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Glass gene chip for simultaneous detection of Group A, B and C human rotaviruses and the prepn and application

A rotavirus and gene chip technology, applied to biochips and diagnostic reagents, glass gene chips and preparation of three groups of human rotaviruses in C, and simultaneous detection of A and B fields, which can solve the problem of three groups of rotaviruses that cannot be detected at one time. Problems such as low virus detection sensitivity

Inactive Publication Date: 2006-05-17
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Aiming at the deficiencies in the prior art, one of the technical problems to be solved in the present invention is to provide a glass gene chip for simultaneously detecting three groups of human rotaviruses A, B, and C, so as to overcome the problem of the prior art that human rotavirus cannot be detected at one time. 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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  • Glass gene chip for simultaneous detection of Group A, B and C human rotaviruses and the prepn and application
  • Glass gene chip for simultaneous detection of Group A, B and C human rotaviruses and the prepn and application
  • Glass gene chip for simultaneous detection of Group A, B and C human rotaviruses and the prepn and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1 Preparation of a glass gene chip for simultaneous detection of A, B, and C three groups of human rotaviruses according to the present invention

[0103] (1) Glass slide treatment: take a conventional glass slide bare chip without any chemical modification and immerse it in a 10% glutaraldehyde aqueous solution for 1 hr with a volume percentage concentration to obtain an aldylated glass slide as the substrate of the chip;

[0104] (2) 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 thereof are:

[0105] ProbeA1: 5′-ATTTATTGAATGCTTCGATAT-3′

[0106] ProbeA2: 5'-ACTGGTGAGTGGATTGTTTGA-3'

[0107] ProbeB1: 5′-ATCCCATTTGAGTAAATTCAG-3′

[0108] ProbeB2: 5′-ATGATAATTCAGCCAAGCC-3′

[0109] ProbeC1: 5′-CTGTATTAGCTACATGACCGT-3′

[0110] ProbeC2: 5′-AGCTATTGGAGTTTGGTAGTT-3′

[0111] 1 positive control prob...

Embodiment 2

[0126] Example 2 Detection of Group A Human Rotavirus by Using the Glass Gene Chip of the Present Invention to Simultaneously Detect Three Groups of Human Rotaviruses A, B and C

[0127] (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 (Tamra-PAu, PAd, Tamra-PBu, PBd, Tamra-PCu, PCd Each 10 μmol / L), 0.5 μl RT product.

[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 in FIG. 2 .

[0130] (...

Embodiment 3

[0134] Example 3 Detection of Group B Human Rotavirus Using the Glass Gene Chip for Simultaneous Detection of Three Groups A, B, and C Human Rotaviruses of the Present Invention

[0135] (1) Establish a PCR amplification system for the human group B human rotavirus target nucleic acid sequence

[0136] 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 (Tamra-PAu, PAd, Tamra-PBu, PBd, Tamra-PCu, PCd Each 10μmol / L), 0.5μl template.

[0137] The PCR program is: 94. Pre-denaturation at ℃ for 2 minutes, followed by a cycle of 94°C for 45 sec, 52°C for 45 sec, and 72°C for 1 min. After 30 cycles, extend at 72°C for 5 min.

[0138] (2) Electrophoretic detection of PCR amplification products

[0139] 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 ....

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Abstract

The glass gene chip for simultaneous detection of Group A, B and C human rotaviruses includes aldehydo treated glass chip, oligonucleotide probe array on the glass chip, contrast and blank spot coating, which includes 6 specific human rotavirus detecting probes in different groups, 1 positive contrast probe, 4 negative contrast probes and black sample application liquid contrast. The detecting probes and the contrast probes are connected with poly(dT)15 as connecting arm, and the probes have C6 radical with amino group in the 5í» end. The present invention also discloses the preparation process and clinical application of the chip. The chip of the present invention adopts multiple PCR amplification mode, may be used in the gene detection and group identification of several groups of human rotaviruses, and has wide latent application foreground in sanitary detection of food, disease prevention and control, and clinical medicine.

Description

technical field [0001] The invention relates to a gene microarray biochip and its preparation method and application, in particular to a glass gene chip for simultaneously detecting three groups of human rotaviruses A, B and C, its preparation method and its application, belonging to the field of biochip and diagnosis Reagent [0002] technology field. Background technique [0003] 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 m...

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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