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Lamp detection primer set and kit for Aeromonas hydrophila

A technology of Aeromonas hydrophila and a detection kit, which is applied in the field of detection of Aeromonas hydrophila, can solve the problems of unavailable on-site detection and expensive equipment, and achieve the effect of simple operation and high sensitivity

Active Publication Date: 2015-08-19
广东双螺旋基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, real-time fluorescent PCR equipment is expensive and cannot be used for on-site detection

Method used

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  • Lamp detection primer set and kit for Aeromonas hydrophila
  • Lamp detection primer set and kit for Aeromonas hydrophila
  • Lamp detection primer set and kit for Aeromonas hydrophila

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1 The establishment of the LAMP primer set and detection kit for Aeromonas hydrophila

[0042] LAMP detection kit for Aeromonas hydrophila, including LAMP primer set, LAMP reaction solution, blocking solution, Bst DNA polymerase, positive control and negative control.

[0043] 1) LAMP primer design: According to the gene sequence of Aeromonas hydrophila (AH) pili retrieved from Genbank (Genbank accession number is CP000462.1) as the target gene, select the conserved sequence, and use the online design software Primer Explorer version4 ( http: / / primerexplorer.jp / e ) designed to detect the specific primers of Aeromonas hydrophila, its sequence is shown in the following table 1:

[0044] Table 1 Primer sequence list

[0045]

[0046] 2) LAMP reaction solution: mix 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 The aqueous solution and the three are mixed in a volume ratio of 8:5:2.

[0047] 3) The positive control is a recombinant plasmid containing ...

Embodiment 2

[0051] Example 2 Detection of Aeromonas hydrophila LAMP

[0052] Utilize the kit of the present invention to detect Aeromonas hydrophila, comprising the following steps:

[0053] 1) Nucleic acid extraction: Bacterial samples were extracted according to the bacterial genome extraction kit (TIANGEN) to obtain nucleic acid extraction solution;

[0054] 2) Establishment of LAMP reaction system: 25 μL reaction system contains: 12.5 μL reaction solution, 1 μL Bst DNA polymerase, AH-FIP1.6 μM, AH-BIP1.6 μM, AH-LF0.8 μM, AH-LB0.8 μM, AH -F30.2μM, AH-B30.2μM, 2μL nucleic acid extraction solution, sterile deionized water, set positive control and negative control; add two drops of blocking solution, shake and centrifuge.

[0055] Set the reaction temperature of the turbidimeter to 63°C, the reaction time is 65min, and keep at 80°C for 5min after the reaction is completed;

[0056] 3) Result judgment:

[0057] Method 1 is turbidity meter detection: set the real-time turbidity meter (L...

Embodiment 3

[0060] Embodiment 3 The specificity test of the LAMP detection method of Aeromonas hydrophila of the present invention

[0061] In order to detect the specificity of the kit of the present invention, the LAMP detection method in the above-mentioned Example 2 was adopted, and Aeromonas hydrophila (ATCC7966), Aeromonas sobria (ATCC43979), Aeromonas victoria (ATCC35624 ), Aeromonas caviae (ATCC15468), Escherichia coli, Edwardsiella tarda, Streptococcus agalactiae, Streptococcus iniae, Staphylococcus aureus, Vibrio cholerae, Bacillus cereus, Shigella, Streptococcus pneumoniae , Pseudomonas aeruginosa, Citrobacter freundii, and Morganella morganii were used as templates, and a negative control using sterile deionized water as a template was set up to conduct a specificity test.

[0062] Test results such as figure 1 Shown: Specific amplification of Aeromonas hydrophila, Aeromonas sobria, Aeromonas victorii, Aeromonas caviae, Escherichia coli, Edwardsiella tarda, Streptococcus agal...

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) detection primer group of aeromonas hydrophila. The loop-mediated isothermal amplification (LAMP) detection primer group comprises a pair of outer primers AH-F3 and AH-B3, a pair of inner primers AH-FIP and AH-BIP, and a pair of loop primers AH-LF and AH-LB, wherein nucleotide sequence of AH-F3 is represented by SEQ ID No.1, nucleotide sequence of AH-B3 is represented by SEQ ID No.2, nucleotide sequence of AH-FIP is represented by SEQ ID No.3, nucleotide sequence of AH-BIP is represented by SEQ ID No.4, nucleotide sequence of AH-LF is represented by SEQ ID No.5, and nucleotide sequence of AH-LB is represented by SEQ ID No.6. The invention also discloses a kit. The kit is convenient and fast for operation, is suitable for on-site detection, and is high in specificity, detection sensitivity, accuracy, and reliability; detection sensitivity can be as high as 46fg / ml; and application prospect is promising.

Description

technical field [0001] The invention belongs to the technical field of Aeromonas hydrophila detection, and in particular relates to a LAMP detection primer set and a kit for Aeromonas hydrophila. Background technique [0002] Aeromonas hydrophila (Aeromonas hydrophila, AH) is widely distributed in the human food chain, water environment and soil. It can infect animal bodies through drinking water, skin wounds, etc. It can infect fish, shrimp, amphibians, reptiles, poultry, and mammals, causing hemorrhagic sepsis and causing a large number of deaths. At the same time, the bacteria can also cause human food poisoning, gastroenteritis, meningitis, pneumonia and sepsis. one of the pathogenic bacteria. Scholars at home and abroad generally believe that comprehensive prevention is a relatively effective method, that is, to detect diseases as early as possible and take many measures to prevent the spread of bacteria. Therefore, establishing a sensitive, accurate, fast and conven...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q2531/113
Inventor 柯浩李嘉彬马江耀马艳平刘振兴郝乐梁志凌
Owner 广东双螺旋基因技术有限公司
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