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Membrane gene chip for simultaneous detection of three groups, A, B and C, of human rotaviruses and its preparation and application

A rotavirus and gene chip technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inability to detect at one time and low sensitivity of three groups of rotavirus detection.

Inactive Publication Date: 2008-08-20
SHANDONG MEDICAL BIO TECH RES CENT
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0010] Aiming at the deficiencies in the prior art, one of the technical problems to be solved in the present invention is to provide a membrane gene chip for simultaneously detecting three groups of human rotaviruses A, B, and C, so as to overcome the problem that the prior art cannot be detected at one time and cause human The three groups of rotaviruses in diarrhea and the defects of low detection sensitivity meet the needs of food hygiene monitoring, disease prevention and control, and clinical medical fields;

Method used

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  • Membrane gene chip for simultaneous detection of three groups, A, B and C, of human rotaviruses and its preparation and application
  • Membrane gene chip for simultaneous detection of three groups, A, B and C, of human rotaviruses and its preparation and application
  • Membrane gene chip for simultaneous detection of three groups, A, B and C, of human rotaviruses and its preparation and application

Examples

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Effect test

Embodiment 1

[0100] Embodiment 1 The preparation of the membrane gene chip that simultaneously detects A, B, C three groups of human rotaviruses according to the present invention

[0101] (1) Determination of probes: According to the gene sequences of three groups of human rotaviruses A, B and C, 6 human rotavirus group-specific detection probes were selected and determined, and the gene sequences are:

[0102] ProbeA1: 5′-ATTTATTGAATGCTTCGATAT-3′

[0103] ProbeA2: 5'-ACTGGTGAGTGGATTGTTTGA-3'

[0104] ProbeB1: 5′-ATCCCATTTGAGTAAATTCAG-3′

[0105] ProbeB2: 5′-ATGATAATTCAGCCAAGCC-3′

[0106] ProbeC1: 5′-CTGTATTAGCTACATGACCGT-3′

[0107] ProbeC2: 5′-AGCTATTGGAGTTTGGTAGTT-3′

[0108] 1 positive control probe, its gene sequence is:

[0109] PbP: 5′-ATTTATTGAATGCTTCGATAT-3′

[0110] The gene sequences of 4 negative control probes are:

[0111] PbEC: 5′-GATGAGAATGTGCCTTCGGGAA-3′

[0112] PbSA: 5′-GAACATATGTGTAAGTAACTGTGC-3′

[0113] PbHBV: 5′-AGAAAGACCTTTAACCTA-3′

[0114] PbPSE: 5′-TA...

Embodiment 2

[0116] Example 2 Detection of Group A Human Rotavirus Using the Membrane Chip for Simultaneously Detecting Three Groups of Human Rotaviruses A, B and C

[0117] (1) Carry out cell culture on human rotavirus standard strains RV5 and Wa strains of human group A, extract viral RNA in the supernatant of the culture medium, and use Takara company RNA PCR (AMV) VER 2.1 kit instructions to reverse the RNA record the response. Then set up the PCR system: 25μl reaction system contains: 1.5μl MgCl 2 , 2.5μl 10×PCR buffer (without Mg -2 ), 2.0 μldNTP (2.5mmol / L), 16.4 μl sterilized double distilled water, 0.125 μl Taq enzyme (5U / μl), 1.0 μl primer mixture (, the upstream primer of digoxin-labeled group A human rotavirus, A Downstream primer for group B human rotavirus, upstream primer for digoxin-labeled group B human rotavirus, downstream primer for group B human rotavirus, upstream primer for digoxin-labeled group C human rotavirus, C The downstream primers of group human rotavirus ...

Embodiment 3

[0125] Example 3 Detection of Group B Human Rotavirus Using the Membrane Gene Chip for Simultaneous Detection of Three Groups A, B, and C Human Rotaviruses According to the Present Invention

[0126] (1) Establish a PCR amplification system for the human group B human rotavirus target nucleic acid sequence

[0127] 25μl reaction system contains: 1.5μl MgCl 2 , 2.5μl 10×PCR buffer (without Mg -2 ), 2.0 μl ldNTP (2.5 mmol / L), 16.4 μl sterilized double distilled water, 0.125 μl Taq enzyme (5U / μl), 1.0 μl primer mixture (upstream primer of group A human rotavirus labeled with digoxin, group A Downstream Primer for Human Rotavirus, Upstream Primer for Digoxigenin-labeled Group B Human Rotavirus, Downstream Primer for Group B Human Rotavirus, Upstream Primer for Digoxigenin-Labeled Group C Human Rotavirus, Group C Downstream primers of human rotavirus (10 μmol / L each), 0.5 μl template.

[0128] The PCR program is: 94°C pre-denaturation for 2 minutes, followed by 94°C for 45 sec, ...

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Abstract

The membrane chip for simultaneous detection of three groups, A, B and C, of human rotaviruses includes positively charged nylon membrane, and dotted coating with oligonucleotide probes, contrasts and blanks distributed in array on glass substrate. The dotted coating has 6 specific detecting probes of different groups of human rotaviruses, 1 positive contrast probe with digoxin marker in its 5' end, and 4 negative contrast probes and blank sample applying liquid contrast. The present invention also discloses the preparation process and application as clinical diagnosis reagent of the chip. The chip of the present invention operates in multiple PCR amplification mode to make the detected nucleic acid carry digoxin marker and obtain hybridized result through immunological coloration, can complete gene detection and group identification of several groups of human rotaviruses, and has wide application foreground in food detection, disease prevention and clinical application.

Description

technical field [0001] The invention relates to a gene microarray biochip and its preparation method and application, in particular to a membrane gene chip for simultaneous detection of three groups of human rotaviruses A, B and C, its preparation method and application, belonging to biochip and diagnosis Reagent technology field. Background technique [0002] Gene chip refers to the use of many specific oligonucleotide fragments or gene fragments as probes, which are regularly arranged and fixed on a solid support, and then hybridized with the labeled nucleic acid sample to be tested according to the principle of base pairing, and then The hybridization signal is detected by a certain detection system, and a computer system is used to analyze and process the data of each hybridization signal, so as to quickly obtain the desired information. Compared with the traditional hybridization technology, this technology has the remarkable characteristics of miniaturization, high se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 黄海燕韩金祥王健伟
Owner SHANDONG MEDICAL BIO TECH RES CENT
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