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Gene chip for inspecting important intestinal tract peccant germ, its inspecting method and reagent kit

A gene chip and pathogenic bacteria technology, which is applied in the field of gene chip and its detection method and detection kit for detecting important intestinal pathogenic bacteria, can solve problems such as cross contamination and false negatives, and achieve strong repeatability and accuracy High, efficient and fast detection and analysis methods

Active Publication Date: 2010-05-05
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, if the reaction conditions are not well controlled, false negatives and cross-contamination are prone to occur. However, the emergence of gene chip technology has solved this problem.

Method used

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  • Gene chip for inspecting important intestinal tract peccant germ, its inspecting method and reagent kit
  • Gene chip for inspecting important intestinal tract peccant germ, its inspecting method and reagent kit
  • Gene chip for inspecting important intestinal tract peccant germ, its inspecting method and reagent kit

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Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment one Probe design and preparation

[0050] 1. Sequence acquisition: The wzx and wzy sequences of type 15 important intestinal pathogens measured in our laboratory and downloaded from public databases such as GenBank were sorted out with Artemis software (Shigella baumannii O16 is orf5).

[0051] 2. Probe design: Import the obtained gene sequence into the OligoArray2.0 software, set the corresponding parameters (the length is 40bp±5bp, the Tm value is 80°C±2°C), and the running program is designed online to detect intestinal important There were 124 oligonucleotide probes for 15 serotypes of pathogenic bacteria.

[0052] 3. Probe selection: carry out probe screening by 683 hybridization experiments, and finally select from the output results to obtain specific and sensitive probes with a length of 40bp ± 5bp, Tm80°C ± 2°C to form the probes required for the gene chip of the present invention. Needles collection.

[0053] In a preferred embodiment of the pre...

Embodiment 2

[0063] Embodiment two Primer design and preparation

[0064] 1. Sequence acquisition: The wzx and wzy sequences of type 15 important intestinal pathogens measured in our laboratory and downloaded from public databases such as GenBank were sorted out with Artemis software (Shigella boaumannii O16 is orf5).

[0065] 2. Design primers: Import the obtained gene sequence into the primer design software Primer Premier 5.0 software, set the corresponding parameters (length: 20bp±2bp, Tm value: 50°C±5°C) and run the program.

[0066] 3. Selection of primers: Select primers with a Tm value of 50°C±5°C and a length of 20bp±2bp from the output results, including the probe sequence used in the gene chip. Individual probes need to be manually adjusted, and the primers should be appropriately lengthened or shortened by a few bases to include the probe, conform to the Tm value of 50°C±5°C, and other parameters.

[0067] In a preferred embodiment of the present invention, in order to adapt...

Embodiment 3

[0074] Embodiment three chip spotting

[0075]Dissolve the synthesized probe dry powder tube (by Aoke Synthetic) first. The steps are as follows: 12000 rpm, centrifuge for 5 minutes (be careful not to open the lid of the dry powder tube before centrifugation), add 50% DMSO (adding 14 μl at 1OD), mix with a shaker and then centrifuge quickly, and the tube wall The liquid on the top is off. After standing at room temperature for 1 hour, take 1 μl and dilute 180 times with 50% DMSO to measure OD, measure the nucleic acid concentration (ng / μl) of the probe (single strand), and then use the formula to dilute the probe to the final concentration 1 μg / μl.

[0076] Formula: supplemented with 50% DMSO -Remaining volume after measuring OD

[0077] Add 52 probes to corresponding positions in the 384-well plate, and add 10 μl of probes to each well. The probes in the 384-well plate were spotted onto the glass slides using an automatic spotting device to form the microarray we desi...

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Abstract

Gene chip for inspecting enteric important pathomycete, its inspection and reagent kit are disclosed. The gene chip consists of solid-phase carrier and oligonucleotide probe fixed on the carrier, which comprises at least one DNA fragment selected from oligose unit treating enzyme gene or glycosylation transferring enzyme of intestinal bacterium O-antigen gene cluster and DNA fragment selected fromcolon bacillus 16S rRNA conservative area, Multiplex PCR amplifying for gene set DNA to be inspected by primer, hybridizing with the gene chip, analyzing and appraising by software Bactarray Analyzer. It's efficient and fast, and has excellent sensitivity and specificity. It can be used to discriminate blood serum type and used in science life, medical inspection, food safety, import-export quarantine.

Description

technical field [0001] The invention relates to a gene chip, a detection method thereof, and a detection kit, in particular to a gene chip for detecting important intestinal pathogenic bacteria, a detection method thereof, and a detection kit. Background technique [0002] Intestinal infectious diseases are one of the important global public health problems, especially in Asia, Africa and Latin America. According to the data of WHO, children under the age of 5 in the above-mentioned areas die from intestinal infectious diseases every year for more than 5 million people, and the incidence is about 750 million to 1 billion person-times. Even in economically developed countries and regions, the above problems also exist. For example, the serious prevalence of hemorrhagic Escherichia coli O157:H7 enteritis in the United States, Japan and other countries can illustrate this fact. An outbreak of hemorrhagic enteritis caused by EHEC O157:H7 was first reported in the United States ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王磊李雅玥刘丹温琳延冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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