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Method for identifying high-homology marine vibrios based on rpoB genes

A marine vibrio and homology technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low identification accuracy and achieve low identification accuracy, cumbersome operation, and long detection cycle Effect

Inactive Publication Date: 2016-09-21
胡成进
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AI Technical Summary

Problems solved by technology

[0004] In order to solve the above technical problems, the present invention provides a detection method based on rpoB gene sequencing to identify marine vibrio, which overcomes the problem of low identification accuracy of conventional 16S rRNA sequencing identification of marine vibrio. The present invention is simple and fast, and has a high identification accuracy

Method used

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  • Method for identifying high-homology marine vibrios based on rpoB genes
  • Method for identifying high-homology marine vibrios based on rpoB genes
  • Method for identifying high-homology marine vibrios based on rpoB genes

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Embodiment 1

[0029] In this example, 50 strains of marine Vibrio were used as the research object, including Vibrio harvei (25 strains), Vibrio parahaemolyticus (10 strains), Vibrio alginolyticus (7 strains), Vibrio mimicus (2 strains), eel Vibrio (2 strains), Vibrio resplendent (2 strains), Vibrio tower (1 strain), Vibrio orientalis (1 strain). The selected microorganisms are listed in Table 1.

[0030] In order to confirm that rpoB gene sequencing has advantages over 16S rRNA sequencing in detecting marine Vibrio with high homology, this example simultaneously performed 16S rRNA and rpoB sequencing on 50 strains of marine Vibrio, and compared the sequencing results.

[0031]

[0032]

[0033] The specific method is:

[0034] 1) Bacterial recovery and culture: Take out the strain to be recovered in the ultra-low temperature refrigerator, check the strain number, pick an appropriate amount of solid frozen material with a sterile toothpick in the 2216E medium, use the three-section...

Embodiment 2

[0052] Two strains of Vibrio harveyi VIB 651 and VIB 642 were used as identification strains, and 16S rRNA and rpoB sequencing were performed respectively. The 16SrRNA and rpoB sequencing processes were the same except for the primers used in PCR amplification.

[0053] 1) Bacterial recovery and cultivation: Take out the strains VIB 651 and VIB642 to be recovered in the ultra-low temperature refrigerator, check the strain number, pick an appropriate amount of solid frozen material with a sterile toothpick on the 2216E medium, and use the three-section line method to inoculate. Place the plate in a 28°C incubator for 16-18 hours.

[0054] 2) Extract bacterial genomic DNA: Add 50µl sterile water to a sterile PCR tube, pick a single colony and mix it well, adjust the bacterial solution to 1-2 turbidity, shake for 15 seconds; boil in boiling water at 100°C for 10 minutes After taking it out, place it on ice for 10 minutes; centrifuge at 13,000 rpm for 5 minutes, and take about 30...

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Abstract

The invention discloses a method for identifying high-homology marine vibrios based on rpoB genes. The method comprises the steps that firstly, marine vibrio rpoB gene specific primers are designed by analyzing an rpoB gene sequence between marine vibrio strains; secondly, genomic DNA of 50 marine vibrios serves as templates, a proper amplification system and proper amplification conditions are selected for PCR amplification with the primers, and marine vibrios are quickly and accurately identified with the combination of the sequencing technique. By means of the method, marine vibrios high in homology can be easily and quickly detected, the accuracy is high, and the problem that the accuracy in 16S rRNA sequencing identification at present is low is solved. The method can be used for monitoring and detecting water body and clinical samples and carrying out epidemiological investigation and evolution analysis on marine vibrios and the like, relates to the fields of clinical diagnosis, aquaculture, food safety and the like, and has profound and lasting social benefits and high economic benefits.

Description

technical field [0001] The invention belongs to the technical field of bacterial detection, and specifically relates to a detection method based on RNA polymerase beta subunit (rpoB) gene, which is used for detection of marine Vibrio with high homology (16S rRNA sequence similarity greater than 99%) which is difficult to identify detection. Background technique [0002] Marine vibrios are the dominant flora present in seawater and marine organisms. Vibrio infection in marine aquatic animals can cause disease, and human beings can also cause infection by eating seafood contaminated by Vibrio or contacting seawater with bacteria. Vibrio identification can be used for the supervision and detection of water bodies and clinical samples, epidemiological investigation and evolution analysis of marine Vibrio, etc., involving clinical diagnosis, aquaculture, food safety and other fields. The epidemiological survey in the United States shows that the infection of marine Vibrio has b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/689C12Q1/686
Inventor 武静胡成进刘晓菲周月霞陈英剑公衔文
Owner 胡成进
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