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Primer for quickly determining enterotoxigenic eschericha coli in feed sample and application for primer

A rapid detection technology for Escherichia coli, which is applied in the detection/testing of microorganisms, material stimulation analysis, biochemical equipment and methods, etc., to achieve the effects of high amplification efficiency, easy operation, and simplified detection procedures

Active Publication Date: 2012-07-11
河北方田农牧科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, there is no report in China based on the SYBR Green I fluorescence quantitative PCR method based on the specific gene of enterotoxic Escherichia coli K88 pili

Method used

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  • Primer for quickly determining enterotoxigenic eschericha coli in feed sample and application for primer
  • Primer for quickly determining enterotoxigenic eschericha coli in feed sample and application for primer
  • Primer for quickly determining enterotoxigenic eschericha coli in feed sample and application for primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Rapid qualitative detection of enterotoxic Escherichia coli K88 in predicted feed samples

[0040] This embodiment uses the gel recovery product of the target amplified fragment as a positive control; Sterile water is used as a negative control; In addition, Enterococcus faecalis, Staphylococcus aureus, and Salmonella are used as samples to carry out.

[0041] A. Preparation method of template DNA

[0042] (1) Take a certain amount of sample to be tested (bacteria liquid is generally 5ml, feed sample is 1000mg), and quickly grind it into powder under liquid nitrogen freezing; put the powder into a 2ml centrifuge tube, and then add 1MTris.Cl buffer 750ul (2) Place the centrifuge tube in a 65°C water bath for 1 hour, and mix gently during the water bath; take out the centrifuge tube, and add an equal volume of phenol-chloroform (V / V=1:1) solution to each tube 700ul, mix well and centrifuge at 10000rpm for 10 minutes; (3) transfer the supernatant to another cen...

Embodiment 2

[0049] Example 2: Prediction of the preparation of the external standard of enterotoxic E. coli K88 in the feed sample and the drawing of the standard curve

[0050] After making a 10-fold serial dilution of enterotoxic Escherichia coli K88 with a known concentration, the concentration of the prepared bacteria was 10 7 -1CFU / ml according to

[0051] The method in embodiment 1 extracts bacterial DNA, and real-time fluorescent PCR detects, and the steps are as follows:

[0052] A. Fluorescent quantitative PCR

[0053] Reaction system 25.0 μL, including: 2×SuperReal PreMix mother solution 12.5 μL, upstream and downstream primers DCf and DCr 0.5 μL (20 μmol / L), DNA template 2.0 μL, 50×ROX Reference Dye 0.5 μL, RNase-free ddH 2 0 to 25 μL system;

[0054] Fluorescence quantitative PCR reaction parameters: pre-denaturation at 95°C for 10 minutes; denaturation at 94°C for 5s, annealing at 60°C for 15s, 40 cycles; storage at 4°C; repeat 3 times for each sample;

[0055] B. Prepara...

Embodiment 3

[0060] Embodiment 3: accuracy, stability and repeatability experiment

[0061] Using samples of the same concentration as templates, the methods in the national standard GB / T 4789.38-2008 and the method in Example 2 were used to detect the samples, and the two methods were compared. In order to evaluate the repeatability and stability of the test, the same batch was repeated twice for fluorescence quantitative reaction. For each group of test samples, 5 gradient reaction tubes were made, and the template concentration was divided into 2×10 7 , 2×10 6 , 2×10 5 , 2×10 4 , 2×10 3 CFU / μL. The results showed that the coefficients of variation of all dilutions were small (CV<5%), indicating that the inter-batch differences were small, and the fluorescent quantitative PCR reaction system established in this experiment had better intra-batch repeatability and stability (Table 1).

[0062] Substituting the Ct value shown in Table 1 into the standard curve in Example 2, the obtain...

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Abstract

The invention belongs to the technical field of feed science and detection of feed additives, and particularly relates to a primer for quickly determining enterotoxigenic eschericha coli K88 in a feed sample and application for the primer. A pair of primers is designed aiming at the pilus specific gene of the enterotoxigenic eschericha coli K88, the gradient dilution of deoxyribonucleic acid (DNA) is used as an external standard substance; and an SYBRGreen I real-time quantitative polymerase chain reaction (PCR) detection method for the enterotoxigenic eschericha coli K88 is established; by the method, the enterotoxigenic eschericha coli K88 in the feed sample can be quantified quickly and specifically; more than or equal to 2*10<2> CFU / g of enterotoxigenic eschericha coli K88 can be detected in the feed sample, and only 5 hours is required by the whole operation process; and compared with the conventional detection method, the method has the advantages of simple detection process, high detection efficiency and accuracy, short detection period and the like, and lays the foundation for development of a kit for quickly and accurately detecting the enterotoxigenic eschericha coli.

Description

technical field [0001] The invention belongs to the technical field of feed science and feed additive detection, and in particular relates to a primer for rapid determination of Enterotoxigenic Escherichia coli K88 in a feed sample and an application thereof. Background technique [0002] Coliform bacteria and the enterotoxin they produce can cause food poisoning in animals and humans, and the most common symptom is diarrhea in animals and humans. The existence of coliform bacteria in the feed not only directly endangers the health of livestock and poultry, but also causes poisoning and death of livestock and poultry in serious cases, causing serious economic losses; pathogenic E. coli mainly includes enterotoxic E. coli (enterotoxigenic E. coli) ( Enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (ELEC) and enteroinvasive Escherichia coli (EIEC). Among them, ETEC is one of the most important pathogens caus...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12Q1/06C12N15/11G01N21/64
Inventor 刘滢谢飞胡婷刘婷彭子欣
Owner 河北方田农牧科技有限公司
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