CN111876528A - PCR kit of bovine papilloma virus oncogene E5 and establishment method thereof - Google Patents
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Abstract
The invention discloses a PCR kit of bovine papilloma virus oncogene E5 and an establishment method, which are specifically carried out according to the following specific steps: designing a PCR primer by referring to oncogene E5 in a bovine papilloma virus genome disclosed in GenBank, amplifying a specific fragment by PCR by using the primer with an enzyme cutting site, connecting a vector of pEGFP-N1 subjected to double enzyme cutting with an E5 oncogene fragment by using T4 ligase, transforming a connecting system into an E.coli DH5 alpha competent cell, screening a positive colony, sequencing, verifying and extracting a plasmid to obtain a positive standard quality particle pEGFP-N1-E5; and (3) carrying out PCR reaction by using the correctly identified pEGFP-N1-E5 standard product as a template to obtain the PCR kit of the bovine papilloma virus oncogene E5. The invention can quickly and effectively diagnose the bovine papilloma disease by detecting the oncogene E5, and has convenient operation and strong specificity.
Description
Technical Field
The invention belongs to the technical field of animal disease diagnosis, and particularly relates to a PCR kit of bovine papilloma virus oncogene E5, and an establishment method of the kit.
Background
Bovine Papilloma (BP) is a chronic proliferative disease caused by Bovine Papilloma Virus (BPVs) to tumorigenesis, the tumor of which usually occurs on the body surface, mucous membrane, etc., and is the most common benign tumor disease of dairy cows. Papillomatosis damages the quality of animal leather, reduces the constitution of animals, and the occurrence of diseases in genitals can damage the reproductive function of bulls and cows, reduce the production performance and cause economic loss. Under the stimulation of auxiliary factors such as environment, except benign tumors, the animal may develop upper digestive tract cancer and urinary bladder cancer, which usually cause the death of animals and finally cause serious economic loss in meat, dairy and leather industries. The Bovine Papilloma Virus (BPV) causing this disease is a double-stranded DNA virus that induces benign hyperproliferation of skin and mucosal epithelium, where BPV-13 belongs to the delta Papilloma Virus (PVs), also commonly referred to as the fibromyalgia virus. The E5 gene is the major oncogene of BPV, which is mainly localized in the membrane and endoplasmic reticulum of the golgi apparatus of host cells, and the E5 protein acts by interfering with cell growth inhibition and cell cycle regulation, interacts with growth factor receptors, and activates cell proliferation.
According to clinical manifestations, epidemiology, pathology observation and other ways, the primary diagnosis of bovine papilloma disease can be easily made; however, bovine papilloma occurs not only in the skin mucosa but also in organs such as the digestive tract and the bladder, and there are some cases of recessive infection of cattle or atypical cattle, for which there is no effective diagnostic method, and the diagnosis must be confirmed by a laboratory. However, since there are many types of BPV and there are differences in tissue specificity, it is necessary to identify BPV by applying molecular biology techniques. The method specifically comprises the detection methods of sequencing, electron microscopy, immunofluorescence and the like, and the confirmed diagnosis can be obtained by combining clinical data. However, these methods have disadvantages of complicated operation and high cost. Therefore, a method for rapidly and effectively detecting and diagnosing bovine papilloma disease is urgently needed. The PCR method has been widely used for rapid detection of many animal diseases because of its advantages such as convenient operation, rapid sensitivity and strong specificity. In conclusion, the invention establishes the PCR diagnostic kit capable of diagnosing bovine papilloma disease rapidly and efficiently.
Disclosure of Invention
The first purpose of the invention is to provide a PCR kit of bovine papilloma virus oncogene E5, which can diagnose bovine papilloma virus rapidly and efficiently.
The second purpose of the invention is to provide a method for establishing the PCR kit of the bovine papilloma virus oncogene E5.
In order to achieve the above object, a first technical solution adopted by the present invention is as follows:
the PCR kit of the bovine papilloma virus oncogene E5 comprises a reaction system and a reaction system, wherein the reaction system comprises a pair of PCR primers designed aiming at the bovine papilloma virus oncogene E5:
the upstream primer BPV-E5-F is:
5’ATGCCGAATCTATGGTTTC-3’；
the downstream primer BPV-E5-R is as follows:
5’-CGAAAAGGCAGACCTGTACAGGA-3’。
the first technical solution adopted by the present invention is further characterized in that,
the reaction system of the PCR kit is as follows: the reaction system is as follows: 2 Xpfu Mix10uL, template 1uL, upstream primer BPV-E5-F0.5 uL, downstream primer BPV-E5-R0.5 uL, H2O, 20uL overall.
