Kit for detecting proviral DNA (deoxyribonucleic acid) of bovine leukemia virus (BLV) and application of kit

Abstract

The invention provides a kit for detecting proviral DNA (deoxyribonucleic acid) of bovine leukemia virus (BLV). The kit comprises (a) fluorescent PCR (polymerase chain reaction) liquid reactant, (b) a fluorescent probe, (c) hot start Tag enzyme, (d) a standard positive template and (e) negative control, wherein the fluorescent PCR liquid reactant contains forward primers and reverse primers; the sequence of the forward primers is shown in SEQ ID NO:2; the sequence of the reverse primers is shown in SEQ ID NO:3; the sequence of the fluorescent probe is shown in SEQ ID NO:4; fluorescent substance is designed at the 5' end of the sequence of the fluorescent probe; cancellation substance is designed at the 3' end of the sequence of the fluorescent probe; and the nucleotide insertion sequence of the standard masculine template is shown in SEQ ID NO:5. The invention further provides application of the kit to detecting proviral DNA of BLV in whole blood. The PCR detection kit provided by the invention can be used for quickly and accurately detecting BLV and is applicable to quick diagnosis of BLV, epidemiological investigation and risk assessment.

Description

technical field

[0001] The invention belongs to the field of biological detection, and in particular relates to a kit for detecting bovine leukemia virus preDNA and application thereof. Background technique

[0002] Epidemic bovine leukemia is a chronic neoplastic disease of cattle caused by bovine leukemia virus (BLV). It is characterized by malignant proliferation of lymphoid cells, progressive cachexia, and high mortality after onset. At present, the disease has spread almost all over the cattle-raising countries in the world. It was first discovered in Hefei, Anhui Province in 1976 in my country, and then occurred successively in various provinces and cities. In recent years, the seropositive rate of the disease in cattle herds in my country has been increasing year by year, from about 4% to 30% to 50%. The Veterinary Epidemic Prevention Guidelines for Raising Dairy Cattle with Pollution-Free Food (NY 5047-2001) and the Epidemic Prevention Guidelines for Raising Beef C...

Examples

Embodiment 1

[0029] Design and synthesis of embodiment 1 primers and probes

[0030] The full-length genome of BLV has an accession number of FJ914764.1 on GenBank, and the present invention is aimed at the pol gene of BLV, which is 2559 bp in full length, and the coding sequence is located at 2320-4878 bp of the full BLV gene, and the sequence is as shown in SEQ ID NO: 1 Show.

[0031] A pair of specific primers were designed according to the BLV pol gene sequence as follows:

[0032] Upstream primer C1: 5'-AACCCCACCTTCCCATGAC-3';

[0033] Downstream primer C2: 5'-TGGTAGGAGGTTAATCTGATTGTGAGT-3';

[0034] Its role is to specifically amplify the pol gene fragment of BLV pre-DNA.

[0035] Fluorescent probes were designed according to the sequence of the BLV pol gene, the sequence of which is as follows:

[0036] FAM-CAGGCCCTTTCTCGAGCCCTCTG-TAMRA

[0037]Among them, in addition to FAM (hydroxyfluorescein), the fluorescent substance at the 5' end of the probe sequence can also be selected...

Embodiment 2

[0039] The preparation of embodiment 2 standard positive template

[0040] 1. Take a blood sample infected with bovine leukemia virus and extract its DNA. For the specific steps, please refer to the instruction manual of AxyPrep Body Fluid Virus DNA / RNA Mini Kit from AXYGEN Company. The detailed steps are as follows:

[0041] a) Take 200 μL of the sample, add DNA lysate Buffer V-L, vortex and oscillate to mix evenly, and let it stand for 5 minutes.

[0042] b) Add 75 μL Buffer V-N, vortex to mix evenly, and centrifuge at 12000×g for 5 minutes.

[0043] c) Transfer the supernatant to a new 2ml centrifuge tube, add 300μL isopropanol (1% glacial acetic acid), invert up and down 6-8 times, and mix well.

[0044] d) Put the preparation tube in a 2ml centrifuge tube, transfer the mixture in step 3 into the preparation tube, and centrifuge at 6000×g for 1 min.

[0045] e) Discard the filtrate, put the preparation tube back into a 2ml centrifuge tube, add 500 μL Buffer W1A, and le...

Embodiment 3

[0053] Embodiment 3 The optimization of fluorescent PCR reaction conditions of this kit

[0054] 1. Optimization of primer concentration:

[0055] Set the final concentration range of a single primer to 50-1000nM, and perform fluorescent PCR experiments with primers of different concentration gradients. The results show that primers in this concentration range can amplify the S-shaped curve, and the effect is the best when the final concentration of each primer is 0.24μM good.

