value of the first fluorescent group detection channel is less than or equal to 38 and an obvious amplification curve exists, judging that the bovine polyoma virus BPyV1 is positive; when the Ct value of the second fluorescent group detection channel is less than or equal to 38 and an obvious amplification curve exists, judging that the bovine polyoma virus BPyV2a or BPyV2b is positive; when the Ct value of the third fluorescent group detection channel is less than or equal to 38 and an obvious amplification curve exists, judging that the bovine polyoma virus BPyV3 is positive;
when the Ct value of the first fluorescent group, the first fluorescent group or the third fluorescent group detection channel is more than 38 and an obvious amplification curve exists, the judgment result is invalid;
when the first fluorescent group detection channel has no Ct value and no obvious amplification curve, judging that the bovine polyoma virus BPyV1 is negative; when the second fluorescent group detection channel has no Ct value and no obvious amplification curve, judging that the bovine polyoma viruses BPyV2a and BPyV2b are negative; and when the third fluorescent group detection channel has no Ct value and no obvious amplification curve, judging that the bovine polyoma virus BPyV3 is negative.
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