The concentrations of the upstream primer BPV-E5-F and the downstream primer BPV-E5-R are both 10 mu M, the template is pEGFP-N1-E5 positive standard, and the negative control sample is ddH2O。
The second technical scheme adopted by the invention is as follows:
the method for establishing the PCR kit of the bovine papilloma virus oncogene E5 is carried out according to the following specific steps:
PCR primers were designed with reference to oncogene E5 in the bovine papilloma virus genome published in GenBank, and the upstream primers were: 5 'ATGCCGAATCTATGGTTTC-3'; the downstream primer is 5' -CGAAAAGGCAGACCTGTACAGGA-3;
Amplifying a specific fragment with the size of 134bp by using a primer with an enzyme cutting site in the step 1 through PCR, connecting a vector of pEGFP-N1 subjected to double enzyme cutting and an E5 oncogene fragment by using T4 ligase, transforming a connecting system into E.coli DH5 alpha competent cells, screening out positive bacterial colonies, performing sequencing verification, and extracting plasmids to obtain a positive standard quality particle pEGFP-N1-E5;
And (3) performing PCR reaction on the genome to be detected by using the group of sequences designed in the step (1) as primers and the pEGFP-N1-E5 standard substance identified correctly in the step (2) as a template to obtain the PCR diagnostic kit of the bovine papilloma virus E5 gene.
The second technical solution adopted by the present invention is further characterized in that,
the optimal primer concentration in the PCR reaction process in step 3 is 0.25 uM/uL.
The optimal primer amount during the PCR reaction in step 3 was 0.5 uL.
The optimal template amount in the PCR reaction process in step 3 is 1 uL.
The optimal annealing temperature during the PCR reaction in step 3 was 60 ℃.
The optimal cycle number in the PCR reaction process in step 3 is 35.
The invention has the beneficial effects that: the PCR kit of the oncogenic gene E5 of the bovine papilloma virus is convenient to operate, low in price, and capable of rapidly, accurately and sensitively detecting the bovine papilloma virus through the oncogenic gene E5, the existing method needs large-scale manpower and material resources to detect, consumes long time, is difficult to achieve the detection accuracy of the invention, and has wide application in the detection of the bovine papilloma virus.
Drawings
FIG. 1 is a schematic diagram showing the result of PCR amplification of E5 gene in the method for constructing the PCR kit for bovine papilloma virus oncogene E5 of the present invention;
FIG. 2 is a diagram showing the colony PCR identification of the pEGFP-N1-E5 recombinant plasmid in the method for constructing the PCR kit for bovine papilloma virus oncogene E5 of the present invention;
FIG. 3 is a diagram showing the restriction enzyme digestion of the pEGFP-N1-E5 recombinant plasmid in the method for constructing the PCR kit for bovine papilloma virus oncogene E5 of the present invention.
Detailed Description
The invention is further elucidated with reference to the drawings and the detailed description.
The invention refers to a bovine papilloma virus (accession number: KM258443) genome E5 oncogene published in GenBank, obtains a gene coding frame thereof by using information analysis software, establishes a rapid PCR detection method of the bovine papilloma virus by establishing and optimizing a PCR reaction system, and verifies the rapid PCR detection method by respectively performing specificity experiments, sensitivity experiments, repeatability experiments and the like.
The method for establishing the PCR kit of the bovine papilloma virus oncogene E5 specifically comprises the following steps of:
PCR primers were designed based on the E5 oncogene in the bovine papilloma virus genome:
the upstream primer BPV-E5-F is:
5’ATGCCGAATCTATGGTTTC-3’；
the downstream primer BPV-E5-R is as follows:
5’-CGAAAAGGCAGACCTGTACAGGA-3’。
A primer with an enzyme cutting site is used for amplifying a specific fragment with the size of 134bp through PCR, the PCR amplification result of the E5 gene is shown in figure 1, and a double-enzyme-cut pEGFP-N1 vector and an E5 oncogene are used as a construct (the total system is 10 uL): pEGFP-N11 uL, E57 uL, T4 DNA ligase 1uL, 10 XT 4 DNA ligase buffer 1uL, deionized water make up to 10uL, placed at 16 ℃, connected overnight. Transforming the connecting system into escherichia coli DH5 alpha competent cells, screening out positive colonies, identifying through colony PCR, finding out a band at a 134bp position amplified in the positive colonies as a result, storing correctly identified positive bacteria into glycerol as shown in figure 2, wherein the storage temperature is-20 ℃, simultaneously carrying out amplification culture on the positive colonies, extracting plasmids, carrying out double enzyme digestion to identify recombinant plasmids, and as shown in figure 3, the plasmid double enzyme digestion identification is correct, which indicates that a positive standard product pEGFP-N1-E5 is successfully prepared.
