Supercharge Your Innovation With Domain-Expert AI Agents!

Primers for pcr detection of Pseudomonas putida and its pcr method

A Pseudomonas putida, nucleotide sequence technology, applied in biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc., can solve problems such as uncertainty, and achieve high accuracy and detection. Method sensitive effect

Inactive Publication Date: 2015-12-30
中华人民共和国沈阳出入境检验检疫局
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, only two methods of morphological identification and biochemical identification are used to detect Pseudomonas putida, 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
  • Primers for pcr detection of Pseudomonas putida and its pcr method
  • Primers for pcr detection of Pseudomonas putida and its pcr method
  • Primers for pcr detection of Pseudomonas putida and its pcr method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Primer design:

[0023] 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:

[0024] Sequence 1: 5'-CTGCATCATGGCCGGTGACAACATTT-3'

[0025] Sequence 2: 5'-GTCGCATGGCTGTCGGTCTTCAGATC-3'

[0026] The amplified target gene fragment of Pseudomonas putida was 161bp.

Embodiment 2

[0028] DNA template extraction of the bacteria liquid to be tested:

[0029] 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.

[0030] 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.

[0031] 3) Extraction of bacterial genomic DNA

[0032] 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

[0034] Establishment of PCR amplification method:

[0035] 1) Preparation of PCR reaction system: See Table 1 for specific composition preparation.

[0036] Table 1

[0037]

[0038] A negative control and a positive control were also set up.

[0039] 2) PCR reaction conditions:

[0040] Put the above-prepared small tube of PCR reaction solution into the PCR instrument, and the reaction conditions are: 94°C pre-denaturation for 1 min; 94°C denaturation for 15 s, 61°C annealing for 15 s, 72°C extension for 15 s, and 30 cycles; 72°C extension 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

No 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 PCR method thereof. 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
no application Login to View More
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
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com