Method for detecting bacillus coli inducing diarrhoea and hydropsy during pig weaning period and reagent kit thereof

A technology of Escherichia coli and weaning period, which is applied in the direction of biochemical equipment and methods, biological testing, microbial measurement/inspection, etc., can solve the problems of inapplicability to a large number of sample detection and limited information in one detection, and achieve high accuracy, Easy to operate and highly reproducible results

Inactive Publication Date: 2008-02-06
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 for detecting bacillus coli inducing diarrhoea and hydropsy during pig weaning period and reagent kit thereof
  • Method for detecting bacillus coli inducing diarrhoea and hydropsy during pig weaning period and reagent kit thereof
  • Method for detecting bacillus coli inducing diarrhoea and hydropsy during pig weaning period and reagent kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Design and preparation of probes

[0035] 1. Sequence acquisition: use Artemis software to sort out the 5 O antigen serotype E. coli (O8, O45, O138, O139 and O157) O antigen synthesis gene cluster sequences downloaded from the GenBank public database (the other 3 O141, O147, O149) The sequence of the O antigen synthesis gene cluster has been sequenced by the inventor’s unit and has applied for a patent), the gene sequence of 11 virulence factors of Escherichia coli, and the gene sequence of Shigella ipaH.

[0036] 2. Probe design: Import the above sequence into OligoArray2.0 software, and design the probe with reference to the instructions of the software.

[0037] In a preferred embodiment of the present invention, 71 probes designed by OligoArray2.0 software are selected, and probes are screened through 156 hybridization experiments, and finally the probes shown in Table 1 are obtained. Among them, the probe SEQ ID NO: 1 is selected from the conservative regi...

Embodiment 2

[0043] Example 2: Design and preparation of primers

[0044] 1. Sequence acquisition: use Artemis software to sort out the 5 O antigen serotype E. coli (O8, O45, O138, O139 and O157) O antigen synthesis gene cluster sequences downloaded from the GenBank public database (the other 3 O141, O147, O149) The O antigen synthesis gene cluster sequence has been sequenced by the inventor’s laboratory and has applied for a patent), the 11 virulence factor gene sequences of Escherichia coli, and the Shigella ipaH gene sequence.

[0045] 2. Design primers: import the above sequence into the primer design software Primer Premier 5.0 software, and refer to the software's instructions for primer design.

[0046] In a preferred embodiment of the present invention, the main E. coli serotypes (O149, O138, O139, O141, O8, O8, O149, O138, O139, O141, O8) O147, O45, O157) and virulence factor genes (F18 (107), F4 (K88), F5 (K99), F6 (987P), F41, intimin, STap, STb, LT, Stx2e, EAST1) DNA primers 30 Y...

Embodiment 3

[0052] Example 3: Gene chip preparation-chip spotting

[0053] 1. Dissolving the probe: Dissolve the probes synthesized in Example 1 in a 50% DMOS solution, so that the final concentration of the probe reaches 1 μg / μl.

[0054] 2. Add plate: add the dissolved probe to the corresponding position of the 384-well plate, 10μl per well.

[0055] 3. Spotting: Place a clean aldehyde-based glass slide (CELAssociates, Inc.) of 57.5mm×25.5mm×1mm (length×width×height) as shown in Figure 1 on the chip spotter (Spotarray 72) Use SpotArray's control software (Tele chem smp3stealty pin) to run the program on the stage on the aldehyde-based glass slide according to the arrangement shown in Figure 2. The low-density DNA micro-matrix has the same array arrangement in the six spots on the slide.

[0056] 4. Drying: Dry the ordered chips overnight at room temperature, and then dry them in an oven at 45°C for 2 hours.

[0057] 5. Cross-linking: cross-link twice with a cross-linker (uvpcl-2000M ultra...

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Abstract

The present invention relates to a gene chip, a testing method and a testing reagent case used for testing E.coli serological type and virulent gene that cause diarrhea and hydropsy of piglets in the ablactation period. The gene chip is fixed on an oligonucleotide probe of a solid phase support, the gene chip is mainly to select DNA fragments from UDPG-glucosyltransferase and Oligosaccharide unit treatment enzyme gene on E.coli serological type O-antigen gene cluster and main E.coil virulent factors that cause diarrhea and hydropsy of piglets in the ablactation period. The gene chip and the designed primer can be used for testing and can be made into the reagent case. The product and the method of the present invention can realize the purpose of testing test the common diarrhea and hydropsy of piglets in the ablactation period which are caused by E.coil serological type and virulent factors. The present invention has the advantages of simple operation, high accuracy and strong repetition; and is of great significance to the study and diagnosis of epidemiology.

Description

Technical field [0001] The invention relates to a detection method and a kit, in particular to a gene chip and a kit for detecting the serotype and virulence factor of Escherichia coli that cause diarrhea and edema in pigs during weaning. Background technique [0002] Post-weaning diarrhea (PWD) and porcine edema disease (ED) are two important infectious diseases that lead to the death of piglets. They have become diseases that are harmful to the pig industry. PWD usually occurs after weaning to 12 weeks of age, especially within 14 days after weaning, and the peak of death is usually 4-10 weeks of age. There are many reasons for this disease, the most important of which is diarrhea caused by pathogenic Escherichia coli. In the course of disease, pathogenic E. coli produces toxins and is absorbed by piglets, causing a series of clinical symptoms and leading to piglet death. ED is a relatively common infectious disease in piglets, which mainly occurs in piglets that are weaned for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48C12Q1/10
Inventor 冯露韩巍青王磊
Owner TIANJIN BIOCHIP TECH CO LTD
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