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Method and kit for detecting Shigella and ipaH pathogenicity island thereof

A Shigella and kit technology, which is applied in the field of real-time quantitative fluorescent polymerase chain reaction technology to detect Shigella contamination, can solve problems such as high cost, difficult clinical detection, PCR product contamination, etc., and achieve the effect of rapid diagnosis

Active Publication Date: 2010-06-30
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gene chip method has high detection efficiency, but the technology is immature and the cost is high, and it is not easy to carry out in general laboratories; although ordinary PCR technology is mature and low in cost, it requires post-processing of PCR products, which can easily lead to contamination of PCR products. Not easy to use for clinical testing

Method used

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  • Method and kit for detecting Shigella and ipaH pathogenicity island thereof
  • Method and kit for detecting Shigella and ipaH pathogenicity island thereof
  • Method and kit for detecting Shigella and ipaH pathogenicity island thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the development of Shigella detection reagent

[0032] 1. Design of primers and probes: Through sequence comparison and analysis of all Shigella nucleic acid sequences already available in the Genbank database and nucleic acid sequences reported in published literature at home and abroad, the ipaH gene was selected to have no secondary structure and was highly conserved. According to the basic principles of primer-probe design, multiple pairs of primers and probes are designed by software and manually.

[0033] 2. Establishment and optimization of the reaction system

[0034] Sample preparation: the standard strain 51313 purchased by the Institute of Biological Products of the Chinese Academy of Sciences was used as a positive standard for Shigella detection; as a negative reference. The genomic DNA of the above-mentioned positive standard product and negative reference product were extracted by boiling the DNA extraction solution for use.

[0035] Scre...

Embodiment 2

[0043] Embodiment 2: Shigella detection kit and its use

[0044] 1. Prepare a kit including the following components: 2 tubes of DNA extraction solution (500 μl / tube), 20 tubes of PCR reaction solution, 1 tube of negative reference product (100 μl / tube), 1 tube of critical positive reference product (50 μl / tube) , Positive quantitative standard (50μl / tube) 4 tubes.

[0045] 2. Specimen collection, transportation and storage

[0046] 2.1 Feces: Collect mucus, pus and blood feces in the early stage of the disease (before treatment) for bedside inoculation. If the inoculation cannot be inoculated in time, it can be placed in glycerin preservation solution or card-cloth transport medium for inspection.

[0047] 2.2 Specimen storage and delivery: Specimens can be used for testing immediately, or can be stored at -20°C for testing, and the storage period is 6 months. Specimens should be transported in a 0°C ice bottle.

[0048] 3. Detection steps

[0049] Add 4 times the volume ...

Embodiment 3

[0052] Embodiment 3: Detecting the specimen for determining the type of Shigella

[0053] The specific steps are the same as in Example 2, except that the samples used are Shigella dysenteriae, Shigella flexneri, Shigella baumannii, and Sonnei from China Microorganism Culture Collection Center and China Institute for the Control of Pharmaceutical and Biological Products. Shigella. After nucleic acid is extracted from the specimen, the kit of the present invention is used for PCR amplification, and the instrument automatically monitors and collects changes in fluorescent signals during the amplification. Analysis after reaction finishes, application kit of the present invention all can detect the specimen of known type (referring to Figure 4 ).

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Abstract

The invention relates to a method and a reagent for detecting the existence of shigella bacteria genus bacteria and ipaH pathogenicity island (PAI) of the shigella in clinical samples and in particular relates to the method and the eagent kit for detecting the shigella with real-time fluorescence quantitative polymerase chain reaction technique.

Description

technical field [0001] The invention relates to a method and a kit for detecting the presence of Shigella (Shigella) bacteria (Shigella) and its ipaH virulence island (PAI) in food, water quality and clinical samples, in particular to real-time quantitative fluorescent polymerase chain The method and kit used to detect Shigella contamination by reaction technology. Background technique [0002] Shigella bacteria, commonly known as Shigella, are a type of Gram-negative intestinal pathogenic bacteria that are highly contagious and harmful to human health. They are the most common pathogens of human bacillary dysentery and can be divided into group A : Shigella dysenteriae (Sh.dysenteriae), commonly known as Shigella dysenteriae; Group B: Shigella flexneri (Sh.flexneri), commonly known as Shigella flexneri; Group C: Shigella baumannii ( Sh.boydii), commonly known as Shigella baumannii; group D: Shigella sonnei (Sh.sonnei), commonly known as Shigella sonnei. Each strain of Shi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12Q1/06G01N21/64
CPCY02A50/30
Inventor 邓中平高秀洁王伟毅程钢何蕴韶
Owner DAAN GENE CO LTD
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