Kit for detecting EB virus/HCMV and application thereof

An EB virus and kit technology, which is applied in the field of in vitro detection of organisms to achieve the effects of convenient clinical practice, wide detection range and simple operation.

Inactive Publication Date: 2021-02-26
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Kit for detecting EB virus/HCMV and application thereof
  • Kit for detecting EB virus/HCMV and application thereof
  • Kit for detecting EB virus/HCMV and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 determines the target protein epitope of Epstein-Barr virus, HCMV

[0038] According to literature review and bioinformatics analysis, the viral protein to be tested that is suitable as the detection target of the nucleic acid aptamer and the specific epitope region on the viral protein are determined.

[0039] A) Select the 400-857 region of the Epstein-Barr virus gp125 protein as an aptamer screening target protein, which has the sequence shown in SEQ ID No.3:

[0040] TTPTSSPPSSPSPPAPSAARGSTPAAVLRRRRRDAGNATTPVPPTAPGKSLGTLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETMCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNEIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNVFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGRNQFVDGLGELMDSLGSVGQSITNLVSTVGGLFSSLVSGFISFFKNPFGGMLILVLVAGVVILVISLTRRTRQMSQQPVQMLYPGIDELAQQHASGEGPGINPISKTELQAIMLALHEQNQEQKRAAQRAAGPSVASRALQAARDRFPGLRRRRYHDPETAAALLGEAETEF(SEQ ID No.3)

[0041] B) Select the ...

Embodiment 2

[0044] Example 2 Construction of aptamer library and primer design

[0045] A) A single-stranded DNA library containing 45 random sequences was synthesized in vitro through the gene synthesis service of a conventional biotechnology service company, and its nucleotide sequence is as follows

[0046] GATGACATTGCACAAGTCAGG-(N45)-GAGTGAATCCTGCTGTTCGA

[0047] B) For the fixed sequence at the 5' end of the nucleic acid adapter, design a synthetic upstream primer P1, which has a sequence as shown in SEQ ID No.5:

[0048] 5'-GATGACATTGCACAAGTCAGG-3' (SEQ ID No.5)

[0049] C) Aiming at the fixed sequence at the 3' end of the nucleic acid aptamer, design a synthetic downstream primer P2, which has the sequence shown in SEQ ID No.6, and a biotin group is attached to the 5' end of the primer:

[0050] 5'-biotin-TCGAACAGCAGGATTCACTC-3'(SEQ ID No.6)

Embodiment 3

[0051] Example 3 Using SELEX screening technology to screen the library for nucleic acid aptamer sequences that can effectively bind to viral target proteins

[0052] A) Dissolve 10 nmol of the initial single-stranded DNA random library in 500 μl of PBS solution, bathe in a constant temperature water bath at 92°C for 5 minutes, then quickly insert it into ice for 10 minutes, and then place the treated initial single-stranded DNA random library Incubate with the corresponding virus target protein on ice for 1h, then add Ni-NTA Magnetic Agarose Beads, and continue to incubate for 1h. After incubation, use a magnetic separator to absorb, remove the supernatant, and wash the Beads with 2ml of PBS. Finally, resuspend the Beads with 10ml of pre-cooled PBS to make them fully blown and mixed, and then in a constant temperature water bath at 92°C for 10 minutes, centrifuge at 13,000g, and collect the supernatant, which is the single-stranded DNA library that specifically recognizes the...

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PUM

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Abstract

The invention provides a nucleic acid aptamer detection kit for EB virus and HCMV, and also provides a preparation method and application of the kit. The kit has the characteristics of simplicity in operation, quick response, high accuracy and high sensitivity during detection, and can be combined with detection devices such as a microplate reader, a flow cytometry and a fluorescence microscope torealize accurate detection of the two viruses. The kit is relatively high in sensitivity, good in specificity, wide in measurement range and simple to operate, can be used for simultaneously detecting two viruses, and has a good market prospect.

Description

technical field [0001] This application relates to in vitro detection, specifically, the present invention relates to a kit for simultaneous detection of Epstein-Barr virus / HCMV, a method for preparing the kit, and the application of the kit in simultaneous detection of Epstein-Barr virus and HCMV samples. Background technique [0002] Epstein-Barr virus (EBV) is a DNA virus belonging to the family Herpesviridae and the genus Lymphotropia virus. Epstein-Barr virus has the biological characteristics of specifically infecting human and some primate B cells in vivo and in vitro. Humans are the main host of EB virus infection, which is mainly transmitted through saliva. Asymptomatic infection mainly occurs in early childhood. According to surveys, more than 90% of children aged 3 to 5 have been infected with EB virus, and more than 90% of adults have virus antibodies. Epstein-Barr virus is the pathogen of infectious mononucleosis. In addition, Epstein-Barr virus is closely rela...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/569
CPCG01N33/56994G01N33/582
Inventor 刘为勇张祺刘芳刘映乐屈三甫梅益
Owner WUHAN UNIV
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