[0056] 2. Mg 2+ Concentration Optimization:

[0057] Set Mg 2+ The final concentration range is 1.5-5.0mM, and the results show that Mg in this concentration range 2+ Both can amplify the S-shaped curve, and when Mg 2+ When the final concentration is 2mM, the amplification effect is the best.

[0058] 3. Optimization of dNTPs concentration:

[0059] The four dNTPs were mixed in equal amounts, and the final concentration range of each dNTPs was set to 0.1-1mM. The results showed that the dNTPs in...

technical field

[0001] The invention belongs to the field of biological detection, and in particular relates to a kit for detecting bovine leukemia virus preDNA and application thereof. Background technique

[0002] Epidemic bovine leukemia is a chronic neoplastic disease of cattle caused by bovine leukemia virus (BLV). It is characterized by malignant proliferation of lymphoid cells, progressive cachexia, and high mortality after onset. At present, the disease has spread almost all over the cattle-raising countries in the world. It was first discovered in Hefei, Anhui Province in 1976 in my country, and then occurred successively in various provinces and cities. In recent years, the seropositive rate of the disease in cattle herds in my country has been increasing year by year, from about 4% to 30% to 50%. The Veterinary Epidemic Prevention Guidelines for Raising Dairy Cattle with Pollution-Free Food (NY 5047-2001) and the Epidemic Prevention Guidelines for Raising Beef C...

Examples

Embodiment 1

[0029] Design and synthesis of embodiment 1 primers and probes

[0030] The full-length genome of BLV has an accession number of FJ914764.1 on GenBank, and the present invention is aimed at the pol gene of BLV, which is 2559 bp in full length, and the coding sequence is located at 2320-4878 bp of the full BLV gene, and the sequence is as shown in SEQ ID NO: 1 Show.

[0031] A pair of specific primers were designed according to the BLV pol gene sequence as follows:

[0032] Upstream primer C1: 5'-AACCCCACCTTCCCATGAC-3';

[0033] Downstream primer C2: 5'-TGGTAGGAGGTTAATCTGATTGTGAGT-3';

[0034] Its role is to specifically amplify the pol gene fragment of BLV pre-DNA.

[0035] Fluorescent probes were designed according to the sequence of the BLV pol gene, the sequence of which is as follows:

[0036] FAM-CAGGCCCTTTCTCGAGCCCTCTG-TAMRA

[0037]Among them, in addition to FAM (hydroxyfluorescein), the fluorescent substance at the 5' end of the probe sequence can also be selected...

Embodiment 2

[0039] The preparation of embodiment 2 standard positive template

[0040] 1. Take a blood sample infected with bovine leukemia virus and extract its DNA. For the specific steps, please refer to the instruction manual of AxyPrep Body Fluid Virus DNA / RNA Mini Kit from AXYGEN Company. The detailed steps are as follows:

[0041] a) Take 200 μL of the sample, add DNA lysate Buffer V-L, vortex and oscillate to mix evenly, and let it stand for 5 minutes.

[0042] b) Add 75 μL Buffer V-N, vortex to mix evenly, and centrifuge at 12000×g for 5 minutes.

[0043] c) Transfer the supernatant to a new 2ml centrifuge tube, add 300μL isopropanol (1% glacial acetic acid), invert up and down 6-8 times, and mix well.

[0044] d) Put the preparation tube in a 2ml centrifuge tube, transfer the mixture in step 3 into the preparation tube, and centrifuge at 6000×g for 1 min.

[0045] e) Discard the filtrate, put the preparation tube back into a 2ml centrifuge tube, add 500 μL Buffer W1A, and le...

Embodiment 3

[0053] Embodiment 3 The optimization of fluorescent PCR reaction conditions of this kit

[0054] 1. Optimization of primer concentration:

[0055] Set the final concentration range of a single primer to 50-1000nM, and perform fluorescent PCR experiments with primers of different concentration gradients. The results show that primers in this concentration range can amplify the S-shaped curve, and the effect is the best when the final concentration of each primer is 0.24μM good.

[0056] 2. Mg 2+ Concentration Optimization:

[0057] Set Mg 2+ The final concentration range is 1.5-5.0mM, and the results show that Mg in this concentration range 2+ Both can amplify the S-shaped curve, and when Mg 2+ When the final concentration is 2mM, the amplification effect is the best.

[0058] 3. Optimization of dNTPs concentration:

[0059] The four dNTPs were mixed in equal amounts, and the final concentration range of each dNTPs was set to 0.1-1mM. The results showed that the dNTPs in...

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