Fluorescent quantitative PCR kit for detecting enterococcus faecium in microbial feed additive

A microbial feed, Enterococcus faecium technology, applied in the field of bioengineering, can solve the problems of uneven quality of commercially available products

Pending Publication Date: 2021-05-18
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no standard method for detecting Enterococcus faecium in feed in my country, and the quality of commercially available products i...

Method used

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  • Fluorescent quantitative PCR kit for detecting enterococcus faecium in microbial feed additive
  • Fluorescent quantitative PCR kit for detecting enterococcus faecium in microbial feed additive
  • Fluorescent quantitative PCR kit for detecting enterococcus faecium in microbial feed additive

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1. Fluorescence quantitative PCR (qPCR) detection

[0020] (1) Mix the qPCR reaction solution, hot-start Taq enzyme and probes in a centrifuge tube according to the number of tested samples n (n=the number of samples to be tested + 2), shake on a vortex shaker, and divide each tube into Installed, cover the tube cover and set aside.

[0021] (2) Add the negative control solution to a dispensing tube, take the DNA of each sample and add it to the corresponding reaction tube; finally, take out the positive control and add it to another reaction tube.

[0022] (3) Fluorescence quantitative PCR reaction system: 10 μL Premix Ex Taq TM (2×), 2μL template, 1μL each of upstream and downstream primers and probes (10μmol / L), 5μL ddH 2 O, 20 μL total.

[0023] (4) Fluorescence quantitative PCR reaction conditions: pre-denaturation at 95°C for 30s, cycle 40 times according to the following parameters: denaturation at 95°C for 10s, fluorescence collection at 62°C for 20s, fluores...

Embodiment 2

[0040] Example 2 Parameter optimization

[0041]Determine the probe concentration: 10 μL Premix Ex TaqTM (2×), 2 μL template DNA, 1 μL (10 μmol / L) each of the upstream and downstream primers, respectively add 0.1, 0.5, 1, 2 μL (10 μmol / L) of double distilled water to make up for the probe 20 μL. The results showed that the amplification of the reaction system was good when the final concentration of the probe was 0.5 μM.

[0042] Determine primer concentration: 10 μL Premix Ex TaqTM (2×), 2 μL template DNA, 1 μL (10 μmol / L) probe, add 1 μL (10 μmol / L) of upstream and downstream primers respectively, 0.5, 1, 2, 4 μL (10 μmol / L) L), make up 20 μL with double distilled water. The results showed that when the final primer concentration was 0.5 μM, the reaction system amplified well.

[0043] Annealing temperature: pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s, extension at 60°C, 62°C, and 65°C for 20s, 40 cycles. The results showed that when the annealing temp...

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Abstract

The invention provides a fluorescent quantitative PCR kit for detecting enterococcus faecium in a microbial feed additive. The fluorescent quantitative PCR kit comprises an upstream primer, a downstream primer and a probe, wherein the upstream primer has a sequence as shown in SEQ ID NO.1, the downstream primer has a sequence as shown in SEQ ID NO.2, and the probe has a sequence as shown in SEQ ID NO.3. The designed primers and probe are used for detecting a real sample, and the primers and the probe are proved not to be crossed with other bacteria, so that detection sensitivity is very high.

Description

Technical field: [0001] The invention belongs to the field of biological engineering, and relates to a detection kit, in particular to a fluorescence quantitative PCR kit for detecting Enterococcus faecium in a microbial feed additive. Background technique: [0002] Microbial feed additives can participate in regulating the microecological balance in the gastrointestinal tract and stimulate immune function, thereby promoting animal growth and improving feed conversion rate. It has a good application prospect in the development of animal husbandry because it has no pollution, no residue, and can be used as an effective substitute for antibiotics. Enterococcus faecium, as one of the approved strains in my country, has good intestinal adhesion, good tolerance to high temperature, high salt, acid and alkali, and also has good tolerance to some antibiotics. It is widely used because it can significantly improve the growth, immunity and digestive function of livestock and poultry...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2563/107C12Q2545/114
Inventor 姜芹孙冰清黄士新张文刚曹莹顾欣商军严凤张浩然潘娟田恺吴剑平黄家莺
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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