Method for detecting gene cassette array in bacterium integron by using EcoRII enzyme digestion restriction map library

A gene cassette and map library technology, which is applied in the field of rapid detection of gene cassette arrays in bacterial integrons, can solve the problems of high cost and long time.

Inactive Publication Date: 2011-09-21
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Aiming at the time-consuming and high-cost deficiencies of commonly used methods in the current research, the purpose of the present invention is to provide a restriction endonuclease EcoRII to digest the PCR amplific

Method used

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  • Method for detecting gene cassette array in bacterium integron by using EcoRII enzyme digestion restriction map library
  • Method for detecting gene cassette array in bacterium integron by using EcoRII enzyme digestion restriction map library
  • Method for detecting gene cassette array in bacterium integron by using EcoRII enzyme digestion restriction map library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Screening of resistant bacteria

[0029] 1. Take a sample of the waste water around the hospital and quickly put it in a medical wide-mouth sampling bottle after autoclaving, the general sampling volume is about 500ml, and the water sample is placed in an ice box and brought back to the laboratory within 4 hours Filter processing.

[0030] 2. If the sampled water sample has a lot of stolen goods, you can first use a sterile qualitative filter paper for rough suction filtration to remove more impurities, so as to avoid extremely slow processing with a sterile filter membrane.

[0031] 3. Pass the coarsely filtered sample water sample through a sterile sand core filter device ( 0.22μm sterile filter membrane) to filter, the bacteria are enriched on the membrane, the filter membrane is transferred to a liquid medium containing 100mL LB (yeast extract 5g, tryptone 10g, NaCl 10g, dissolved in 800ml distilled water, adjust the pH to 7. 2-7.5, dilute to 1L, autoclave at ...

Embodiment 2

[0038] Example 2. Extraction of 247 strains of drug-resistant bacterial genomic DNA by CTAB method

[0039] 1. Bacterial cell culture: Inoculate 247 test bacteria in LB liquid medium and shake culture at 37°C for 16-18h to obtain sufficient cells.

[0040] 2. Take 1.5ml of bacteria liquid and centrifuge at 8000rmp / min for 2min in an EP tube to collect the bacteria.

[0041] 3. Add 500ulTE to resuspend the bacteria (instrument: vortex oscillator) and centrifuge at 8000rpm / min for 2min.

[0042] 4. Remove the supernatant and take care to absorb the excess water. Add 680ul TE buffer to the precipitate, repeatedly pipette to resuspend the bacteria. After mixing, add 36ul 10% SDS and mix to avoid DNA degradation by nuclease and mechanical vibration. Add proteinase K on ice and incubate at 37°C for more than 1 hour until the solution becomes clear. After clarification, add 120ul 5mol / l NaCl, mix well, then add 96ul CTAB / NaCl solution preheated, and after mixing, warm bath at 65℃ for 10min,...

Embodiment 3

[0049] Example 3. The genomic DNA of 247 strains of drug-resistant bacteria obtained by screening was used as a template to carry out PCR amplification, and the amplified products were taken to detect whether the drug-resistant bacteria contained type 1 integrase by agarose gel electrophoresis. This determines whether the bacteria are resistant bacteria containing type 1 integrons. The amplification primers used in the above PCR are:

[0050] Upstream primer intI1-F: 5’-GTTCGGTCAAGGTTCTGG-3’&

[0051] Downstream primer intI1-R: 5'-CGTAGAGACGTCGGAATG-3'.

[0052] The reaction system and procedure of PCR are as follows:

[0053]

[0054] Test results: Among 247 strains of resistant bacteria, 146 strains tested positive for integrase, that is, resistant bacteria containing type 1 integrons.

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Abstract

The invention discloses a method for detecting a gene cassette array in a bacterium integron by using an EcoRII enzyme digestion map library, which comprises the following steps of: firstly, carrying out enzyme digestion on the gene cassette array by adopting the restriction enzyme EcoRII when a new screened gene cassette array is determined; comparing the gene cassette array with the constructed EcoRII enzyme digestion map library so as to determine the novelty of the gene cassette array; and selecting connection, transformation and sequencing analysis so as to realize purposeful sequencing if maps in the library are not matched with the gene cassette array. The method is accurate, rapid and economic and is convenient for information sharing. The method is more suitable for relevant departments such as hospitals, quarantine inspection, forecast, preventive treatment and the like to realize the effective monitoring and prevent the outbreak prevalent of drug-resistant bacteria caused by the horizontal transferring and spreading of the drug-resistant bacteria containing the integron under the condition that antibiotic misuse is not well restrained.

Description

Technical field [0001] The invention belongs to the field of molecular biology technology and drug-resistant bacteria monitoring. Specifically, the present invention relates to a restriction endonuclease EcoRII to digest the PCR amplified products of the gene cassette array in bacterial integrons to construct an enzyme digestion map library, and realize rapid detection of bacterial integrons through the EcoRII digestion map library. Gene cassette array method. Background technique [0002] With the advancement of medical standards, a wide variety of antibiotics are increasingly being developed and put on the market. It is widely used in clinical and veterinary medicine, and even in animal husbandry, it is often used to promote growth. It is the wide application of these antibiotics, especially the unreasonable use, which has caused the problem of bacterial resistance and even multi-drug resistance. The drug mechanism is becoming more and more complex. Among them, the main dura...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 徐海国宪虎夏蕊蕊
Owner SHANDONG UNIV
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