Method for detecting aquatic animal pathogenic bacteria by using 23S ribosome gene probe array

A ribosomal gene, aquatic animal technology, applied in biochemical equipment and methods, biological testing, material inspection products, etc., can solve problems such as unsatisfactory identification effect and difficulty in identifying probes

Inactive Publication Date: 2006-12-13
NANKAI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is difficult to design similar species identification probes using the di...

Method used

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  • Method for detecting aquatic animal pathogenic bacteria by using 23S ribosome gene probe array
  • Method for detecting aquatic animal pathogenic bacteria by using 23S ribosome gene probe array
  • Method for detecting aquatic animal pathogenic bacteria by using 23S ribosome gene probe array

Examples

Experimental program
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Effect test

Embodiment 1

[0145] Sample: A certain batch of exported live fish. Suspected colonies of Aeromonas salmonicida were detected by routine microbial culture and biochemical tests.

[0146] 1. DNA extraction

[0147] Take one gram each of the epidermis, kidney tissue, liver tissue, and heart of the sample fish scales, mix and shake after homogenization, take 1 mL of the homogenate solution and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, Discard the supernatant, add 100 μL lysozyme solution, incubate at 37°C for 10 minutes, add 500 μl TE buffer, shake and mix well. Add the same volume of Tris saturated phenol with a pH value of 8.0, shake vigorously at 12,000 rpm, centrifuge for 3 minutes, absorb the supernatant, and repeat the phenol extraction. Aspirate the supernatant, add 0.1 times the volume of sodium acetate (2mol / L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, and ce...

Embodiment 2

[0183] Sample: A certain batch of exported live fish. Suspected colonies of Aeromonas hydrophila were detected by routine microbial culture and biochemical tests.

[0184] 1. DNA extraction: with embodiment 1

[0185] 2.PCR amplification of 23S gene fragment: same as Example 1

[0186] 3. Hybridization of the target gene amplification product and the probe on the nylon membrane: the same as in Example 1

[0187] 4. ELISA color development of hybridization positive spots

[0188] The ELISA of the hybridization spots was performed according to the instructions of Roche Digoxigenin DNA Labeling and Detection Kit.

[0189] * Interpretation of results Interpretation of hybridization results Probe site 3 (Aeromonas general probe AFp1) and probe site 5 (Aeromonas hydrophila probe Ahyr1) on the nylon membrane are all dark blue spots, which means positive hybridization The results, indicating that the sample contained the Aeromonas hydrophila probe, see image 3 .

[0190] After 2...

Embodiment 3

[0192] Sample: A certain batch of imported live fish. Suspected colonies of Aeromonas caviae were detected by routine microbial culture and biochemical tests.

[0193] 1. DNA extraction: with embodiment 1

[0194] 2.PCR amplification of 23S gene fragment: same as Example 1

[0195] 3. Hybridization of the target gene amplification product and the probe on the nylon membrane: the same as in Example 1

[0196] 4. ELISA color development of hybridization positive spots

[0197] The ELISA of the hybridization spots was performed according to the instructions of Roche Digoxigenin DNA Labeling and Detection Kit.

[0198] * Interpretation of results Interpretation of hybridization results Probe site three (Aeromonas general probe AFp1) and probe site six (Aeromonas caviae probe Acav1) on the nylon membrane are all dark blue spots, which are positive hybridization results. Indicates that the sample contains Aeromonas caviae, see Figure 4 .

[0199] After 5 days, routine microb...

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Abstract

The invention discloses a bacteria detecting technology which comprises designing special probes with biological information, including designing 34 pieces oligonucleotide as filtered probes and the corresponding PCR and hybridizing reacting conditions; enlarging via PCR and marking bacteria 23srRNA gene internal sub array with Digoxin, and hybridizing the PCR products and a group of special oligonucleotide probes; after enzyme-linked immunoassay color-rendering, identifying whether has aquatic infectious pathogeny bacteria from the sample.

Description

technical field [0001] The present invention relates to a kind of bacterium inspection technique, specifically is to use nucleic acid molecular hybridization reaction to detect pathogenic bacteria, especially aquatic animal pathogenic bacteria (comprising Aeromonas salmonicida, Aeromonas hydrophila, Aeromonas caviae, Clostridium perfringens, Clostridium botulinum, Edwardsiella catfish, Enterococcus piscicida, Streptococcus, Flexibacter psychrophilus, Flexibacter columnar, Pasteurella multocida, Pasteur piscicida Bacteria, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas syringae, Vibrio cholerae, Vibrio mimicus, Vibrio harveii, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio riverina, Freundii Vibrio, Vibrio alginolyticus and other 23 kinds of bacteria) technology. Background technique [0002] At present, the detection methods of pathogenic microorganisms in aquatic animals basically rely on biochemical identification and culture identification. These identific...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N21/78C12Q1/04
Inventor 黄熙泰侯艳梅刘寅张立怀郑泽军高旗利周浩董志珍李永君王玉玲魏晓娜霍蕾
Owner NANKAI UNIV
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