Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Lamp primer set, kit and rapid detection method for detecting Enterobacter cloacae

A technology of Enterobacter cloacae and primer set, applied in the field of molecular biology detection, can solve the problems of long time and complicated operation, and achieve the effects of low probability, high sensitivity and high specificity

Active Publication Date: 2020-09-15
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These time-consuming, cumbersome and expensive instruments and equipment have limited the development of on-site detection of Enterobacter cloacae. Therefore, the development of a simple and fast detection method for Enterobacter cloacae has great application prospects.

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
  • Lamp primer set, kit and rapid detection method for detecting Enterobacter cloacae
  • Lamp primer set, kit and rapid detection method for detecting Enterobacter cloacae
  • Lamp primer set, kit and rapid detection method for detecting Enterobacter cloacae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Kit for detecting Enterobacter cloacae

[0039] The kit for detecting Enterobacter cloacae includes the following components: (1) LAMP primer set; (2) DNA polymerase; (3) reaction solution; (4) color reagent; (5) fluorescent dye; (6) Blocking solution; (7) Positive control and negative control.

[0040] (1) LAMP detection primer set

[0041] According to multiple specific target genes of Enterobacter cloacae, multiple sets of LAMP primers were designed. After multiple sensitivity and specificity experiments, dnaJ (GenBank accession number: AB272638.1) was screened as the final target gene and the final preferred primer The group has high amplification efficiency, excellent sensitivity and specificity. It includes a pair of outer primers, a pair of inner primers and a pair of loop primers. The nucleotide sequences are as follows:

[0042] dnaJ-F3: 5'-CATGTCCGCACTGTCATG-3' (SEQ ID NO:1);

[0043] dnaJ-B3: 5'-TTCACAGTAGAGGTTGTTGC-3' (SEQ ID NO: 2);

[0044] dnaJ-FIP: 5'-...

Embodiment 2

[0055] Example 2. Constant temperature fluorescence amplification detection method of Enterobacter cloacae

[0056] Using the kit of Example 1 to detect Enterobacter cloacae, the specific steps are as follows:

[0057] (1) Extract the DNA of the sample to be tested;

[0058] (2) Use the LAMP primer set to perform constant temperature fluorescence amplification of the DNA of the sample to be tested:

[0059] Add 25μL system of constant temperature amplification into the reaction tube, which contains: dnaJ-F3 0.02μM, dnaJ-B3 0.02μM, dnaJ-FIP 0.16μM, dnaJ-BIP 0.16μM, dnaJ-LF 0.08μM, dnaJ-LB 0.08μM, Reaction solution 12.5μL, DNA polymerase 2μL, sample DNA 2μL, fluorescent dye 0.5μL, make up to 25μL with ultrapure water, after mixing, add 20μL sealing solution, and cap the reaction tube tightly;

[0060] Set up the qPCR instrument, select the FAM channel, the reaction program is 63°C for 30s, take 63°C for 15s, 63°C for 45s as a cycle, collect the fluorescence signal at 63°C for 45s, and re...

Embodiment 3

[0062] Example 3. Constant temperature amplification detection method of Enterobacter cloacae

[0063] Using the kit of Example 1 to detect Enterobacter cloacae, the specific steps are as follows:

[0064] (1) Extract the DNA of the sample to be tested;

[0065] (2) Use the LAMP primer set for constant temperature amplification of the DNA of the sample to be tested:

[0066] Add 25μL system of constant temperature amplification into the reaction tube, which contains: dnaJ-F3 0.02μM, dnaJ-B3 0.02μM, dnaJ-FIP 0.16μM, dnaJ-BIP 0.16μM, dnaJ-LF 0.08μM, dnaJ-LB 0.08μM, 12.5μL of reaction solution, 2μL of DNA polymerase, 2μL of DNA polymerase to be tested, make up to 25μL with ultrapure water, mix well, add 20μL of sealing solution, and finally add 1μL of color developing solution to the cap of the tube, and close the cap of the reaction tube; React at 63°C for 60 minutes in a thermostat.

[0067] After the reaction is over, spin the chromogenic solution on the reaction tube cap into the tub...

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 LAMP primer group for detecting enterobacter cloacae, a kit and a rapid detection method. The LAMP primers are six specific primers designed according to a dnaJ gene of enterobacter cloacae. By applying the six primers, specific amplification can be realized, the enterobacter cloacae can be effectively identified, and the lowest detection limit can reach 1 DNA copy; a DNA sample can be detected within 60min; and the detection method can be flexibly selected according to actual conditions, and the primer group is convenient and rapid and has a very wide application prospect.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology detection, and specifically relates to a primer set, a kit and a rapid detection method for detecting Enterobacter cloacae. Background technique [0002] There are currently 15 species of Enterobacter, of which Enterobacter cloacae is the most common one. It is part of the normal intestinal flora. It is believed to not cause diarrhea. It is widely present in the natural environment. It is found in human and animal feces, water and soil. It can be detected in plants, but it can cause a variety of extra-intestinal pathogenic infections, such as urinary tract, respiratory tract and wound infections, as well as bacteremia and meningitis. [0003] The traditional detection of Enterobacter cloacae is mainly through biochemical identification and ordinary PCR. Among them, the biochemical identification requires pure culture of the bacteria and the preparation of a considerable concentration of bacter...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119Y02A50/30
Inventor 芮勇宇杨秋李思
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com