LAMP detection primer combination of aeromonas sobria and kit

A detection kit and technology for Aeromonas, applied in the determination/inspection of microorganisms, microorganisms, recombinant DNA technology, etc., can solve the problems of LAMP detection primers and kits for Aeromonas temperatus, and achieve high specificity performance, easy operation, and easy identification

Active Publication Date: 2014-07-16
广州百博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there are no LAMP detection primers and kits for detecting Aeromonas temperatus on the market.

Method used

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  • LAMP detection primer combination of aeromonas sobria and kit
  • LAMP detection primer combination of aeromonas sobria and kit
  • LAMP detection primer combination of aeromonas sobria and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Establishment of Aeromonas temperii LAMP Detection Primer Set and Detection Kit

[0051] LAMP Detection Kit for Aeromonas temperii, including LAMP Primer Set, LAMP Reaction Solution, Mineral Oil, Bst DNA Polymerase, Positive Control and Negative Control, Product Instruction Manual, the kit can also contain color reagent SYBR Green I.

[0052] 1) LAMP primer design: According to the sequence of Aeromonas temperii (AS) zipA gene (Genbank accession number is JN830311.1) retrieved from the American gene database as the target gene, the conserved sequence was selected, and the online design software Primer Explorer version4( http: / / primerexplorer.jp / e ) are designed to detect the specific primers of Aeromonas temperatus, and its sequence is shown in the following table 1:

[0053] Table 1 Primer sequence list

[0054]

[0055] 2) LAMP reaction solution: mix 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 The aqueous solution and the three are mixed in ...

Embodiment 2

[0058] Example 2 Detection of Aeromonas temperii LAMP

[0059] Utilize the kit of the present invention to detect Aeromonas temperis, comprising the following steps:

[0060] 1) Bacterial DNA extraction: Bacterial samples were extracted according to the Bacterial Genome Extraction Kit (TIANGEN).

[0061] 2) Establishment of LAMP reaction system: 25 μL reaction system contains: 12.5 μL reaction solution, 1 μL Bst DNA polymerase, AS-FIP1.6 μM, AS-BIP1.6 μM, AS-LF0.8 μM, AS-LB0.8 μM, AS -F30.2μM, AS-B30.2μM, 2μL DNA extract solution to be tested, make up to 25μL with sterile deionized water, set positive control and negative control; add two drops of mineral oil, shake and centrifuge, set the reaction temperature of the turbidimeter to 63°C, the reaction time is 65min, after the reaction is completed, keep at 80°C for 5min.

[0062] 3) The results can be judged by any one of the following three methods:

[0063] ①Turbidimeter detection: Set the real-time turbidimeter (LA-320C)...

Embodiment 3

[0066] Embodiment 3 The specificity test of the LAMP detection method of Aeromonas temperii of the present invention

[0067] 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 sobria (ATCC43979), Aeromonas hydrophila (ATCC7966), Aeromonas victoria (ATCC35624 ), Aeromonas caviae (ATCC15468), Escherichia coli, Edwardsiella tarda, Streptococcus agalactiae, Streptococcus iniae, Staphylococcus aureus, Vibrio cholerae, Bacillus cereus, Shigella, Krei pneumoniae The nucleic acids of Burdock bacteria, Pseudomonas aeruginosa, Citrobacter freundii, and Morganella morganii were used as templates, and negative controls using sterile deionized water as templates were set up for specificity tests.

[0068] Test results such as figure 1Shown: Specific amplification of Aeromonas hydrophila, Aeromonas sobria, Aeromonas victorii, Aeromonas caviae, Escherichia coli, Edwardsiella tarda, St...

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Abstract

The invention discloses an LAMP detection primer combination of aeromonas sobria and a kit. The LAMP detection primer combination of aeromonas sobria comprises a pair of outer primers AS-F3 and AS-B3, a pair of inner primers AS-FIP and AS-BIP and a pair of loop primers AS-LF and AS-LB. the kit comprises the detection primer combination, BstDNA polymerase, an LAMP reaction solution, confining liquid mineral oil, a positive control and a negative control. The invention also discloses a method for detecting aeromonas sobria by the utilization of the above LAMP detection kit of aeromonas sobria. The kit provided by the invention is simple and rapid to operate, is suitable for field test, has high specificity, high detection sensitivity and high and reliable accuracy, and has a wide application prospect. The detection sensitivity of the kit can reach 320fg / mL.

Description

technical field [0001] The invention belongs to the technical field of detection of Aeromonas temperii, and in particular relates to a LAMP detection primer set and a kit for Aeromonas temperii. Background technique [0002] Aeromonas sobria (Aeromonas sobria, AS) belongs to Vibriaceae, Aeromonas genus, is a Gram-negative bacillus. It is widely distributed in fresh water, sewage, soil and human food chain. It is one of the most harmful pathogenic bacteria in aquaculture. The disease often breaks out and has a high mortality rate. It can cause hemorrhagic sepsis and mainly harms soft-shelled turtles and Brazilian turtles. , tilapia, carp, eel, eel, loach, crab. At the same time, it can also infect people through contaminated aquatic products, livestock and poultry meat and water sources, causing acute diarrhea in patients, and may also cause meningitis, septic arthritis and sepsis. It not only causes serious economic losses to the aquaculture industry, but also brings harm ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q2531/119
Inventor 柯浩李嘉彬马艳平刘振兴郝乐马江耀梁志凌
Owner 广州百博生物科技有限公司
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