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Detection reagent kit and detection method of aeromonas bacteria

A detection kit, Aeromonas technology, applied in the direction of botany equipment and methods, microbe-based methods, biochemical equipment and methods, etc., can solve whether the detection sensitivity is consistent cannot be proved, does not provide detection sensitivity, operation Complicated problems, to achieve good application prospects, prevent fish diseases, and high sensitivity

Active Publication Date: 2014-06-25
TONGWEI AGRI DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional physiological and biochemical detection methods can accurately identify the strains to be tested, but the operation is complicated, costly and time-consuming
Pan Xiaoyi et al., "Establishment of a dual-PCR detection method for Aeromonas and Aeromonas hydrophila", Journal of Huazhong Agricultural University, 2011, 30(6):759-763. The disclosed PCR detection can overcome the limitations of physiological and biochemical detection methods. However, this method does not provide detection sensitivity for reference and reference, and it cannot be proved whether the detection sensitivity of the two pairs of primers used is consistent, so there are defects in pathogenic diagnosis and disease epidemiology research

Method used

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  • Detection reagent kit and detection method of aeromonas bacteria
  • Detection reagent kit and detection method of aeromonas bacteria
  • Detection reagent kit and detection method of aeromonas bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Primer Design

[0031] 1. Experimental method

[0032] 1. PCR primer design and synthesis

[0033] According to experience, the common glycerophospholipid cholesterol acyltransferase gene (gene number: HM245310.1, HM245308.1, GQ856318.1, CP000644.1, AF268080.1) and 16s rRNA (gene number: HM245310 .1, HM245308.1, GQ856318.1, CP000644.1, AF268080.1) designed two pairs of primers respectively, and sent them to Shanghai Sangong for synthesis after Blast comparison.

[0034] Table 1PCR primer information

[0035]

[0036] 2. Template preparation

[0037] Streak inoculate Aeromonas hydrophila TW-YYS-26 and Aeromonas caviae ATCC15468 stored at -20°C on LB solid medium plate, culture at 28°C, pick a single colony after growing a single colony and inoculate it into LB Liquid culture medium, cultivate overnight at 28°C in a shaker to activate the bacteria. The bacterial culture solution was taken, and DNA was extracted using a genomic DNA extraction kit (TIANGEN...

Embodiment 2

[0049] Embodiment 2 primer specificity test

[0050] 1. Test method

[0051] 1. PCR primers

[0052] Take the primer pair 1 in Example 1.

[0053] 2. Template preparation:

[0054] The bacteria listed in Table 1 were respectively activated with culture medium before the test. After the bacteria were activated, DNA was extracted according to the operating instructions of the Bacterial Genomic DNA Extraction Kit (TIANGEN) and used as a template for PCR detection.

[0055] 3. PCR amplification

[0056] Take the genomic DNA obtained in step 2 as a template, and the template for the negative control is ddH 2 O, amplify according to the amplification procedure in Example 1.

[0057] 4. Result detection

[0058] Take 5 μL of the amplified product, use 1% agarose gel, electrophoresis at a constant voltage of 80V for 60 min, and place it in a gel imaging system to observe the PCR amplification result.

[0059] 2. Results

[0060] The result is as figure 2 As shown, only Aero...

Embodiment 3

[0062] Embodiment 3 primer sensitivity test

[0063] 1. Test method

[0064] 1. PCR primers

[0065] Take the primer pair 1 in Example 1.

[0066] 2. Template preparation:

[0067] Aeromonas hydrophila TW-YYS-26 and Aeromonas caviae ATCC15468 were respectively activated with medium. After the bacteria were activated, DNA was extracted according to the operating instructions of the Bacterial Genomic DNA Extraction Kit (TIANGEN);

[0068] Determine the concentration of the DNA sample, and follow the steps of 10 for the DNA sample -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 Perform serial dilutions.

[0069] 3. PCR amplification

[0070] The genomic DNA of Aeromonas hydrophila and Aeromonas caviae in a gradient dilution was used as a template, and amplified according to the amplification procedure in Example 1.

[0071] 4. Result detection

[0072] Take 5 μL of the amplified product, use 1% agarose gel, electrophoresis at a constant voltage of 80V f...

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Abstract

The invention discloses a detection reagent kit of aeromonas bacteria, which comprises a primer pair 1 with a sequence shown as SEQ ID No. (sequence identification number) 1-2, which is used for amplifying genes from the aeromonas bacteria. The invention further discloses a detection method of the aeromonas bacteria. With the adoption of the detection reagent kit and the detection method, the aeromonas bacteria can be detected accurately and effectively; the specificity is good; the sensitivity is high; consumed time is short; the detection is quick; the reagent kit and the detection method can be applied to quick detection and forecast of a culture water body; fish diseases are prevented; and the economic benefit is improved.

Description

technical field [0001] The invention relates to a detection technology for bacteria, in particular to a detection kit and a detection method for Aeromonas bacteria. Background technique [0002] With the development of modern molecular genetics, the genus Aeromonas has been expanded to include Aeromonas hydrophil, Aeromonas caviae, [0003] Aeromonas sobria, Aeromonas salmonicida, Aeromonas media, Aeromonas veronii, Aeromonas jandaei) and other 14 Aeromonas phenotype species and 16 gene species, the type species is Aeromonas hydrophila, and Aeromonas caviae is a very common Aeromonas. [0004] Aeromonas bacteria are widely distributed clinically and in the environment, and are distributed in water, soil and air. There are many kinds of aquaculture animal diseases caused by Aeromonas bacteria, which not only bring serious economic losses to the aquaculture industry, but also infect humans and animals through aquatic animals and aquatic products, causing diarrhea and food po...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/54C12N15/10C12R1/01
Inventor 肖丹黄冠军刘天强刘衍鹏李茂
Owner TONGWEI AGRI DEV CO LTD
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