PCR (Polymerase Chain Reaction) detection primer and method of pseudomonas putida
What is Al technical title?
Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
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
View PDF1 Cites 4 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
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
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more
Image
Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
Examples
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:
[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.
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
PUM
Login to view more
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
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
Application Information
Patent Timeline
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more 
Login to view more