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Fluorescent quantitative PCR detection method for lymphocyte EB virus nucleic acid

A technology of EB virus and fluorescence quantification, applied in the biological field, can solve the problems of inability to eliminate virus latent infection, inability to accurately and sensitively reflect EB infection, inability to detect the virus itself, and achieve the effect of convenient operation

Inactive Publication Date: 2019-02-01
济南星齐医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned humoral immune system can prevent exogenous virus infection, but cannot eliminate the latent infection of the virus
[0004] At present, ELISA detection of EB antibodies is one of the important indicators for clinical diagnosis of EB infection, but it actually detects the state of the human immune response to EB infection, and cannot detect the virus itself, so it cannot accurately and sensitively reflect the situation of EB infection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The technical solution adopted by the present invention to solve the technical problems is: a method for detection of lymphocyte EB virus nucleic acid fluorescence quantitative PCR, comprising the following steps:

[0031] Step 1. Extraction of sample RNA

[0032] (1) Pretreatment of the tissue to be tested

[0033] Take the tissue to be tested, add 1ml of TRIZOL reagent to lyse, and obtain the lysed sample, take 1ml of the lysed sample and add 0.2ml of chloroform, vibrate the tube body manually for 10S, incubate at 15°C for 2min, and then put the liquid in a centrifuge , centrifuge at 8000 for 10min at 0°C;

[0034] (2) Incubate the tissue to be tested to obtain a colloidal pellet containing RNA

[0035] After centrifugation, the mixed liquid will be divided into the red phenol chloroform phase in the lower layer, the middle layer, and the colorless water phase in the upper layer. All the RNA of the test tissue will be distributed in the water phase. Transfer the upp...

Embodiment 2

[0052] The technical solution adopted by the present invention to solve the technical problems is: a method for detection of lymphocyte EB virus nucleic acid fluorescence quantitative PCR, comprising the following steps:

[0053] Step 1. Extraction of sample RNA

[0054] (1) Pretreatment of the tissue to be tested

[0055] Take the tissue to be tested, add 5ml of TRIZOL reagent to lyse, and obtain the lysed sample, take 1ml of the lysed sample and add 0.3ml of chloroform, shake the tube body vigorously by hand for 15S, incubate at 25°C for 3min, and then put the liquid in a centrifuge , centrifuge at 10,000rpm for 15min at 2°C;

[0056] (2) Incubate the tissue to be tested to obtain a colloidal pellet containing RNA

[0057] After centrifugation, the mixed liquid will be divided into the red phenol chloroform phase in the lower layer, the middle layer, and the colorless water phase in the upper layer. All the RNA of the test tissue will be distributed in the water phase. Trans...

Embodiment 3

[0074] The technical solution adopted by the present invention to solve the technical problems is: a method for detection of lymphocyte EB virus nucleic acid fluorescence quantitative PCR, comprising the following steps:

[0075] Step 1. Extraction of sample RNA

[0076] (1) Pretreatment of the tissue to be tested

[0077] Take the tissue to be tested, add 10ml of TRIZOL reagent to lyse to obtain the lysed sample, take 1ml of the lysed sample and add 0.5ml of chloroform, shake the tube body vigorously by hand for 15S, incubate at 30°C for 3min, and then put the liquid in a centrifuge , centrifuge at 12000rpm for 15min at 4°C;

[0078] (2) Incubate the tissue to be tested to obtain a colloidal pellet containing RNA

[0079] After centrifugation, the mixed liquid will be divided into the red phenol chloroform phase in the lower layer, the middle layer, and the colorless water phase in the upper layer. All the RNA of the test tissue will be distributed in the water phase. Trans...

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PUM

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Abstract

The invention relates to a fluorescent quantitative PCR detection method for lymphocyte EB virus nucleic acid, the method is as follows: a tissue to be detected is taken, and is subjected to crackingand centrifugation, an upper-layer aqueous phase of a mixed liquid is transferred into a clean centrifugal tube, equal volume of isopropanol is added, after uniform mixing, and the mixture is centrifuged to obtain a colloidal precipitation block on the bottom and the side wall of the tube; deionized water which is free of RNA enzyme is added to obtain a RNA solution; the RNA solution is taken, anda reverse transcription buffer, upstream and downstream primers, DEPC water and a reverse transcriptase are added to obtain a reverse transcription final solution as a DNA solution; reaction tubes are put into a fluorescent quantitative PCR instrument, fluorescent group species, sample names and types are set, and amplification is carried out according to conditions. The method has the beneficialeffects that a sample is subjected to amplification on a target region under the action of polymerase, the accumulation of amplification products is monitored in real time by utilizing a fluorescentprobe technology; the existence of the EB virus nucleic acid is judged according to the detection of the amplification products; and the method has the advantages of being specific, sensitive and rapid, convenient to operate and the like.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a detection method for fluorescent quantitative PCR of lymphocyte EB virus nucleic acid. Background technique [0002] Epstein-Barr virus can only proliferate in B lymphocytes, which can transform them and pass them down for a long time. Cells infected by the virus have the genome of EBv and can produce various antigens, which have been identified: EBv nuclear antigen, early antigen (ea), membrane antigen (ma), capsid antigen (vca), lymphocyte recognition membrane antigen (lydma). [0003] Epstein-Barr virus stays latent in lymphocytes for a long time, dissociates in the cytoplasm in the form of circular DNA, and integrates into chromosomes. Epstein-Barr virus is widely infected in the crowd, and most children have no obvious symptoms after infection, or cause mild pharyngitis and upper respiratory tract infection. Primary infection occurs in adolescence, and infectious mo...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/705C12Q2563/107C12Q2545/114
Inventor 戴永刚
Owner 济南星齐医学检验有限公司