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Gene chip for detecting enterohemorrhagic escherichia coli, use method and detection kit

A technology of Escherichia coli and gene chip, which is applied in the field of gene chip and its use method and detection kit, can solve the problems of complicated operation, missed detection or false positive result, easy to be affected by human factors, etc. Accurate effect

Inactive Publication Date: 2010-06-30
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current testing standards for Escherichia coli in exported food adopted by China’s State Commodity Inspection Bureau are roughly equivalent to the US FDA’s standard method, which is also subject to great limitations in detection accuracy and efficiency.
[0012] China's current national standard method for the detection of ETEC is also through a series of steps such as isolation and culture, biochemical reactions, serotyping, and toxin determination. The entire process takes about 4-5 days. Affected by human factors
In addition, most sera are extremely difficult to produce, and are prone to missed or false positive results in serotype testing

Method used

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  • Gene chip for detecting enterohemorrhagic escherichia coli, use method and detection kit
  • Gene chip for detecting enterohemorrhagic escherichia coli, use method and detection kit
  • Gene chip for detecting enterohemorrhagic escherichia coli, use method and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Probe design and preparation

[0056] 1. Sequence acquisition

[0057] The genes involved in the present invention include the 16S rRNA gene of bacteria, the oligosaccharide unit processing enzyme genes wzx and wzy of Escherichia coli of 19 different O serogroups (Escherichia coli O8 corresponds to wzt, wzm genes), and ETEC unique The enterotoxin coding genes elt, estA (including human estA (h) and porcine est (p)), estB genes. In addition, due to the high sequence similarity between the wzx and wzy genes of Escherichia coli O148 and the corresponding genes of Shigella dysenteriae 1 (Shigella dysenteriae 1), the rfpB gene region unique to Shigella dysenteriae 1 Two probes were designed to distinguish Escherichia coli O148 and Shigella dysenteriae type 1. The sources of these gene sequences are detailed in Table 1.

[0058] Table 1 Description of sequence sources

[0059]

[0060]

[0061]

[0062] 2. Probe Design

[0063] Enter the O antigen ol...

Embodiment 2

[0075] Example 2 Gene Chip Preparation - Chip Spotting

[0076] 1. Dissolving the probe: Dissolve the probe dry powder tube (synthesized by Aoke) synthesized in Example 1 above. The steps are as follows: centrifuge the above-mentioned probe dry powder tube at 12000 rpm for 10 minutes (be careful not to open the lid of the dry powder tube before centrifugation), then add 1xSpotting Buffer (add 20μl to 1OD), and mix well with a shaker Centrifuge quickly to remove the liquid on the tube wall. After standing at room temperature for 1 hour, take 1.5 μl to measure OD, measure the nucleic acid concentration (ng / μl) of the probe (single strand), and then use the following formula to dilute the probe to a final concentration of 1 μg / μl.

[0077] formula:

[0078] 2. Adding plates: add the 51 dissolved probes to the corresponding positions in the 384-well plate, and add 10 μl of probe solution to each well.

[0079] 3. Spotting: Spot the probes in the above-mentioned 384-well p...

Embodiment 3

[0088] Example 3 Rapid detection of O serogroup and virulence factor types of enterotoxigenic Escherichia coli using the prepared gene chip

[0089] In the method for detecting enterotoxigenic Escherichia coli using the above-mentioned prepared gene chip, the present invention has made a series of experiments on factors such as detection steps and detection conditions, such as the ratio of each component in the whole genome random primer PCR reaction mixture, The template concentration, the hybridization temperature of the chip hybridization reaction, the hybridization time, and the main reagent formulations, such as hybridization solution, washing solution, etc. (refer to the following formula for details), are all excellent ratios obtained after gradient experiments. The experimental procedure of the gene chip for the detection of enterotoxigenic Escherichia coli is also the result of optimization. The following is a specific description of a preferred embodiment of the de...

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Abstract

The invention relates to a gene chip for detecting enterohemorrhagic escherichia coli, comprising a solid-phase carrier and an oligonucleotide specific probe fixed on the solid-phase carrier, wherein the oligonucleotide specific probe fixed on the solid-phase carrier has at least one of the following gene sequences: at least one DNA fragment selected from oligosaccharide unit treatment enzyme gene sequences in O-antigen gene clusters of different sero-group escherichia coli; a DNA fragment selected from coding genes of enterohemorrhagic escherichia coli enterotoxin; a DNA fragment selected from shigella dysenteriae type-1 genes; and a complementary DNA or RNA fragment of the previous DNA fragments. The invention also relates to a use method of the gene chip and a kit comprising the gene chip. The gene chip and the kit can achieve the purpose of detecting the enterohemorrhagic escherichia coli and have simple and convenient operation, high accuracy and strong repeatability.

Description

technical field [0001] The present invention relates to a gene chip, its use method and detection kit, in particular to a gene chip for detection of enterotoxigenic Escherichia coli O serogroup and virulence gene, its detection method and detection kit. Background technique [0002] Escherichia coli (E.coli), commonly known as Escherichia coli, is a facultative anaerobic bacterium in the intestinal tract of humans and many animals, and is the most dominant normal resident bacterium. It can ferment a variety of sugars, produce acid and gas, enter the intestinal tract with breastfeeding after the baby is born, and stay with people for life. , can also synthesize vitamin B and K, and colicin with bactericidal effect. [0003] Escherichia coli under normal living conditions in the intestinal tract is not pathogenic. When the body's immunity is low, or when it invades other parts of the intestine, it can cause intestinal or extraintestinal diseases, so E. coli is an opportunist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10
CPCY02A50/30
Inventor 王磊王荃王苏日古嘎冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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