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Pulsed field gel electrophoresis method for S.paratyphi A

A technology of gel electrophoresis and paratyphoid fever, applied in the direction of material analysis, instruments, and measuring devices through electromagnetic means, can solve the problem of insufficient typing ability, difficulty in tracing the source of pathogenic molecules, non-epidemic strains and non-epidemic strains Distinguishing problems such as separation to achieve the effect of improving the ability to distinguish

Inactive Publication Date: 2012-01-04
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This standardized scheme is not strong enough in typing when analyzing Salmonella paratyphi A, and cannot effectively distinguish epidemic strains from non-epidemic strains. It is difficult to carry out molecular traceability of pathogens when paratyphi A outbreaks
Therefore this standardized protocol is not suitable for Salmonella paratyphi A

Method used

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  • Pulsed field gel electrophoresis method for S.paratyphi A
  • Pulsed field gel electrophoresis method for S.paratyphi A
  • Pulsed field gel electrophoresis method for S.paratyphi A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Carry out PFGE to Salmonella paratyphi A in Table 4, comprising the following steps:

[0036] [1]. Preparation of rubber block

[0037] [11] Add 2ml of cell suspension (CSB) into the Falcon 2054 tube, wet the inoculation loop with CSB, evenly suspend the Salmonella paratyphi A sample isolated and cultured in CSB, measure its OD value, and adjust to 3.6~4.5;

[0038] [1.2] Take 400 μl of the bacterial suspension obtained in step [11] into a corresponding 1.5ml test tube, and incubate in a water bath at 37°C for 5 minutes; place the remaining bacterial suspension on ice until the gel block is prepared and put it away in a water bath shaker;

[0039] [1.3] Take out the test tube obtained in step [1.2] from the water bath, add 20 μl proteinase K with a storage solution concentration of 20 mg / ml to each tube and mix well to make the final concentration 0.5 mg / ml;

[0040] [1.4] Add 1g of commercially available SeaKem Gold Agarose agarose (SKG) to the solution prepared by ...

Embodiment 2

[0074] Carry out the first PFGE according to the method of embodiment 1 earlier, then take the bacterium that obtains with same batch of separation and cultivation as sample, with subselection enzyme Xba I and corresponding electrophoresis parameter (pulse time is 1.5-29s, total electrophoresis time 20h) According to the process described above, the second PFGE treatment was performed to finally obtain gel blocks after electrophoresis.

[0075] After the second electrophoresis, take out the gel and put it in a tray containing 400ml of EB solution (the concentration of EB stock solution is 10mg / ml, diluted 1:10,000, that is, add 40μl of stock solution to 400ml of water). Shake the tray on a shaker for 25-30 minutes. Drain the TBE in the electrophoresis tank, wash the electrophoresis tank with 1-2L pure water, and pour off the liquid. Put on gloves and carefully pour the used EB solution into a marked brown bottle, add 400-500ml of pure water to the tray, put it on a shaker for...

Embodiment 3

[0077] Carry out 2 times of PFGE according to the method of embodiment 2 first, then take the bacterium obtained by the same batch of isolation and culture as a sample, with the third enzyme Xho I and its corresponding electrophoresis parameters (pulse time is 2.2-29s, total electrophoresis time 20h) according to the process, the third PFGE treatment was carried out to finally obtain the gel block after electrophoresis.

[0078] After the third electrophoresis, take out the gel and place it in a tray containing 400ml of EB solution (the concentration of EB stock solution is 10mg / ml, diluted 1:10,000, that is, add 40μl of stock solution to 400ml of water). Shake the tray on a shaker for 25-30 minutes. Drain the TBE in the electrophoresis tank, wash the electrophoresis tank with 1-2L pure water, and pour off the liquid. Put on gloves and carefully pour the used EB solution into a marked brown bottle, add 400-500ml of pure water to the tray, put it on a shaker for 60-90 minutes,...

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Abstract

The invention provides a pulsed field gel electrophoresis method for S.paratyphi A. S.paratyphi A which is obtained through separation is used as an analysis sample, and the method sequentially comprises the following steps of: preparing a gel block, performing lysis, washing the gel block, performing enzyme digestion on deoxyribonucleic acid (DNA) in the gel block, and injecting the sample and performing electrophoresis. The steps of preparing the gel block, performing lysis, washing the gel block, and injecting the sample are performed according to the conventional pulsed field gel electrophoresis method; in the step of performing enemy digestion on the DNA in the gel block, Spe I is used as a preferred enzyme, Xba I is used as a secondarily selected enzyme, and Xho I is used as the third kind of enzyme; in the electrophoresis parameters, the pulse time corresponding to Spe I enzyme is 1 to 20s, 19 to 20h; the pulse time corresponding to Xba I enzyme is 1.5 to 29s, 19 to 20h; and the pulse time corresponding to the Xho I enzyme is 2.2 to 29s, 19 to 20h. Compared with the prior art, the method has the advantage that the resolution ratio of S.paratyphi A is improved obviously, and can be effectively applied to the detection of molecular classification or genome variation of S.paratyphi A.

Description

technical field [0001] The invention relates to a gel electrophoresis method, in particular to a pulsed field gel electrophoresis method for Salmonella paratyphi A. Background technique [0002] The incubation period of Salmonella paratyphi A (S.paratyphi A) is long, usually 8-10 days. When an outbreak of Salmonella paratyphi A occurs, it is very difficult to trace the source. If the distinguishing ability of pulsed field gel electrophoresis (PFGE) typing method is not strong, it will increase the difficulty of tracing the source. Therefore, it is necessary to develop a PFGE method for Salmonella paratyphi A with strong applicability. [0003] Previous studies have proved that many factors can affect the results of PFGE, such as gel block preparation, cell lysis, gel block washing time, restriction endonuclease and electrophoresis parameters, etc., but the most important factor is the enzyme and parameters. choose. The genetic relationship between the strains was judged ...

Claims

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Application Information

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IPC IPC(8): G01N27/447
Inventor 韩辉李海山陈春霞胡群徐宝梁姚李四杨宇王艳李新张晓龙
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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