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EB virus gene quick diagnosis kit based on loop-mediated equal-temperature amplification technology

A technology for rapid diagnosis of Epstein-Barr virus, which is applied in the field of biological detection reagents, can solve the problems of detection of Epstein-Barr virus with warm amplification technology, and achieve the effect of simple identification method, high yield and high detection rate

Inactive Publication Date: 2007-08-01
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no methods and diagnostic kits for the detection of Epstein-Barr virus using the loop-mediated isothermal amplification technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The preparation of embodiment 1 kit

[0024] (1) Synthesize oligodeoxynucleic acid primers with a DNA synthesizer according to the following sequence.

[0025] Outer Primer 1: TCAACAGATAATCCACCCGCGC

[0026] Outer Primer 2: TAGTAAATTGCAGGCCCAGG

[0027] Inner primer 1: TCAAACATGCCAGAGGCGTTGGTTTTTGGCGTGGAAGTTAATGTCC

[0028] Inner primer 2: GCGACCACCAGCTCTGTCATTTTTGAGCCTTGCACCCAACAAG

[0029] (2) Purchase DNA polymerase: Bst DNA polymerase (Large Fragment). Put in container.

[0030] (3) Prepare the reaction solution: make a solution containing 2mM dNTP, 25mM Tris-Cl, 12.5mM potassium chloride, 12.5mM ammonium sulfate, 10mM magnesium sulfate, 0.125%TritonX-100, 1M betaine. Add 2M each of the inner primers of the above (1), and 0.25M each of the outer primers

[0031] (4) Preparation of Lysis Solution 1: 0.2M NaOH; Lysis Solution 2: 0.4M Tris-HCl[pH7.5])

[0032] (5) Purchase a color developing solution (1000×SYBR Green I) and place it in a container.

[0033] (6)...

Embodiment 2

[0035] The preparation of embodiment 2 kit

[0036] (1) Synthesize oligodeoxynucleic acid primers with a DNA synthesizer according to the following sequence.

[0037] Outer Primer 1: TCAACAGATAATCCACCCGCGC

[0038] Outer Primer 2: TAGTAAATTGCAGGCCCAGG

[0039] Inner primer 1: TCAAACATGCCAGAGGCGTTGGTTTTTGGCGTGGAAGTTAATGTCC

[0040] Inner primer 2: GCGACCACCAGCTCTGTCATTTTTGAGCCTTGCACCCAACAAG

[0041] Other steps are with embodiment 1

Embodiment 3

[0042] Example 3: Application of Epstein-Barr Virus Gene Rapid Diagnostic Kit: Detection of Human Blood Samples

[0043] 1. Sample collection

[0044] 1. Take 1ml of the patient's blood sample and let it stand at room temperature for coagulation;

[0045] 2. Centrifuge at 2500rpm for 8min to separate serum;

[0046] 3. Serum samples are immediately used for DNA extraction or stored at -20°C

[0047] 2. Epstein-Barr virus DNA extraction steps:

[0048]1. Take 50 μl of lysate 1 into a 1.5ml sterile centrifuge tube, add 50 μl of the serum sample to be tested, shake and mix;

[0049] 2. Place the tube in a water bath at 80°C for 10 minutes at a constant temperature, take it out, and put it on ice for 10 minutes;

[0050] 3. Centrifuge at 10000rpm for 1min, take 50μl of the supernatant into a new 1.5ml sterile centrifuge tube

[0051] 4. Add 50 μl Lysis Solution 2, shake and mix;

[0052] 5. Take 2 μl of the above mixture as the reaction template;

[0053] 3. The reaction pr...

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Abstract

The invention relates to a kit checking EB virus with mediated isothermal amplification technology, and belongs to biological checking agent field. The kit comprises a set of primer, DNA polyase, reacting liquid, cracking liquid (1) and (2), coloured solution and positive contrast liquid; said a set of promer comprises two pair of primers, and the squence is: outer primer1: GCATGTTTGAGTCCCGCTG, outer primer 2: CCATTGCTGTGGAAGTGGC;inner primer 1: CACCCAACAAGGCAGGACGCTTTTGCTGAACATTAGCATCCCGG; inner primer2: GTGCCTGGGCCTGCAATTTACTTTTGCGATCTGGTGGGCATTC, or outer primer 1: TCAACAGATAATCCACCCGC, outer primer 2: TAGTAAATTGCAGGCCCAGG; inner primer 1: TCAAACATGCCAGAGGCGTTGGTTTTTGGCGTGGAAGTTAATGTCC; inner primer 2: GCGACCACCAGCTCTGTCATTTTTGAGCCTTGCACCCAACAAG. The kit needs no special agent and device, can fast detect EB virus in the sample, and is characterized by high speciality, high sensitivity, simple determination method and high correcting rate.

Description

technical field [0001] The invention relates to a biological detection reagent, in particular to a diagnostic reagent for detecting Epstein-Barr virus based on a loop-mediated isothermal amplification technique. Background technique [0002] At present, there are many detection methods for pathogenic microorganisms, ranging from the national standard (GB / T4789.7-2003) focusing on the isolation and identification of pathogenic microorganisms, morphological identification and automatic biochemical identification, to the immunological detection technology of specific proteins, nucleic acid Molecular biological detection methods such as probes and polymerase chain reaction (PCR) technology. Among them, the detection of pathogenic nucleic acid has great advantages in terms of rapidity, safety, accuracy and sensitivity. Separation and purification, and rapid detection of genes and gene products directly with the sample or the enrichment solution of the sample, and combined with m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 聂国辉肖德明王大平付林徐小平陈洵杜正平李心晖石磊曹以诚
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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