Primer, reagent kit and method for detecting EB (Epstein-Barr) virus

An Epstein-Barr virus and kit technology, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve the effect of saving time, fast detection process, and simple operation.

Inactive Publication Date: 2011-08-31
宁波基内生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are several kits for detecting EBV-DNA on the market abroad. The detection method is mainly fluorescent immunohybridization (HIGH). The ELISA method is mainly used for domestic detection of EBV. There is no kit approved for the detection of EBV-DNA on the market.

Method used

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  • Primer, reagent kit and method for detecting EB (Epstein-Barr) virus
  • Primer, reagent kit and method for detecting EB (Epstein-Barr) virus
  • Primer, reagent kit and method for detecting EB (Epstein-Barr) virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 : DNA sample preparation

[0041] In this embodiment, a human blood sample is used to prepare a DNA sample used in the detection method of the present invention, which is described in detail as follows.

[0042] 1. Extraction reagent:

[0043] Solution A: cell lysate, 15ml / bottle, 200μl each time.

[0044]

[0045] Component Final Concentration

[0046]

[0047] Tris-HCl (pH8.0) 0.01mol / l

[0048] NaCl 0.1mol / l

[0049] EDTA (pH8.0) 0.01mol / l

[0050] SDS 2% (g / ml)

[0051]

[0052] Solution B: Nucleic acid binding solution, 25ml / bottle, 400μl each time.

[0053]

[0054] Component Final Concentration

[0055]

[0056] GuSCN 5mol / l

[0057] Tris-HCl (pH6.4) 0.05mol / l

[0058] EDTA(pH8.0) 0.02mol / l

[0059] Triti...

Embodiment 2

[0083] Example 2 : Determination of internal reference primer sequence

[0084] In this example, two pairs of DNA amplification primers designed according to the human DNA conserved sequence β-globin-specific gene were used to amplify the DNA sample prepared in Example 1, and the amplification effects of the two pairs of primers were detected by gel electrophoresis to screen out Internal reference primers.

[0085] 1. Single primer PCR amplification

[0086] 1) Prepare a 25 μl reaction system in a 200 μL Eppendorf tube as follows, in which the extracted DNA samples are DNA from 10 different blood samples:

[0087]

[0088] Component Volume / μl

[0089]

[0090] wxya 2 O 18.3

[0091] 5U / μl Taq enzyme 0.2

[0092] Taq enzyme 10×buffer 2.5

[0093] 10μM internal reference upstream and downstream primers 0.5 each

[0094] 2.5mM dNTP2

[0095] Extracted DNA sample 1

[0096] ...

Embodiment 3

[0127] Example 3 : Amplify DNA using a primer mix

[0128] In this example, the primer composition provided by the present invention was used to amplify the DNA sample extracted in Example 1, and gel electrophoresis was used to detect the correctness of the PCR product.

[0129] 1. PCR amplification with mixed primers

[0130] a) Prepare a 25 μl reaction system in a 200 μL Eppendorf tube:

[0131]

[0132] Composition Volume (μl)

[0133]

[0134] wxya 2 O 10.3

[0135] 2×buffer 12.5

[0136] Primer composition 1.2

[0137] Extracted clinical sample DNA 1

[0138]

[0139] Note: 2×buffer contains Tris-HCl (pH 8.4) 40mM, MgCl 2 20mM, KCl 50mM, (NH) 2 SO 4 10mM, each dNTP 0.2mM, Taq DNA polymerase 0.08U / μL, the primer composition contains: B297 upper and lower primers 0.2μM each, EBV upper and lower primers 0.4μM each. The specific seque...

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Abstract

The invention provides a primer, reagent and method for detecting EB (Epstein-Barr) virus. A forward primer nucleotide sequence of the primer is SEQ ID NO: 1, and a reverse primer nucleotide sequence of the primer is SEQ ID NO: 2. The invention also provides a combination, tool, reagent and method for detecting the EV virus, wherein the combination comprises the primer. Through the combination, the tool, the reagent and the method, rapid detection can be performed at the gene level, sensitivity is high and specificity is good, and the advantages of time saving, mature technology and stable detection results are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to primers, kits and methods for detecting Epstein-Barr virus (Epstein-Barr virus, EBV, referred to herein as EBV). Background technique [0002] Epstein-Barr virus (EBV), a member of the Gammaherpesvirus subfamily, is a specific human lymphotropic herpes virus, and has been considered to be the pathogenic factor of some serious human diseases and tumors. , are closely related to the occurrence of many diseases, threatening human health. EBV is mainly transmitted through human saliva, so the respiratory tract is the largest place where EBV hides. The survey results show that more than 90% of the world's population has latent infection of EBV, or become EBV carriers. More importantly, EBV infection is related to more and more human malignant tumors. The application of in situ hybridization has proved that carrying high copies of EBV can not only transform...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 倪剑锋赵珊珊杨文辉谢育媛邓其涛
Owner 宁波基内生物技术有限公司
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