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Gene chip for high-flux detection of pathogens and application thereof

A technology for detecting gene chips and pathogens, applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, DNA/RNA fragments, etc.

Inactive Publication Date: 2012-07-04
李越希
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although methods such as PCR, molecular hybridization technology, and immunoassay can save detection time and have good specificity and sensitivity, they can only detect one or a small number of pathogenic bacteria each time, and a large number of samples are required to be tested. work, even difficult to achieve

Method used

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  • Gene chip for high-flux detection of pathogens and application thereof
  • Gene chip for high-flux detection of pathogens and application thereof
  • Gene chip for high-flux detection of pathogens and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0511] Example 1. Detection of Vibrio cholerae by gene chip for high-throughput detection of pathogens

[0512] Cholera is an acute intestinal infectious disease caused by Vibrio cholerae of O1 and O139 serogroups. It is characterized by acute onset, rapid spread, wide spread, and the ability to cause a pandemic; O157:H7 is a new type that can produce Enteropathogenic bacteria with toxins very similar to toxin cords. Gene chips were prepared with synthetic gene probes to detect Vibrio cholerae O157:H7.

[0513] 1. Extraction and labeling of sample nucleic acid

[0514] Dilute the bacteria doubly and measure the concentration as 10 4 Bacteria / ml, 10 3 Bacteria / ml, 5×10 2 Bacteria / ml, 10 2 Bacteria / ml, take 200ul each, use the Bacterial Genomic DNA Extraction kit kit, and extract DNA according to the instructions. Take 0.6ul as a template for whole genome amplification and klenow fragment random primer labeling, the reaction system is as follows:

[0515] Whole Genome...

Embodiment 2

[0534] Example 2, Pathogen High-throughput Detection Gene Chip Simultaneously Detects Four Kinds of Microorganisms

[0535] Infectious diseases caused by pathogenic bacteria not only rank first in terms of morbidity, but are also an important cause of human death. With the continuous emergence of emerging infectious disease pathogens, recombinant pathogens and unknown pathogens, it poses a major threat to human health. Compared with traditional detection methods, gene chips have unique advantages in the detection of infectious diseases. In this experiment, a gene chip for high-throughput detection of pathogens was established, and the detection conditions of the chip were optimized. Samples were tested.

[0536] 1. Chip Preparation

[0537] A silanized glass slide was selected as the chip carrier, soaked in 5% glutaraldehyde for 50 min, ultrasonically cleaned twice with ddH2O, and placed in a dry place for later use.

[0538] The synthesized 173 probes were dissolve...

Embodiment 3

[0545] Example 3. Gene chips for high-throughput detection of pathogens simultaneously detect five drug-resistant gene-resistant strains

[0546] In recent decades, with the widespread and unreasonable use of broad-spectrum antibiotics in public health and animal husbandry, severe bacterial resistance can be caused, treatment is difficult, and the mortality rate is high. Rapid, accurate, and high-throughput detection of drug resistance genes is the key to the prevention and control of infectious diseases. We have established a bacterial resistance gene detection chip, which can detect Escherichia coli DH5α containing kanamycin resistance gene plasmid, Escherichia coli DH5α containing chloramphenicol resistance gene plasmid, Escherichia coli containing bleomycin resistance gene plasmid DH5α and some samples of Escherichia coli DH5α containing tetracycline resistance gene plasmid were detected and compared.

[0547] 1. Bacterial whole genome extraction and labeling

[0548...

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Abstract

The invention relates to a gene chip for high-flux detection of pathogens and application thereof. The gene comprises (1) a combination of 174 oligonucleotide probes of pathogen variety specific genes, toxin genes and drug-resistant genes; and (2) a probe array, which is formed by curing the oligonucleotide probes on a carrier material by arm molecules. The gene chip comprises 174 gene probes, namely 32 pathogen variety specific gene probes of the following 8 pathogens of Burkholderia mallei, Burkholderia pseudomallei, Brucella, salmonella, Yersinia pestis, Bacillus anthracis, comma bacillus and the like, 25 toxin gene probe of the following 7 toxins of diphtheria toxin, Shiga toxin, staphylococcus enterotoxin, choleratoxin and the like, and 117 drug-resistant gene probes of 17 drug-resistant genes of extended-spectrum beta-lactamase, cephalosporinase, carbapenemase, integrase gene, common gene engineering carrier drug-resistant gene and the like. The gene chip can be used to detect multiple pathogen variety specific genes, toxin genes and drug-resistant genes.

Description

Technical field [0001] The altitude detection of genetic chips and its application involves a high -throughput detection gene chip and application of a variety of pathogens and drug -resistant genes.The gene chip contains 174 gene probes, including Plome Berk Holde and Pullid -like Berk Horde, Brucelli, Salmonella, Plague Jearson, Anthramacia, TurkraFrancis, Veosor, and E. EIEC (EIEC), and cholera vaginal species have 32 species of different types of pathogens;Total 7 types of toxin genes of glycotic toxins, glyphobacterial bowel toxins and cholera toxins; ultra -wide -spectrum β lactamase, cephalosporinase, carbon moldne enzymes, integrated enzyme genes, tetracycline family resistance genes, drug -resistant genes, and tetracycline family resistance genes, and tetracycline family resistance genes.Amino glycosides, drug resistant genes, disinfection agent genes, erythromycin -resistant genes, macroexer outer extract genes, vancopycin resistance genes, multi -drug resistance outer p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCY02A50/30
Inventor 李越希潘英傅雅丽潘明洁
Owner 李越希
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