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Kit for detecting ST251-type virulent aeromonas hydrophila and application

A technology based on Aeromonas hydrophila and ST251, which is applied in the direction of microorganism-based methods, measurement/inspection of microorganisms, microorganisms, etc., can solve the problems of indistinguishability, instability, laborious accuracy, etc., and achieve strong pertinence and high accuracy The effect of specificity and high sensitivity

Active Publication Date: 2016-09-28
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, such a detection technique cannot distinguish Aeromonas hydrophila type ST251 from other groups of Aeromonas hydrophila
[0006] Traditional phenotype-based bacterial identification and detection techniques have obvious technical disadvantages, such as time-consuming, labor-intensive and unstable accuracy
The PCR detection schemes introduced by many literature reports or patented technologies are blank in the identification of pathogenic Aeromonas hydrophila, especially there is no PCR detection technology specifically for ST251 pathogenic Aeromonas hydrophila

Method used

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  • Kit for detecting ST251-type virulent aeromonas hydrophila and application
  • Kit for detecting ST251-type virulent aeromonas hydrophila and application
  • Kit for detecting ST251-type virulent aeromonas hydrophila and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A test kit for detecting pathogenic Aeromonas hydrophila, the test kit contains:

[0058] a.PCR premix: a PCR premix freeze-dried powder, including high-fidelity DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancer and stabilizer;

[0059] b. Primer pair;

[0060] c. Standard positive DNA template;

[0061]d. Standard negative DNA template;

[0062] e. Sterilized double distilled water.

[0063] The upstream primer of the primer pair is 5'-GCGGCGTAAGCGGATTTGTAGC-3';

[0064] The downstream primer of the primer pair is 5'-CCGGTCGGATTGGTCAGGTTCAT-3'.

[0065] The specific operation process is as follows:

[0066] 1) Genomic DNA extraction of 15 strains of bacteria: using a bacterial genome extraction kit, such as Promega's Genomic DNA Purification Kit, operated according to the instructions.

[0067] 2) The PCR reaction system uses PCR Master Mix, and then add the following components

[0068]

[0069]

[0070] After the above components are...

Embodiment 2

[0076] A kit for the rapid detection of pathogenic Aeromonas hydrophila for the detection of target bacteria that may be present in tissue samples from aquaculture animals. To achieve this purpose, the sample must first be pretreated, the total DNA in the sample is extracted, and then the kit provided by the invention is used for PCR amplification analysis.

[0077] Application of a PCR kit for detecting Aeromonas hydrophila ST251 in the preparation of drugs for treating or preventing outbreaks of freshwater cultured fish. Its operation steps are:

[0078] 1) By aseptic operation, take the internal organs such as the liver, kidney and spleen of the animal, or the blood of the animal.

[0079] 2) Use QIAGEN's DNeasy Blood & Tissue Kit to extract all DNA in the tissue, including bacterial genomic DNA, and operate according to the instructions.

[0080] 3) The PCR reaction system adopts the PCR Premix in the kit of the present invention, and then adds the following components ...

Embodiment 3

[0087] A kit for rapid detection of pathogenic Aeromonas hydrophila, which is used to detect target bacteria that may exist in non-biological samples (water samples, sediment samples, feed samples). To achieve this purpose, the sample must first be pretreated, the total DNA in the sample is extracted, and then the kit provided by the invention is used for PCR amplification analysis.

[0088] A PCR kit for detecting Aeromonas hydrophila ST251 is used in the preparation of treatment or prevention of freshwater cultured fish

[0089] Application of drugs for outbreak-like diseases. Its operation steps are:

[0090] 1) For water samples: take 1 liter of water samples, perform aseptic operation, centrifuge at 12800×g for 20 minutes at 4°C, and collect the precipitate for DNA extraction.

[0091] 2) For solid or semi-solid samples such as pond sediment, feed, or animal intestines: First, mash them thoroughly under sterile conditions, then take an appropriate amount and dilute 1:2 ...

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Abstract

The invention discloses a PCR kit for detecting ST251-type aeromonas hydrophila and application. The kit comprises a PCR Premix, a primer pair, a standard positive DNA template, a standard negative DNA template and sterilization double distilled water. An upstream primer sequence is 5'-GCGGCGTAAGCGGATTTGTAGC-3', a downstream primer sequence is 5'-CCGGTCGGATTGGTCAGGTTCAT-3', the annealing temperature is 63 DEG C, a correct amplification product is 826bp in size, the standard positive DNA template is genomic DNA extract of an ST251-type aeromonas hydrophila XS91-4-1 strain, and the DNA concentration is 500 nanograms per microliter; the negative DNA template is genomic extractive of an aeromonas veronii IB340 strain, and the DNA concentration is 500 nanograms per microliter. The kit is applicable to all kinds of gene amplification instruments, high in detection specificity and sensitivity, quick, accurate and good in stability. The kit can be used for quickly diagnosing freshwater fish outbreak diseases and blood poisoning caused by aeromonas hydrophila of other fishes and quickly detecting and identifying bacteria and then is widely applied to disease prediction and forecast and epidemiologic studies.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a kit for rapidly detecting ST251 pathogenic Aeromonas hydrophila, and also relates to the use of a kit for detecting ST251 pathogenic Aeromonas hydrophila, It can be used for rapid differential diagnosis and epidemiological research of outbreaks of freshwater farmed fish and sepsis caused by Aeromonas hydrophila in other freshwater fish, and can also provide technical support for related basic research. Background technique [0002] Aeromonas (aeromonads) is the most common pathogenic bacteria in freshwater cultured fish in my country, which can attack almost all kinds of aquaculture animals. They exist in various ecological niches in aquaculture systems and are one of the main resident bacteria in the gut of healthy aquatic animals. Classification belongs to γ-proteobacteria, Aeromonas family, and Aeromonas genus. There are at least 25 recognized species, and new s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/686C12Q1/689C12Q2545/101
Inventor 李爱华刘天强彭波张倩倩
Owner INST OF AQUATIC LIFE ACAD SINICA
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