High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof

A quantitative detection and kit technology, applied in the field of DNA detection, can solve the problems of poor detection effect and low sensitivity

Pending Publication Date: 2017-02-08
林勤 +2
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] The present invention provides a highly sensitive EBV DNA quantitative detection kit based on ddPCR and its use method. Using the kit provided by the present invention can solve the problems of poor detection effect and low sensitivity of existing detection kits; at the same time, the extraction of EBV DNA Standardize the method and PCR amplification process to achieve international comparability of test results and quantitative units

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  • High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof
  • High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof
  • High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof

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Embodiment 1

[0044] The highly sensitive EBV DNA quantitative detection kit based on ddPCR (Droplet digital PCR) provided in the embodiment of the present invention includes DNA extraction buffer, PCR primers and probes, PCR reaction solution, and EBV negative and positive controls.

[0045] Wherein, the DNA extraction buffer includes proteinase K buffer, buffer1 (buffer), buffer2, buffer3 and buffer4; the PCR reaction solution includes 5×PCR reaction buffer, and the concentration is 0.8-1.2mmol / L deoxyribonucleoside three Phosphoric acid, H-Taq DNA polymerase (Thermusaquaticus, H-thermostable DNA polymerase) at a concentration of 1-7U / μl; PCR primers and probes include a concentration of 700nmol / L-1.0μmol / L for amplifying the target The upstream and downstream primers of the polynucleotide and the probe used to detect the target polynucleotide at a concentration of 0.2mol / L-0.5μmol / L; the EBV negative control is negative artificial serum, and the EBV positive control is B958 cell culture s...

Embodiment 2

[0055] The highly sensitive EBV DNA quantitative detection kit based on ddPCR provided in the embodiment of the present invention can be applied to detect the content of EBV DNA in plasma and serum samples. The specific steps of the detection operation are as follows:

[0056] 1. Preparation of samples to be tested

[0057] 1. Plasma sample preparation

[0058] Centrifuge EDTA anticoagulated whole blood for 3 minutes within 2 hours at a rotational speed of 2000r / min, take the separated supernatant, and centrifuge the supernatant at a rotational speed of 20000g / min for 10min to remove broken cells For fragments, collect the supernatant to obtain a plasma sample. If the plasma sample is not used temporarily, store the plasma sample at -20°C for future use.

[0059] 2. Serum sample preparation

[0060] Let fresh EDTA anticoagulated whole blood stand at room temperature for 30 minutes, then centrifuge at a speed of 20,000 g / min for 3 minutes, and take the separated supernatant, ...

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Abstract

The invention provides a high-sensitivity EBV DNA quantitative detection kit based on ddPCR and a using method thereof. The kit comprises DNA extraction buffer solution, PCR primers and probes, a PCR reaction solution and EBV positive and negative control. When the kit provided by the invention is used for detecting ddPCR, the influences of PCR equipment, standard substances and the like can be effectively eliminated, and further high-sensitivity absolute quantification is further realized. The EBV DNA quantitative detection kit provided by the invention is capable of performing high-sensitivity accurate quantification on EBV in serum and plasma through a ddPCR technology, and providing a reference basis for diagnosis and curative effect monitoring of EBV related diseases. According to the kit, a function relationship between the international unit and common unit of EBV DNA international standard substances is determined to be 1IU/ml=2.5Copy/ml, the international traceability of EBV DNA is realized, and the international comparability between the detection results and quantification unit is realized.

Description

technical field [0001] The invention relates to the field of DNA detection, in particular to a ddPCR-based highly sensitive EBV DNA quantitative detection kit and a use method. Background technique [0002] Epstein-Barr virus (EBV), also known as human herpesvirus type Ⅵ (Human herpesvirus type 4), belongs to the genus Lymphofollicular virus of the subfamily Gammaherpesviridae, and its carrier rate in the population is as high as 90%. carry. EBV mainly infects human nasopharyngeal epithelial cells and B lymphocytes. Studies have shown that EBV settles in human B lymphocytes for life after the initial infection. EBV-related diseases have a wide range and can be roughly divided into two categories: blood system and non-blood system. EBV-related blood system diseases mainly include infectious mononucleosis (Infectious mononucleosis), Burkitt lymphoma, primary lymphoma, T-cell lymphoma, etc.; EBV-related non-hematological diseases mainly include gastric cancer, nasopharyngeal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/705C12Q1/6851C12Q2521/537C12Q2527/125C12Q2563/159
Inventor 林勤胡斌孙鸣洪国粦骆启聪廖希一王紫晶
Owner 林勤
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