Primers, probe and kit

A technology of kits and detection kits, applied in the field of probes and kits, PCR primers, can solve the problems of small fragments, uneven distribution, low content, etc., achieve small physiological variation, ensure positive rate, and increase detection rate Effect

Pending Publication Date: 2021-03-02
CANCER CENT OF GUANGZHOU MEDICAL UNIV
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Problems solved by technology

However, when plasma is used as a test sample, since the Epstein-Barr virus DNA in plasma is fragmented free viral DNA, it has the characteristics of small fragments, low content, and uneven distribution of different gene fragment concentrations in plasma.
Therefore, the establishment of quantitative PCR detection method has high requirements on the size of the amplification target region, primers, probes and amplicons, and the detection methods designed for different amplification target regions may have inconsistent detection results when detecting low-concentration samples. Case
3. Epstein-Barr virus gene copy number variation: There are multiple multi-copy gene regions in the Epstein-Barr virus genome, such as the BamHI-W region, etc., and there are 5-11 repetitions in the DNA sequence in these regions, so when PCR amplifies the target region When located in such an area, Epstein-Barr virus DNA can theoretically be detected more sensitively, but the naturally occurring copy number difference will affect the accuracy of quantitative results and is not conducive to the establishment of a unified clinical diagnosis threshold
4. The problem of detection sensitivity: In view of the fact that the detection of Epstein-Barr virus DNA in plasma has high application value for nasopharyngeal carcinoma screening, prognosis and efficacy evaluation, it is expected that plasma Epstein-Barr virus DNA can be detected as sensitively as possible in clinical practice
However, when the PCR amplification target region is selected in the single-copy gene region, although it can be accurately quantified in theory, the detection sensitivity is not as good as that of the target detection region designed in the multi-copy region

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Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Epstein-Barr virus EBNA1 gene conservation region screening and sequence feature analysis

[0022] Search the NCBI website (https: / / www.ncbi.nlm.nih.gov) to obtain the reference sequence of the EB virus EBNA1 gene (NC_007605.1), and analyze its gene structure. It can be seen that the size of the EBNA1 gene is 86KB, accounting for about 20% of the EB virus genome. Half of them are located at positions 11305-97654 of the genome, overlapping with multiple genes such as EBNA2, EBNA3a and EBNA3b / 3c. There are multiple repeat sequence regions and sequence variation regions on the gene, such as repeat sequence regions 21217-24288, 82124-82201, etc., most of which are located in the front of the gene, and hypervariable sequence regions 33127-40536, 33356-40162, 86839-89829 etc. In view of the structural characteristics of the above-mentioned EB virus EBNA1 gene, the invention selects exon 21 of the EBNA1 gene located at 96654-97654 as the target amplification region, ...

Embodiment 2

[0023] Embodiment 2: Epstein-Barr virus EBNA1 fluorescent quantitative PCR primer and probe design

[0024] A set of primers and probes were designed in the 95654-95929 and 96636-97654 regions of exon 21 of the EBNA1 gene using Oligo 7.0 software, see Table 1 for details ( figure 2 ). In order to ensure that the two sets of primer and probe systems can be successfully constructed as a dual PCR detection system in the later stage, the following principles must be ensured when designing and screening primers and probes: 1. Good specificity of primers and probes; 2. Key parameters such as the Tm value of primers and probes are close, so that the closest amplification performance can be obtained under the same PCR reaction conditions; 3. False mismatches and secondary structures between each primer and probe are as small as possible In order to ensure the maximum possible reduction of the interaction between the dual PCR systems, the performance of the two systems after the comb...

Embodiment 3

[0025] Example 3: Preliminary establishment of Epstein-Barr virus DNA fluorescent quantitative PCR detection system

[0026] Use Shanghai Zhijiang DNA Extraction Reagent to extract the DNA of 1 Epstein-Barr virus DNA-positive plasma sample according to the instructions for future use. Use the PCR amplification primer configuration reaction system of system 1 and system 2 in Table 1 to carry out PCR amplification on the extracted EB virus DNA positive plasma sample DNA on the ABI gradient PCR instrument, and identify by 1.5% agarose gel electrophoresis. Reaction system: Green Mix 10 μl, upstream and downstream primers 1 μl (10 μM), sample DNA 2 μl, pure water 6 μl. Reaction conditions: 94°C for 3min; 94°C for 5s, (58°C, 60°C, 62°C, 64°C) for 30s, a total of 40 cycles. Electrophoresis results ( image 3 A) It shows that System 1 and System 2 can effectively amplify a single fragment within the annealing and extension temperature range of 58-64°C, and the size of each single fr...

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Abstract

The invention relates to the technical field of biology, and discloses primers, a probe and a kit for EB virus DNA detection. The kit takes EB virus DNA as a template and comprises PCR amplification primers, probes, a quantitative standard substance and a PCR reaction solution. An EB virus genome single-copy gene EBNA1 conservative coding region is used as a detection target region, two sets of PCR amplification primers and probes are designed to be used for amplifying different sites in the detection target region, and the two sets of PCR amplification primers and probes are placed in the same PCR reaction system to form a double PCR amplification system to achieve detection of free EB virus DNA in a plasma sample. The problem that quantitative results of different patients are incomparable due to copy number variation of different detection target regions of EB viruses is solved, and the quantitative results of different laboratories can be conveniently standardized to facilitate formation of a universal clinical diagnosis boundary value; meanwhile, the design of double target spots and double probes is adopted, so that the detection sensitivity is effectively improved on the premise of ensuring the accuracy. The kit has the advantages of sensitivity, accuracy, quickness and simplicity and convenience in operation.

Description

technical field [0001] The invention relates to the field of biotechnology, and discloses a PCR primer, a probe and a kit that can be used for the quantitative detection of Epstein-Barr virus DNA. Background technique [0002] Epstein-Barr virus belongs to the gamma-herpesvirus subfamily Lymphocyptivirus, also known as human herpesvirus type 4 (HHV-4). It was first isolated from Burkitt lymphoma cells in 1964 and the virus was observed under an electron microscope. particles. Epstein-Barr virus is a linear double-stranded DNA virus. The virus genome size is about 172KB, with 100 coding genes. The virus genome contains multiple repeat regions and there are thousands of SNV mutations, indel mutations and other types of mutations in the genomes of different strains. . Epstein-Barr virus infection is ubiquitous in the population, about 90% of individuals in China have been infected with Epstein-Barr virus and many individuals have been infected in infancy. The infection of EB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6851C12Q2563/107C12Q2531/113
Inventor 郑国沛罗凯张志杰贺智敏董静王倩
Owner CANCER CENT OF GUANGZHOU MEDICAL UNIV
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