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

A rotavirus and gene chip technology, applied in biochemical equipment and methods, microbe measurement/inspection, etc., can solve the problems of not being able to detect at one time, low detection sensitivity of three groups of rotaviruses, etc.

Inactive Publication Date: 2006-08-02
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Preparation of a membrane gene chip for simultaneous detection of three groups of human rotaviruses A, B, and C according to the present invention

[0100] (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:

[0101] ProbeA1: 5′-ATTTATTGAATGCTTCGATAT-3′

[0102] ProbeA2: 5'-ACTGGTGAGTGGATTGTTTGA-3'

[0103] ProbeB1: 5′-ATCCCATTTGAGTAAATTCAG-3′

[0104] ProbeB2: 5′-ATGATAATTCAGCCAAGCC-3′

[0105] ProbeC1: 5′-CTGTATTAGCTACATGACCGT-3′

[0106] ProbeC2: 5′-AGCTATTGGAGTTTGGTAGTT-3′

[0107] 1 positive control probe, its gene sequence is:

[0108] PbP: 5′-ATTTATTGAATGCTTCGATAT-3′

[0109] The gene sequences of 4 negative control probes are:

[0110] PbEC: 5′-GATGAGAATGTGCCTTCGGGAA-3′

[0111] PbSA: 5′-GAACATATGTGTAAGTAACTGTGC-3′

[0112] PbHBV: 5′-AGAAAGACCTTTAACCTA-3′

[0113] PbPSE: 5′-TAGGT...

Embodiment 2

[0115] Example 2 Detection of Group A Human Rotavirus Using the Membrane Gene Chip for Simultaneous Detection of Three Groups A, B and C Human Rotaviruses of the Present Invention

[0116] (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 ), 2.0μldNTP (2.5mmol / L), 16.4μl sterile double distilled water, 0.125μl Taq enzyme (5U / μl), 1.0μl primer mix (DIG-PAu, PAd, DIG-PBu, PBd, DIG-PCu, PCd Each 10 μmol / L), 0.5 μl RT product.

[0117] (2) Electrophoretic detection of PCR amplification products

[0118] Take 5 μl of the PCR product, and observe the results after electrophoresis with 1% agarose gel containing ethidium bromide, as shown in FIG. 2 .

[0119] (3) T...

Embodiment 3

[0124] 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

[0125] (1) Establish a PCR amplification system for the human group B human rotavirus target nucleic acid sequence

[0126] 25μl reaction system contains: 1.5μl MgCl 2 , 2.5μl 10×PCR buffer (without Mg -2 ), 2.0μldNTP (2.5mmol / L), 16.4μl sterile double distilled water, 0.125μl Taq enzyme (5U / μl), 1.0μl primer mix (DIG-PAu, PAd, DIG-PBu, PBd, DIG-PCu, pCd Each 10μmol / L), 0.5μl template.

[0127] The PCR program was: 94°C pre-denaturation for 2 minutes, followed by 94°C for 45 sec, 52°C for 45 sec, and 72°C for 1 min as a cycle, and after 30 cycles, extended at 72°C for 5 min.

[0128] (2) Electrophoretic detection of PCR amplification products

[0129] Take 5 μl of the PCR product, and observe the results after electrophoresis with 1% agarose gel containing ethidium bromide, as shown ...

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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 5í» 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 Applications(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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