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Gene chip and detection method for detecting FMDV, VSV, SVDV, PPRV and BTV

A gene chip and chip technology, which is applied in the field of biochips, can solve the problems that cannot fully meet the requirements of fast, accurate and high-throughput detection of imported animal diseases, increase the difficulty of pathogen diagnosis and instability, and achieve stability and save time and effort. , The hybridization signal is stable and the stability is strong

Inactive Publication Date: 2015-06-10
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional pathogen isolation and identification and serological methods are time-consuming and require professional operators, which cannot fully meet the requirements of rapid, accurate and high-throughput detection of imported animal diseases.
With the development of molecular biology, polymerase chain reaction (PCR) is widely used in the detection of animal diseases, but there is currently a lack of PCR methods that can distinguish several viruses
In addition, the five viruses of FMDV, VSV, SVDV, PPRV, and BTV are all RNA viruses, and RNA is easily degraded and unstable to a certain extent, which increases the difficulty of pathogenic diagnosis

Method used

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  • Gene chip and detection method for detecting FMDV, VSV, SVDV, PPRV and BTV
  • Gene chip and detection method for detecting FMDV, VSV, SVDV, PPRV and BTV
  • Gene chip and detection method for detecting FMDV, VSV, SVDV, PPRV and BTV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1, the present invention is based on cell proliferation of foot-and-mouth disease virus, vesicular stomatitis virus, porcine vesicular disease virus, Peste des Petits Ruminants virus and bluetongue virus, and extracts virus genomes. Select different specific conserved sequences to design 8 oligonucleotide probes, which can specifically bind to the corresponding viral cDNA PCR products. When performing genome amplification by PCR, the 5′ end of the universal upstream primer is labeled with Cy3 fluorescent dye , the probe can hybridize with this product at 50°C. According to the position and intensity of the fluorescent signal, the hybridization result is judged to identify foot-and-mouth disease virus, vesicular stomatitis virus, porcine vesicular disease virus, Peste des Petits Ruminants virus and bluetongue disease virus. PCR primers SEQ ID NO.11, SEQ ID NO. 12. SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, S...

Embodiment 2

[0097] Example 2, see figure 1 , the gene chip used for the detection of foot-and-mouth disease virus, vesicular stomatitis virus, porcine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus, adopts the gene chip micro-spotting technology, and combines the probes of the samples to be tested with various quality control The probes were immobilized on the chemically modified aldehyde substrate to form a microarray of 7 rows×8 columns. The distribution of probes on the microarray from top to bottom is as follows: spotting position quality control partition P1: 1 row × 8 points, hybridization positive quality control partition P2: 1 row × 4 points, foot-and-mouth disease virus type A detection probe partition 1: 1 line × 4 points, FMD virus Asian type detection probe division 2: 1 line × 4 points, FMD virus general detection probe division 3: 1 line × 4 points, FMD virus O-type detection probe division 4: 1 line ×4 points, vesicular stomatitis virus det...

Embodiment 3

[0100] Embodiment 3, reagent preparation

[0101] 1) Chip washing solution

[0102] According to needs and actual conditions, prepare washing solution I and washing solution II according to the following proportions.

[0103] Washing solution Ⅰ: the final concentration of SSC is 2×, and the final concentration of SDS is 0.2%. For example, 500mL lotion I = 440mL distilled water + 50mL 20×SSC + 10mL 10% SDS, or prepare in proportion as required.

[0104] Washing solution Ⅱ: The final concentration of SSC is 0.2×. Such as 500mL lotion II = 495mL distilled water + 5mL 20 × SSC, or according to the need to prepare in proportion.

[0105] If 10% SDS produces white flocculent precipitates, please dissolve and mix in a 42°C water bath to prepare a lotion.

[0106] 2) absolute ethanol.

[0107] 3) Ice-water mixture.

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Abstract

The invention discloses a gene chip and a detection method for detecting FMDV, VSV, SVDV, PPRV and BTV. The detection method comprises the step of detecting foot and mouth disease viruses (type A, Asian type I and type O), vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus. The method comprises the following specific steps: designing a PCR primer by virtue of sequence analysis of standard strain genome, and performing cloning and sequence analysis on target genes; designing a specific probe, and simultaneously detecting the foot and mouth disease viruses, vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus. The invention aims at establishing a method for detecting the foot and mouth disease viruses, vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus by adopting a microarray chip which is high in sensitivity and high in specificity and through which the time and labor are saved and the result is easily observed.

Description

technical field [0001] The invention belongs to the field of biological chips. Specifically, the present invention relates to gene chips of foot-and-mouth disease virus, vesicular stomatitis virus, porcine vesicular disease virus, Peste des Petits Ruminants virus and bluetongue virus and detection methods thereof. Background technique [0002] Foot-and-mouth disease (FMD), vesicular stomatitis (VS), swine vesicular disease (SWD), peste des petits ruminants (PPR) and bluetongue ( Bluetongue disease (BT) consists of Foot-and-mouth disease virus (FMDV), Vesicular stomatitis virus (VSV), Swine vesicular disease virus (SVDV), Peste des petits ruminants Acute and severe viral infectious diseases caused by peste des petits ruminants virus (PPRV) and bluetongue virus (BTV). Five viruses, FMDV, VSV, SVDV, PPRV, and BTV, are widely prevalent all over the world, have a wide range of hosts, and have a high incidence rate. They are all important diseases that hinder the development of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C12R1/93
CPCC12Q1/70C12Q1/6888C12Q1/705C12Q2600/16C40B40/06
Inventor 聂福平李应国王昱杨俊保雨王国民艾军张强
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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