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Fluorescent quantitative PCR method for detecting escherichia coli

A technology of Escherichia coli and fluorescence quantification, applied in the field of conventional molecular biology, can solve the problems of cumbersome and inaccurate results, and achieve the effect of simple operation.

Inactive Publication Date: 2017-07-25
WUHAN IGENEBOOK BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Escherichia coli (Escherichia coli), commonly known as Escherichia coli, was discovered by Escherich in 1885. For a long period of time, it has been regarded as a component of normal intestinal flora and considered to be non-pathogenic bacteria ; until the middle of the 20th century, it was recognized that some special serotypes of Escherichia coli were pathogenic to humans and animals, especially for infants and young animals (poultry), often causing severe diarrhea and sepsis. It is a common prokaryote, According to different biological characteristics, pathogenic Escherichia coli can be divided into 6 categories: enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterohaemorrhagic Escherichia coli (EHEC), enteroadhesive Escherichia coli (EAEC) and diffusely adherent Escherichia coli (DAEC); Escherichia coli belongs to Gram-negative bacteria (G-); there are many detection methods, but most of them are cumbersome and the results are inconsistent. Inaccurate, the purpose of the present invention is to provide a method that can quickly and effectively detect Escherichia coli in the target

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A fluorescent quantitative PCR method for detecting Escherichia coli, comprising the steps of:

[0037] S1. Design specific primers and fluorescent probes according to the target gene sequence as follows:

[0038] Primer A: 5'-TGGCATGACGTTATAGGCTACAAT-3';

[0039] Primer B: 5'-AGCTTGTTCTAACTGGGCTAATCCT-3';

[0040] Primer C: 5'-TGCTATTCTAACTGGGCTAATAAG-3';

[0041] Primer D: 5'-AGCTATTCTATCTCTGCTAATAAC-3';

[0042] Probe P: 5'-FAM-ATAGGATGACAAATATCTGCGCTGCT-BHQ1-3';

[0043] S2. Select the Escherichia coli sample and extract the DNA / RNA of the sample. The specific steps are as follows:

[0044] B1. Select Escherichia coli samples in a period of vigorous growth;

[0045] B2. Remove the medium, add 1 μL TRNzol and store at -70°C for 4 hours;

[0046] B3. After adding chloroform, centrifuge at 2200rpm for 40s, and let stand to obtain the organic layer located in the lower layer and the water sample layer located in the upper layer;

[0047] B4. Collect the water sam...

Embodiment 2

[0061] A fluorescent quantitative PCR method for detecting Escherichia coli, comprising the steps of:

[0062] S1. Design specific primers and fluorescent probes according to the target gene sequence as follows:

[0063] Primer A: 5'-TGGCATGACGTTATAGGCTACAAT-3';

[0064] Primer B: 5'-AGCTTGTTCTAACTGGGCTAATCCT-3';

[0065] Primer C: 5'-TGCTATTCTAACTGGGCTAATAAG-3';

[0066] Primer D: 5'-AGCTATTCTATCTCTGCTAATAAC-3';

[0067] Probe P: 5'-FAM-ATAGGATGACAAATATCTGCGCTGCT-BHQ1-3';

[0068] S2. Select the Escherichia coli sample and extract the DNA / RNA of the sample. The specific steps are as follows:

[0069] B1. Select Escherichia coli samples in a period of vigorous growth;

[0070] B2. Remove the medium, add 1 μL TRNzol and store at -70°C for 5 hours;

[0071] B3, after adding chloroform, centrifuge at 2300rpm for 40s, and let stand to obtain the organic layer located in the lower layer and the water sample layer located in the upper layer;

[0072] B4. Collect the water sam...

Embodiment 3

[0086] A fluorescent quantitative PCR method for detecting Escherichia coli, comprising the steps of:

[0087] S1. Design specific primers and fluorescent probes according to the target gene sequence as follows:

[0088] Primer A: 5'-TGGCATGACGTTATAGGCTACAAT-3';

[0089] Primer B: 5'-AGCTTGTTCTAACTGGGCTAATCCT-3';

[0090] Primer C: 5'-TGCTATTCTAACTGGGCTAATAAG-3';

[0091] Primer D: 5'-AGCTATTCTATCTCTGCTAATAAC-3';

[0092] Probe P: 5'-FAM-ATAGGATGACAAATATCTGCGCTGCT-BHQ1-3';

[0093] S2. Select the Escherichia coli sample and extract the DNA / RNA of the sample. The specific steps are as follows:

[0094] B1. Select Escherichia coli samples in a period of vigorous growth;

[0095] B2. Remove the medium, add 1 μL TRNzol and store at -70°C for 7 hours;

[0096] B3, after adding chloroform, centrifuge at 2350rpm for 40s, let stand to obtain the organic layer located in the lower layer and the water sample layer located in the upper layer;

[0097] B4. Collect the water sample ...

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Abstract

The invention discloses a fluorescent quantitative PCR method for detecting escherichia coli. The fluorescent quantitative PCR method comprises the following steps: S1. designing a specific primer and a fluorescent probe according to target gene sequence; S2. selecting an escherichia coli, and extracting DNA / RNA of the sample; S3. converting the extracted RNA into cDNA; and S4. detecting the extract of the last step by using an ultraviolet spectrophotometer. The fluorescent quantitative PCR method has the following advantages: 1. being capable of accurately and efficiently extracting high-purity escherichia coli DNA, and beneficial to improvement of experimental results; 2. constructing a standard curve equation by utilizing a double-standard curve standard, so as to calculating the number of escherichia coli in the sample; and 3. providing a rapid and high-efficiency detection system which is flexible, simple and convenient to operate, and can be used for qualitative and quantitative detection of escherichia coli.

Description

technical field [0001] The invention relates to the technical field of conventional molecular science, in particular to a fluorescent quantitative PCR method for detecting Escherichia coli. Background technique [0002] Escherichia coli (Escherichia coli), commonly known as Escherichia coli, was discovered by Escherich in 1885. For a long period of time, it has been regarded as a component of normal intestinal flora and considered to be non-pathogenic bacteria ; until the middle of the 20th century, it was recognized that some special serotypes of Escherichia coli were pathogenic to humans and animals, especially for infants and young animals (poultry), often causing severe diarrhea and sepsis. It is a common prokaryote, According to different biological characteristics, pathogenic Escherichia coli can be divided into 6 categories: enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterohaemorrhagic Esc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12R1/19
CPCC12Q1/6851C12Q2531/113C12Q2563/107
Inventor 李泽卿骆杰
Owner WUHAN IGENEBOOK BIOTECH CO LTD
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