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PCR (Polymerase Chain Reaction) detection primer and method of pseudomonas putida

A technology of Pseudomonas putida, detection methods, applied in the directions of biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc.

Inactive Publication Date: 2014-07-02
中华人民共和国沈阳出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there are only two methods to detect Pseudomonas putida by morphological identification and biochemical identification, and the results of the above two detection methods have great uncertainty

Method used

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  • PCR (Polymerase Chain Reaction) detection primer and method of pseudomonas putida
  • PCR (Polymerase Chain Reaction) detection primer and method of pseudomonas putida
  • PCR (Polymerase Chain Reaction) detection primer and method of pseudomonas putida

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Primer design:

[0024] Check the complete gene sequence of Pseudomonas putida on Gengbank, and design specific primers for the conserved genes of Pseudomonas putida. The specific nucleotide sequence is:

[0025] Sequence 1: 5'-CTG CAT CAT GGC CGG TGA CAA CAT TT- 3'

[0026] Sequence 2: 5'-GTC GCA TGG CTG TCG GTC TTC AGA TC-3'

[0027] The amplified target gene fragment of Pseudomonas putida was 161bp.

Embodiment 2

[0029] DNA template extraction of the bacteria liquid to be tested:

[0030] 1) Add 1g (mL) sample into 100mL normal saline and mix evenly by aseptic operation, pipette 1 loop of bacteria and inoculate 5 Pseudomonas chromogenic medium plates continuously, and incubate at 30°C for 24h. During the sampling process, a Pseudomonas chromogenic medium plate was placed next to the sample as a blank control.

[0031] 2) Pick 1-3 single colonies on the plate and transfer them to nutrient broth in a constant temperature incubator at 35±1°C for 18h-24h.

[0032] 3) Extraction of bacterial genomic DNA

[0033] Take 200 ul of the above-mentioned cultured bacterial solution, use a commercial bacterial genomic DNA extraction kit, and refer to the instructions for specific extraction operations to extract bacterial genomic DNA.

Embodiment 3

[0035] Establishment of PCR amplification method:

[0036] 1) Preparation of PCR reaction system: See Table 1 for specific composition preparation.

[0037] Table 1

[0038]

[0039] A negative control and a positive control were also set up.

[0040] 2) PCR reaction conditions:

[0041] Put the above-prepared small tube of PCR reaction solution into the PCR instrument. The reaction conditions are: 94 °C pre-denaturation for 1 min; 94 °C for 15 s, 61 °C for 15 s, 72 °C for 15 s, and 30 cycles; 72 °C for 5 min.

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) detection primer and method of pseudomonas putida. The primer is that the nucleotide sequence includes a sequence 1 and a sequence 2. The detection method comprises the following steps of: 1) mixing the primer as disclosed in claim 1, a DNA template of bacterium solution to be detected, an Mg<2+> containing PCR buffer solution for PCR amplification, dNTP (diethyl-Nitrophenyl Thiophosphate) and Taq DNA polymerase to obtain a PCR reaction system; 2) transferring the PCR reaction system to a PCR instrument, and then amplifying according to the preset amplification condition; and 3) detecting an amplified segment through electrophoresis after the amplification is done so as to determine the structure. According to the detection method, the PCR method is carried out for detecting the gene segment of pseudomonas putida, thus the accuracy of the detection result is improved; the detection method is sensitive and only needs common PCR instrument and electrophoresis apparatus to reach the purpose of detection; and the detection result can be issued within 2 to 3 hours only.

Description

technical field [0001] The invention relates to the field of biological detection, and in particular provides a primer for PCR detection of Pseudomonas putida and a detection method. Background technique [0002] Pseudomonas putida is ubiquitous in nature and is an environmentally friendly microbial agent that can remove environmental pollution. The United States, Japan and other countries have produced the bacteria in large quantities and made products to be distributed to countries all over the world. In order to increase the quality and safety of imported environmentally friendly microbial agents and protect my country's natural environment from the damage of foreign bacteria, it is necessary to establish a detection method for Pseudomonas putida in environmentally friendly microbial agents as soon as possible. [0003] At present, there are only two methods for detecting Pseudomonas putida by morphological identification and biochemical identification, and the results ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q2531/113
Inventor 耿庆华王芳王金玲张莹林颖
Owner 中华人民共和国沈阳出入境检验检疫局
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