Rapid detection primer for aeromonas salmonicida subsp.salmonicida and application of rapid detection primer

A technology for Aeromonas salmonicida and primer detection, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of precision and cost, limited technology universal applicability, unfavorable grassroots promotion and Application and other problems, to achieve the effect of rapid detection

Inactive Publication Date: 2015-02-18
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional isolation and culture methods are cumbersome to operate and have a long detection cycle, which generally takes 3-5 days to complete, which cannot meet the needs of rapid detection; although in recent years, PCR and real-time fluorescent PCR molecular biology detection methods have been more and more used in the detection of pathogenic bacteria. The wider the range, but these methods have high detection costs, require sophisticated and expensive thermal cycle instruments, and have strict requirements on the experimental environment and operator skills, which limits the general applicability of the technology and is not conducive to the promotion and promotion at the grassroots level. application
By searching existing documents, it is found that there are no reports related to the specific LAMP detection method and nucleic acid primers of Aeromonas salmonicida of the present invention

Method used

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  • Rapid detection primer for aeromonas salmonicida subsp.salmonicida and application of rapid detection primer
  • Rapid detection primer for aeromonas salmonicida subsp.salmonicida and application of rapid detection primer
  • Rapid detection primer for aeromonas salmonicida subsp.salmonicida and application of rapid detection primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Acquisition of Specific LAMP Primers for Aeromonas salmonicida subspecies

[0033] According to the fscT sequence of Aeromonas salmonicida subsp. salmonicida recorded on the NCBI website (GenBank accession number AY505565), a set of specific LAMP primers were designed using the online software PrimerExplorer V4 (http: / / primerexplorer.jp / e / ) . The nucleotide sequences of the primers are as follows:

[0034] F3: 5'-ACATAGGCGATGCCCTCA-3';

[0035] R3: 5'-GCCATGGGGATGGAGCT-3';

[0036] FIP: 5'-GCGGATCTTGATCTCGCTGCG-AGCATGTGCCGGGTGTC-3';

[0037] BIPs:

[0038] 5'-GCGACTACACACTGCTGCTGAT-TCGAAGTCATTGCCAAAGGA-3';

[0039] LF: 5'-GGGGTTGGTGCCTATGATGT-3';

[0040] LB: 5'-CAACGGCAAACGGGTCAAC-3'.

Embodiment 2

[0041] Example 2 Specific Detection of Aeromonas salmonicida subsp. salmonicida LAMP Primers

[0042] 1. Genomic DNA extraction of Vibrio parahaemolyticus, Edwardsiella cathari, Aeromonas victoria, Vibrio splendidus, Listonia anguillarum, Staphylococcus epidermidis, Proteus vulgaris, Streptococcus agalactiae:

[0043] Bacterial strain DNA was extracted according to the manual guideline of the Ezup Column Bacterial Genomic DNA Extraction Kit (Shanghai Sangong) (the public can obtain detailed information from Shanghai Sangong), as shown in Table 1, dissolved in 50 μL TE buffer ( pH7.8), and stored at -20°C for later use.

[0044] Table 1

[0045] strain name

Strain number

Vibrio parahaemolyticus

ATCC17802

Edwardsiella catfish

ATCC33202

Aeromonas vernerii

ATCC9071

vibrio brilliant

ATCC25914

Listonia anguillaris

ATCC14181

Staphylococcus epidermidis

ATCC14990

Proteus vulgaris

ATCC6380

...

Embodiment 3

[0053] Example 3 Sensitivity Test of Aeromonas salmonicida Subspecies Salmonicide Specific Primer LAMP Detection Method

[0054] Cloning of the fstC gene from Aeromonas salmonicida subsp. 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 (copies / μL) of the plasmid. Carry out by adding other LAMP reaction reagents except the DNA template in Example 1, and visually detect the amplification product, such as figure 2 shown. The primer can detect 10 or more copies of the plasmid template, which proves that the primer has good sensitivity. image 3 The electrophoresis results are shown.

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Abstract

The invention relates to a rapid detection primer for aeromonas salmonicida subsp.salmonicida and an application of the rapid detection primer and belongs to the technical field of biological detection. According to the rapid detection primer, the fstC gene is taken as a target gene; three pairs of DNA primers are designed and put into an amplification buffer solution with a DNA template to be detected for amplification in a constant-temperature environment of 63 DEG C, and then the reaction is terminated in a constant-temperature environment of 80 DEG C and the amplification product is detected. The rapid detection primer for the aeromonas salmonicida subsp.salmonicida, namely the LAMP detection primer, has the advantages of good specificity, high sensitivity, short consumed time, and simple and convenient operations. The primer can be applied to the detection of the aeromonas salmonicida subsp.salmonicida, and has a certain application prospect in the aspects of identification and monitoring of pathogen species, disease quarantine and the like of aquatic animals.

Description

technical field [0001] The present invention relates to a rapid detection primer for Aeromonas salmonicida subspecies salmonicida and its application, in particular to a LAMP detection primer for detecting Aeromonas salmonicida subspecies salmonicida and its application, belonging to biological detection technology field. Background technique [0002] Aeromonas salmonicida subsp.salmonicida, also known as Aeromonas salmonicida subsp.salmonicida, is one of the earliest described fish pathogens, mainly causing scabies in salmonid fish. The disease was first reported by Emmerich and Weibel in 1894 in fish farms in Germany, and the bacteria was isolated from the brown trout in the hatchery, which was called Phytophthora trout at that time. In the 7th edition of "Bergey's Handbook of Identifying Bacteriology" published in 1957, the bacterium was officially included in the genus Aeromonas, and in the 9th edition of "Bergey's Handbook of Identifying Bacteriology" it killed Aeromon...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q2531/119
Inventor 梁利国谢骏习丙文周群兰潘良坤
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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