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Target sequence for detection of yersinia enterocolitica and kit

A technology of Yersinia inflammatoryis and kits, which is applied in the field of real-time fluorescent PCR detection kits, can solve the problems that virF is prone to false negatives, plasmids are easy to lose, and the specificity of inV and vst sites is not strong.

Inactive Publication Date: 2010-06-23
SHANGHAI FOSUN PHARMA (GROUP) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] From the perspective of various pathogenic strains at home and abroad, the pathogenicity of Yersinia enterocolitica is located in the gene loci of inV, ail, yst and virF, the ail locus is related to the invasiveness of bacteria, and the yst And the virF gene locus located on the pYv plasmid is related to the virulence of the bacteria. Because the plasmid is easily lost during the culture process, the amplification of virF by polymerase chain reaction is prone to false negatives, while the specificity of inV and vst loci is not strong , Therefore, there is an urgent need in this field to develop new methods and kits for the detection of Yersinia enterocolitica with high specificity, so as to be able to effectively detect Yersinia enterocolitica

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Isolation of Yersinia enterocolitica DNA for PCR Analysis

[0056] In order to isolate Y. enterocolitica DNA from test samples for PCR detection, simple methods that do not interfere with the PCR must be used. First, use sterilized physiological suspension of clinical samples (such as feces, etc.), centrifuge the rinse solution at 15,000rpm for 15 minutes and discard the supernatant. The precipitate may contain Yersinia enterocolitica; after adding DNA lysate to the precipitate, Oscillating and mixing, 100 ° C boiling water bath for 15 minutes, lysing Yersinia enterocolitica to release DNA. Finally, centrifuge at 15,000 rpm for 15 minutes, dissolve the DNA into the supernatant, and take the supernatant as Yersinia enterocolitica DNA for PCR analysis.

Embodiment 2

[0057] The preparation of embodiment 2 detection kits

[0058] A fluorescent PCR kit for rapid quantitative detection of Yersinia enterocolitica is prepared by a conventional method, and the kit includes a) DNA lysate, b) fluorescent quantitative reaction solution, c) quantitative calibrator, d) positive control product, e) negative control product. in:

[0059] 1) Fluorescence quantitative reaction solution contains PCR buffer, dNTPs solution, Taq DNA polymerase, sterile double distilled water, MgCl 2 Solution, the primer sequences for specifically amplifying the ail gene genetic markers are SEQ ID NO: 3 and 4, the fluorescent probe sequence is SEQ ID NO: 5, and the fluorescent reporter group labeled at the 5' end of the fluorescent probe is FAM, 3 The fluorescent quencher group labeled at the 'end is TAMRA. (Amplification product size is 150bp)

[0060] Specifically, the fluorescence quantitative reaction solution contains 50mM KCl, 10mM Tris-HCl pH8.3 (25°C), 2mM MgCl ...

Embodiment 3

[0064] Example 3 Carrying out polymerase chain reaction with Yersinia enterocolitica

[0065] Take 26 μl of the fluorescent reaction solution in Example 2 and add them to different PCR reaction tubes, add 4 μl of the DNA and quantitative calibrator obtained in Example 1 to the fluorescent quantitative reaction solution, with a total volume of 30 μl, and perform real-time fluorescent quantitative Amplification detection was started on the PCR machine (ABI 7500). The amplification parameters were set as 50°C for 2 minutes, 94°C for 5 minutes, and cycled 40 times as 94°C for 10 seconds → 60°C for 45 seconds. The fluorescence detection was programmed to detect at a wavelength of 530 nm at the result of the annealing step of each cycle.

[0066] After the cycle is over, use the instrument supporting software to read the copy number of the sample to be tested. The result is: Quantitative calibrator 5×10 6 copies / μl, 5×10 5copies / μl, 5×10 4 The copies / μl are 22.13, 25.65, and 29...

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Abstract

The invention provides a genetic marker and a method for detection of yersinia enterocolitica, in particular a nucleotide sequence for detection of the ail gene of the yersinia enterocolitica, oligonucleotide primers and a probe. The oligonucleotide primers and the probe are designed based on the nucleotide sequence. The invention also provides a method for rapid and specific detection of the yersinia enterocolitica with the primers. Moreover, the invention can detect the yersinia enterocolitica conveniently, rapidly, accurately, qualitatively and quantitatively.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to a method for amplifying pathogenic ail gene by polymerase chain reaction to specifically detect Yersinia enterocolitica in clinical samples with high sensitivity and using the method Obtained real-time fluorescent PCR detection kit. Background technique [0002] Enterocolitis Yersiniasis (Yersiniasis) is a new intestinal infectious disease that has been paid attention to in the 1980s. It has been found in all continents of the world and is the main disease of diarrhea in some European countries. In many areas, gastroenteritis and severe diarrhea caused by Yarrowia are more than dysentery. In addition to intestinal symptoms, it can also cause respiratory, cardiovascular system, bone, connective tissue and systemic diseases, and the case fatality rate reaches more than 30% when sepsis occurs. The disease was only discovered in my country in 1981, which attracted national atte...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 沈维祥夏懿吴大治
Owner SHANGHAI FOSUN PHARMA (GROUP) CO LTD
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