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Method, gene chip and detection reagent kit for detecting common cow pathogenic escherichia coli

A technology of Escherichia coli and gene chip, which is applied in the field of detection of Escherichia coli serotypes that cause bovine septicemia and diarrhea, can solve the problems of limited information in one detection, inapplicability of a large number of samples, etc., and achieve high accuracy and easy operation , highly reproducible effect

Inactive Publication Date: 2011-02-09
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR method is a relatively mature method, fast, simple, and sensitive. Its disadvantage is that the amount of information in one detection is limited, and it is not suitable for the detection of a large number of samples. The developing gene chip technology can make up for these shortcomings.

Method used

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  • Method, gene chip and detection reagent kit for detecting common cow pathogenic escherichia coli
  • Method, gene chip and detection reagent kit for detecting common cow pathogenic escherichia coli
  • Method, gene chip and detection reagent kit for detecting common cow pathogenic escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Design and preparation of probe

[0045] 1. Sequence acquisition:

[0046] (1) Obtaining the bacterial 16s rDNA sequence: Download the 16s rDNA sequence from the GenBank public database.

[0047] (2) Obtaining the O antigen synthesis gene cluster sequence of Escherichia coli O8, O9, O15, O26, O86: The 5 O antigen serotype E. coli O antigen synthesis gene cluster sequences downloaded from the GenBank public database.

[0048] (3) Obtaining the O antigen-specific gene sequences of E. coli O35, O78, O101, O115 and O119: The O antigen synthesis gene cluster sequences of E. coli O35, O78, O101, O115 and O119 have been sequenced by the inventor's unit.

[0049] 2. Probe design:

[0050] Import the above sequence into OligoArray2.0 software, and design the probe according to the instructions of the software.

[0051] In a preferred embodiment of the present invention, 43 probes designed by OligoArray 2.0 software are selected, and probes are screened through hybridization exper...

Embodiment 2

[0058] Example 2 Design and preparation of primers

[0059] 1. Sequence acquisition: same as the sequence of the designed probe.

[0060] 2. Design primers:

[0061] Import the above sequence into the primer design software Primer Premier 5.0 software, and refer to the software's instructions for primer design.

[0062] In the preferred embodiment of the present invention, the main Escherichia coli serotypes (O8, O9, O15, O26, O35, O78, O86) that both contain probes and are suitable for Multiplex PCR are selected in the preferred embodiment of the present invention. , O101, O115 and O119) 11 pairs of primers, in order to adapt to Multiplex PCR, through the preliminary screening of bioinformatics and a large number of Multiplex PCR experiments, the suitable primers as shown in Table 2 were selected.

[0063] Table 2 The primer sequences used in PCR amplification for detection of 10 common bovine pathogenic E. coli

[0064]

[0065]

[0066] 3. Primer synthesis: commission the primer syn...

Embodiment 3

[0067] Example 3 Preparation of gene chip-chip spotting

[0068] 1. Dissolving the probes: the probes synthesized in Example 1 were respectively dissolved in 50% DMSO solution, and diluted to make the final concentration of the probe reach 1 μg / μl.

[0069] 2. Add plate: add the dissolved probe to the corresponding position of the 384-well plate, 10μl per well.

[0070] 3. Pointing: Will be as figure 1 The shown 57.5mm×25.5mm×1mm (L×W×H) clean aldehyde-based glass slide (CEL Associates, Inc.) is placed on the stage of the chip spotter (Spotarray 72), and SpotArray is used Control software (Tele chem smp3 stealty pin), run the program, press figure 2 The arrangement shown is dotted in a 4.5mm×4.5mm spot area on the aldehyde-based glass slide, forming a medium and low density DNA micro-matrix. The six dot matrix areas on the glass slide have the same array arrangement. The size of the dot matrix area is 3mm×2.25mm, the dot pitch in the dot matrix is ​​250μm, the matrix: 12×9, 12×250...

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Abstract

The invention provides a method, a gene chip and a detection reagent kit for detecting common cow pathogenic escherichia coli. The gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises DNA (Deoxyribonucleic Acid) fragments selected from a glycosyl transferase gene and an oligose unit processing enzyme gene on a main escherichia coli serotype O-antigen gene cluster relative to cow septicemia and diarrhea. The gene chip and a designed primer are utilized for detecting and can be prepared into the reagent kit. The products and the method of the invention are utilized to reach the purpose for detecting the serotype of the common cow pathogenic escherichia coli and have the advantages of simple and convenient operation, high throughput, high accuracy, strong repeatability and important significance on the research and the diagnosis of epidemiology.

Description

Technical field [0001] The invention relates to a method for detecting Escherichia coli, a gene chip and a detection kit, in particular to a method for detecting the serotype of Escherichia coli causing bovine sepsis and diarrhea, a gene chip and a detection kit Background technique [0002] Escherichia coli is an important bovine pathogenic bacteria that can cause diseases such as bovine sepsis and diarrhea, and has become a serious threat to the livestock industry. It is an important type of pathogenic bacteria that causes diarrhea in young animals and infants worldwide. Enteropathogenic E. coli infections cause huge losses to the animal husbandry every year, especially the incidence and incidence of diarrhea in young animals caused by it. The mortality rate has been on the rise in recent years. [0003] The main E. coli serotypes that cause bovine sepsis and diarrhea are: O78 and O35. E. coli O78 can be isolated in many lesions caused by E. coli, and it can cause a variety of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C12Q1/68C12R1/19
Inventor 王磊刘斌郭丹
Owner TIANJIN BIOCHIP TECH CO LTD
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