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Plasmid standard molecules for detecting Shiga-toxin-producing Escherichia coli and application thereof

A technology for producing Shiga toxin and Escherichia coli, which is applied in the field of bioengineering, can solve the problems of lack of unified value determination methods, great differences in detection results, and poor accuracy of plasmid DNA molecular value determination.

Active Publication Date: 2015-09-30
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the actual real-time fluorescent PCR of Escherichia coli, most of the units use the plasmid DNA molecules designed and constructed by themselves as standard products, and the specific fragments of the target genes are different. The accuracy is poor, resulting in great differences in test results between laboratories and lack of comparability

Method used

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  • Plasmid standard molecules for detecting Shiga-toxin-producing Escherichia coli and application thereof
  • Plasmid standard molecules for detecting Shiga-toxin-producing Escherichia coli and application thereof
  • Plasmid standard molecules for detecting Shiga-toxin-producing Escherichia coli and application thereof

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preparation example Construction

[0128] In order to solve the above-mentioned technical problems, the fourth technical solution adopted by the present invention is: a method for preparing a plasmid standard molecule of Shiga toxin-producing Escherichia coli as described above, comprising the following steps:

[0129] ① Artificially synthesizing the virulence factor stx1 and stx2 gene sequences of Shiga toxin-producing Escherichia coli, the virulence factor stx1 and stx2 gene sequences are shown in SEQ ID NO: 1 and NO: 2 in the sequence listing;

[0130] ② Cloning the virulence factor stx1 and stx2 gene sequences of Shiga toxin-producing Escherichia coli obtained in step ① into a cloning vector to obtain a plasmid standard molecule of Shiga toxin-producing Escherichia coli.

[0131] The artificial synthesis method described in step ① is preferably: the sequence is obtained by whole gene synthesis or PCR primer amplification.

[0132] The plasmid standard molecular construction method of the present invention p...

Embodiment 1

[0146] Example 1 Construction of Plasmid Standard Molecules

[0147] Experimental reagents and experimental equipment:

[0148] A large number of plasmid extraction kits (OMEGA), other biochemical reagents are imported aliquots or domestic analytically pure biochemical reagents; experimental equipment includes centrifuges, constant temperature water baths, constant temperature culture shakers, pipette guns, etc.

[0149] The experimental method includes the following steps:

[0150] 1. Search the gene virulence factors stx1 and stx2 gene sequences of Shiga toxin-producing Escherichia coli in GenBank,

[0151] 2. Analyze the above sequence, select a suitable sequence and a suitable restriction site, the lengths of the genovirulence factors stx1 and stx2 gene sequences are 1227bp and 1236bp respectively, and BamHI restriction sites are added at both ends;

[0152] 3. Send the processed sequence to Treasure Bioengineering (Dalian) Co., Ltd., which will be responsible for the ar...

Embodiment 2

[0170] Example 2 Homogeneity Test of Plasmid Standard Molecules PLW03 and PLW04

[0171] Homogeneity is the consistent state of structure or composition that characterizes one or more properties in a substance. By measuring samples of specified size taken from different packaging units (such as bottles, bags, etc.) or from different locations of the same packaging unit, if the measurement results fall within the specified uncertainty range, it can be considered that the reference material has a certain effect on the specified characteristic quantity. is even. Homogeneity is a basic property of reference materials, which is used to describe the spatial distribution characteristics of reference materials. Uniformity evaluation must be carried out during the development (production) of reference materials to prove that they have good uniformity. The value of plasmid standard molecules with good uniformity will not be affected by factors such as aliquoting, and there is little d...

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Abstract

The invention discloses plasmid standard molecules PLW03 and PLW04 for detecting Shiga-toxin-producing Escherichia coli and application thereof. The invention particularly discloses a polynucleotide for Shiga-toxin-producing Escherichia coli PCR detection and a primer pair matched with the polynucleotide. The polynucleotide sequence is prepared into the standard plasmid molecules by using an appropriate framework plasmid. When being matched with the primer pair to perform real-time fluorescent PCR detection, the standard plasmid molecules have the advantages of excellent specificity, excellent sensitivity and favorable stability.

Description

technical field [0001] The invention relates to a plasmid molecule in the technical field of bioengineering, in particular to a plasmid standard molecule suitable for real-time fluorescent quantitative PCR detection of Shiga toxin-producing Escherichia coli and its construction method, quantitative method and application. Background technique [0002] Shiga toxin-producing Escherichia coli is a pathogenic Escherichia coli closely related to public health, which can cause hemorrhagic colitis, hemolytic uremic syndrome and other diseases. Since 1982 when Riley et al first isolated the strain in the United States, outbreaks of Shiga toxin-producing E. coli have been reported in the United Kingdom, Canada, Japan and my country. As a zoonotic pathogen, Shiga toxin-producing Escherichia coli can circulate and spread in livestock and poultry, and spread through animal-derived food. At present, domestic diagnosis of Shiga toxin-producing E. coli mainly relies on bacterial culture, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/31C12N15/11C12N15/10C12R1/19
Inventor 刘刚梁文许丽李妍闻艳丽李兰英任淑贞
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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