Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

A bovine leukemia and kit technology, which is applied to the determination/inspection of microorganisms, fluorescence/phosphorescence, biochemical equipment and methods, etc., can solve the problems of cumbersome procedures, inaccurate quantification, complicated steps, etc., and achieves simple and fast methods and results. Ease of judgment and strong specificity

Inactive Publication Date: 2014-08-06
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional BLV detection generally adopts the method of nested PCR amplification, target fragment sequencing and sequence comparison. This method has been widely used in the field of PCR technology, but its own shortcomings are also obvious: time-consuming and laborious, cumbersome procedures, and complicated steps , the cycle is long, the labor cost is high, and it is not conducive to the detection of a large number of samples; there are still many limitations in the application of clinical genetic diagnosis, mainly in the inaccurate quantification and false positives due to cross-contamination; and high personnel requirements, sequencing The results require personnel with certain professional knowledge to perform sequence comparison to finally judge the results, while the judgment of fluorescent PCR results is simple and intuitive

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Kit for detecting proviral DNA (deoxyribonucleic acid) of bovine leukemia virus (BLV) and application of kit
  • Kit for detecting proviral DNA (deoxyribonucleic acid) of bovine leukemia virus (BLV) and application of kit
  • Kit for detecting proviral DNA (deoxyribonucleic acid) of bovine leukemia virus (BLV) and application of kit

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 李凯航鞠厚斌刘佩红周锦萍王建陈冈
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com