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

A technology of Enterobacter aerogenes and primer set, which is applied in the field of molecular biology detection, can solve the problems of cumbersome operation and long time consumption, and achieve the effect of high sensitivity, low probability and high specificity

Active Publication Date: 2020-09-15
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, the kit for detecting Enterobacter aerogenes

[0040] A kit for detecting Enterobacter aerogenes, including the following components: (1) LAMP primer set; (2) DNA polymerase; (3) reaction solution; (4) chromogenic reagent; (5) fluorescent dye; (6) ) blocking solution; (7) positive control and negative control.

[0041] (1) LAMP detection primer set

[0042]Multiple sets of LAMP primers were designed according to multiple species-specific target genes of Enterobacter aerogenes, and ThdF (GenBank accession number: EU569333.1) was screened out as the final target gene after multiple sensitivity and specificity experiments, and the final optimal one The primer set has high amplification efficiency, excellent sensitivity and specificity, including a pair of outer primers, a pair of inner primers and a pair of loop primers, and their nucleotide sequences are as follows:

[0043] ThdF-F3: 5'-GCGATCGTCACCGATATC-3' (SEQ ID NO: 1);

[0044] ThdF-B3: 5'-GGTAATCGGC...

Embodiment 2

[0056] Embodiment 2, the isothermal fluorescence amplification detection method of Enterobacter aerogenes

[0057] Utilize the kit of embodiment 1 to detect Enterobacter aerogenes, concrete steps are as follows:

[0058] (1) Extract the DNA of the sample to be tested;

[0059] (2) Use the LAMP primer set to perform constant temperature fluorescence amplification of the DNA of the sample to be tested:

[0060] Add 25 μL of constant temperature amplification system into the reaction tube, which contains: ThdF-F3 0.02 μM, ThdF-B3 0.02 μM, ThdF-FIP 0.16 μM, ThdF-BIP 0.16 μM, ThdF-LF 0.08 μM, ThdF-LB 0.08 μM, Reaction solution 12.5 μL, DNA polymerase 2 μL, sample DNA to be tested 2 μL, fluorescent dye 0.5 μL, make up to 25 μL with ultrapure water, mix well, add 20 μL sealing solution, and cap the reaction tube tightly;

[0061] Set up the qPCR instrument, select the FAM channel, and the reaction program is 63°C for 30s, 63°C for 15s and 63°C for 45s as a cycle, collect the fluore...

Embodiment 3

[0063] Embodiment 3, the isothermal amplification detection method of Enterobacter aerogenes

[0064] Utilize the kit of embodiment 1 to detect Enterobacter aerogenes, concrete steps are as follows:

[0065] (1) Extract the DNA of the sample to be tested;

[0066] (2) Use the LAMP primer set to carry out constant temperature amplification of the DNA of the sample to be tested:

[0067] Add 25 μL of constant temperature amplification system into the reaction tube, which contains: ThdF-F3 0.02 μM, ThdF-B3 0.02 μM, ThdF-FIP 0.16 μM, ThdF-BIP 0.16 μM, ThdF-LF 0.08 μM, ThdF-LB 0.08 μM, Reaction solution 12.5 μL, DNA polymerase 2 μL, sample DNA to be tested 2 μL, make up to 25 μL with ultrapure water, mix well, add 20 μL of sealing solution, and finally add 1 μL of chromogenic solution to the tube cap, and cap the reaction tube tightly; React in a constant temperature device at 63°C for 60 minutes;

[0068] After the reaction, through instantaneous centrifugation, throw the chrom...

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Abstract

The invention discloses a LAMP primer group for detecting enterobacter aerogenes, a kit and a rapid detection method. The LAMP primers are six specific primers designed according to a ThdF gene of enterobacter aerogenes. By applying the six primers, specific amplification can be realized, the enterobacter aerogenes can be effectively identified, and the lowest detection limit can reach 1 DNA copy; a DNA sample can be detected within 60min; 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 in particular relates to a primer set, a kit and a rapid detection method for detecting Enterobacter aerogenes. Background technique [0002] There are currently 15 species of Enterobacter, among which Enterobacter aerogenes is the most common one. It is part of the normal flora of the intestinal tract and is not considered to cause diarrhea. It can be detected in soil and plants, but it can cause a variety of opportunistic infections outside the intestinal tract, such as urinary tract, respiratory tract and wound infections, and can also cause bacteremia and meningitis. [0003] The traditional detection of Enterobacter aerogenes is mainly through biochemical identification and ordinary PCR. Among them, the biochemical identification requires pure culture of the bacteria, and then the bacterial solution of a considerable concentration is added to a commercial biochemical...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12Q1/689C12Q1/10C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119Y02A50/30
Inventor 芮勇宇杨秋李思
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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