Step 3.1 determination of optimal primer concentration
PCR reaction was performed with the positive template pEGFP-N1-E5 in the following reaction scheme (20 uL overall): 2 Xpfu Mix10uL and a template 1uL, respectively adding 0.5uL of upstream and downstream primers with different concentrations of 5uM, 10uM and 20uM, adding water to supplement the water to 20uL, uniformly blowing, placing the mixture in a PCR instrument for reaction under the conditions of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10s, annealing at 62 ℃ for 10s, extension at 72 ℃ for 30s and 30 cycles, comparing the amplification efficiency of different primer concentrations, and finally obtaining the primer concentration of 10uM as the optimal primer concentration through comparison.
Step 3.2 determination of optimal primer amount
And (3) carrying out PCR reaction by using a positive template pEGFP-N1-E5 and a test positive sample, wherein the reaction system is (20 uL in total): 2 Xpfu Mix10uL and 1uL of template, sequentially adding upstream and downstream primers of 0.5, 1.0 and 1.5uL, adding water to supplement 25uL, blowing, uniformly mixing, placing in a PCR instrument for reaction under the conditions of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10s, annealing at 62 ℃ for 10s, and extension at 72 ℃ for 30s for 30 cycles. And comparing the amplification efficiency of different primer amounts, and finally obtaining the primer amount of 0.5uL as the optimal primer amount through comparison, so that the amplification effect is optimal.
Step 3.3 determination of the optimal template amount
And (3) carrying out PCR reaction by using a positive template pEGFP-N1-E5 and a test positive sample, wherein the reaction system is (20 uL in total): 2 Xpfu Mix10uL, upstream and downstream primers of 0.5uL, template sequentially adding 0.5, 1.0, 1.5uL, adding water to supplement to 20uL, blowing and mixing uniformly, placing in a PCR instrument for reaction under the conditions of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10s, annealing at 62 ℃ for 10s, and extension at 72 ℃ for 30s, 30 cycles. And comparing the amplification efficiency of different template amounts, and finally obtaining the template amount of 1.0uL as the optimal template amount through comparison, so that the amplification effect is optimal.
Step 3.4 determination of the optimum annealing temperature
PCR reaction was performed with the positive template pEGFP-N1-E5 in the following reaction scheme (20 uL overall): 2 Xpfu Mix10uL, a template 1uL, an upstream primer and a downstream primer are respectively 0.5uL, water is added to supplement 20uL, the mixture is uniformly blown and placed in a PCR instrument for reaction, the reaction conditions are 94 ℃ pre-denaturation for 3min, 94 ℃ denaturation for 10s, annealing sequentially at 55 ℃, 60 ℃, 65 ℃ for 10s, 72 ℃ extension for 30s and 30 cycles, the amplification efficiencies of different annealing temperatures are compared, the 60 ℃ annealing temperature is finally obtained through comparison and is the optimal annealing temperature, and the amplification effect is optimal.
5. Determination of optimum number of cycles
The PCR reaction was carried out with the positive template pEGFP-N1-E5 and test positive samples in the following reaction scheme (20 uL overall): 2 Xpfu Mix10uL, a template 1uL, an upstream primer and a downstream primer are respectively 0.5uL, water is added to supplement 20uL, the mixture is uniformly blown and placed in a PCR instrument for reaction, the reaction conditions are pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10s, annealing at 62 ℃ for 10s, extension at 72 ℃ for 30s, the cycle times are 30, 35 and 40 in sequence, and the optimal cycle times are obtained by comparison, and the optimal cycle times are 35 cycle times, so that the amplification effect is optimal.
