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A rapid typing method for fish-derived streptococci by pulsed-field gel electrophoresis

A technology of gel electrophoresis and typing method, which is applied in the direction of material analysis, material analysis, and measuring devices through electromagnetic means, which can solve the problems of difficult distinction and comparison of small fragments of DNA, time-consuming, etc., and achieve strong discrimination ability and low cost time-shortening effect

Inactive Publication Date: 2014-10-08
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of plasmid typing is that the detection object is the plasmid, not the chromosome with higher genetic stability; the disadvantage of REA technology is that it is difficult to distinguish and compare a large number of small fragments of DNA with traditional agarose electrophoresis; the advantage of PCR technology is that it is fast and convenient, and the disadvantage is that Only partial fragments of chromosomes are detected; however, the pulsed field gel electrophoresis typing method is widely used, with high repeatability and stability. It detects the entire chromosomal DNA and is not affected by the variability of phenotypic traits. It is considered to be a microbial molecule. The gold standard for typing
However, the current international experimental procedure of streptococcal pulsed field gel electrophoresis generally takes 6 to 8 days, which takes a long time

Method used

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  • A rapid typing method for fish-derived streptococci by pulsed-field gel electrophoresis
  • A rapid typing method for fish-derived streptococci by pulsed-field gel electrophoresis
  • A rapid typing method for fish-derived streptococci by pulsed-field gel electrophoresis

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Embodiment 1

[0052] Embodiment 1: A rapid typing method of fish-derived streptococci by pulse-field gel electrophoresis, using streptococci with numbers 1 to 14 in Table 1 as analysis samples, specifically including the following process steps:

[0053] (1) Block preparation:

[0054] (1-1) After culturing the streptococcus sample on the blood plate medium for 24 hours, scrape a single colony with a sterile inoculation loop and resuspend it in 2ml of TE buffer to obtain a bacterial suspension, adjust the bacterial suspension The OD value of the liquid is 3.6; the components of the TE buffer are: 100mM Tris, 100mM EDTA, and the pH is 7.6;

[0055] (1-2) Take 185 μL of bacterial suspension into a 1.5ml centrifuge tube, add 5 μL of lysozyme with a concentration of 10 mg / ml, and mix well; put the mixed solution of bacterial suspension and lysozyme in a 37°C water bath Incubate for 15 minutes; remove the centrifuge tube from the water bath, add 5 μL of proteinase K at a concentration of 20 mg / ...

Embodiment 2

[0086] Embodiment 2: A rapid typing method of fish-derived streptococci by pulse-field gel electrophoresis, using streptococci with numbers 15 to 21 in Table 1 as analysis samples, including the following process steps:

[0087] (1) Block preparation:

[0088] (1-1) After culturing the streptococcus sample on the blood plate medium for 18 hours, scrape a single colony with a sterile inoculation loop and resuspend it in 2ml of TE buffer to obtain a bacterial suspension, adjust the bacterial suspension The OD value of the liquid is 4.5; the components of the TE buffer are: 100mM Tris, 100mM EDTA, and the pH is 7.6;

[0089] (1-2) Take 160 μL of bacterial suspension in a 1.5ml centrifuge tube, add 10 μL of lysozyme with a concentration of 10 mg / ml, and mix well; put the mixed solution of bacterial suspension and lysozyme in a 37°C water bath Incubate for 30 minutes; remove the centrifuge tube from the water bath, add 10 μL of proteinase K at a concentration of 20 mg / ml and 20 μL...

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Abstract

The invention relates to a rapid pulse field gel electrophoresis typing method for streptococcus fish. The typing method takes streptococcus from separation and culture as an analysis sample, and comprises the following steps of gel piece preparation, cell lysis, gel piece washing, digestion of DNA (Deoxyribonucleic Acid) in a gel piece, sample feeding, electrophoresis and image acquisition in sequence, wherein the steps of gel piece preparation, cell lysis, gel piece washing, sample feeding and image acquisition are all implemented according to an ordinary pulse field gel electrophoresis method, and parameters are optimized; the water bath temperature in the gel washing is 50 DEG C; the digestion of the DNA in the gel piece is performed by SmaI enzyme; and in electrophoresis parameters, the voltage is 6V / cm, the corresponding pulse time is 10s-45s, the included angle of an electricity field is 120 degrees, the electrophoresis temperature is 14 DEG C, and the total electrophoresis time is 22h. The method has stronger classification capability on the streptococcus fish, and the time of a whole experimental process is shortened by 3 to 4 days, so that the method has great significance to monitoring of streptococcus fish diseases, infection source tracing, transmission route investigation and recognition and the like.

Description

technical field [0001] The invention relates to a pulse-field gel electrophoresis typing method, in particular to a pulse-field gel electrophoresis rapid typing method for streptococcus fish origin. Background technique [0002] Streptococcus is an opportunistic pathogen that widely exists in nature. Since 2009, streptococcus disease caused by Streptococcus agalactiae and Streptococcus inia has seriously restricted the tilapia industry in my country. And there are new trends such as the expansion of the disease area, the increase of the morbidity and mortality rate, and the expansion of the size range of susceptible tilapia year by year. The outbreak of infectious diseases requires typing of pathogenic microorganisms, and the typing methods include phenotypic methods based on phenotypic feature analysis and genotypic methods based on genotypic features. Currently popular genotyping methods include plasmid typing, restriction endonuclease analysis (REA), pulsed field gel elec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
Inventor 杨弘祝璟琳邹芝英单航宇韩珏
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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