After primers BPV-E5-F and BPV-E5-R of the bovine papilloma virus E5 gene designed in the step 1 are used, an optimal reaction condition is designed through an experiment in the step 3, a pEGFP-N1-E5 standard substance identified correctly in the step 2 is used as a template, and a BPV-E5 primer is used for carrying out PCR amplification on a genome;
and 5, the results of the negative control and the positive control prove that the primers can be used for amplifying target bands of the bovine papilloma virus, the size of target band fragments is 134bp, no non-specific fragments are generated, and the result shows that the establishment of the PCR detection method for the bovine papilloma virus is successful.
Claims (9)
1. The PCR kit of the bovine papilloma virus oncogene E5 is characterized in that the reaction system comprises a pair of PCR primers designed aiming at the oncogene E5 of the bovine papilloma virus:
the upstream primer BPV-E5-F is as follows:
5’ATGCCGAATCTATGGTTTC-3’；
the downstream primer BPV-E5-R is as follows:
5’-CGAAAAGGCAGACCTGTACAGGA-3’。
2. the PCR kit of bovine papilloma virus oncogene E5 according to claim 1, wherein the reaction system of the PCR kit is: the reaction system is as follows: 2 Xpfu Mix10uL, template 1uL, upstream primer BPV-E5-F0.5 uL, downstream primer BPV-E5-R0.5 uL, H2O, 20uL overall.
3. The PCR kit for the oncogene E5 of bovine papilloma virus according to claim 2, wherein the concentrations of the forward primer BPV-E5-F and the reverse primer BPV-E5-R are 10. mu.M, the template is a positive standard pEGFP-N1-E5, and the negative control sample is ddH2O。
4. The method for establishing the PCR kit of the bovine papilloma virus oncogene E5 of claim 1, comprising the following steps:
step 1, design of PCR primer
Designing PCR primers by referring to oncogene E5 in bovine papilloma virus genome published in GenBank, wherein the upstream primers are: 5 'ATGCCGAATCTATGGTTTC-3'; the downstream primer is 5' -CGAAAAGGCAGACCTGTACAGGA-3;
step 2, preparation of positive standard substance pEGFP-N1-E5
Amplifying a specific fragment with the size of 134bp by using a primer with an enzyme cutting site in the step 1 through PCR, connecting a vector of pEGFP-N1 subjected to double enzyme cutting and an E5 oncogene fragment by using T4 ligase, transforming a connecting system into E.coli DH5 alpha competent cells, screening out positive bacterial colonies, performing sequencing verification, and extracting plasmids to obtain a positive standard quality particle pEGFP-N1-E5;
step 3, establishment of PCR reaction system
And (3) performing PCR reaction on the genome to be detected by using the group of sequences designed in the step (1) as primers and the pEGFP-N1-E5 standard substance identified correctly in the step (2) as a template to obtain the PCR diagnostic kit of the bovine papilloma virus E5 gene.
5. The method of claim 4, wherein the optimal primer concentration during the PCR reaction in step 3 is 0.25 uM/uL.
6. The method of claim 4, wherein the optimal primer amount in the PCR reaction process of step 3 is 0.5 uL.
7. The method of claim 4, wherein the optimal template amount in the PCR reaction process of step 3 is 1 uL.
8. The method of claim 4, wherein the optimal annealing temperature for the PCR reaction in step 3 is 60 ℃.
9. The method of claim 4, wherein the optimal number of cycles in the PCR reaction of step 3 is 35.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992020784A1 (en) * | 1991-05-16 | 1992-11-26 | Yale University | Keratinocyte or epithelial cell line which expresses human papillomavirus e5 gene |
| WO2001051660A2 (en) * | 2000-01-14 | 2001-07-19 | Vera Genics Ltd. | Method and kit for diagnosis of neoplastic transformation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992020784A1 (en) * | 1991-05-16 | 1992-11-26 | Yale University | Keratinocyte or epithelial cell line which expresses human papillomavirus e5 gene |
| WO2001051660A2 (en) * | 2000-01-14 | 2001-07-19 | Vera Genics Ltd. | Method and kit for diagnosis of neoplastic transformation |
Non-Patent Citations (2)
| Title |
|---|
| G.CHAMBERS等: "Sequence variants of bovine papillomavirus E5 detected in equine sarcoids", 《VIRUS RESEARCH》 * |
| 庞峰等: "牛乳状瘤病毒13型pEGFP-E5-N1真核表达载体的构建及其在HEK293细胞中的表达", 《中国畜牧兽医》 * |
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Application publication date: 20201103 |