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Escherichia coli O157 nucleic acid detection plasmid DNA reference sample as well as preparation method and application thereof

A technology of Escherichia coli and reference samples, which is applied in the field of microbial detection, can solve problems such as lack of stability and safety, inability to evenly cover detection targets, and inability to stably trace the source of the value, etc., to achieve the effect of clear sources

Pending Publication Date: 2020-11-03
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Clinically positive samples are not easy to obtain, and lack stability and safety
Genomic reference products (gDNA) extracted from a single strain usually cannot evenly cover all detection targets, nor can they be stably traced in terms of quantity
Self-developed DNA references contain only small fragments of individual genes and are not compatible with different kits

Method used

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  • Escherichia coli O157 nucleic acid detection plasmid DNA reference sample as well as preparation method and application thereof
  • Escherichia coli O157 nucleic acid detection plasmid DNA reference sample as well as preparation method and application thereof
  • Escherichia coli O157 nucleic acid detection plasmid DNA reference sample as well as preparation method and application thereof

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preparation example Construction

[0036] Artificial DNA synthesis technology provides a new idea for the preparation of reference materials. In this study, complete genes were obtained through artificial synthesis, the complete forms of these genes under natural conditions were simulated, and the structural genes were connected in series to achieve a quality standard reference covering multiple detection target genes. In order to obtain a detection nucleic acid reference product with a wide range of applicability (covering genotype, virulence and invasion identification).

[0037]The present invention aims to prepare a plasmid DNA reference sample, which contains 6 representative full-length structural gene sequences (udiA, rfbE, flicH7, Stx1, Stx2 and eaeA) of Escherichia coli O157, to improve the quality control and quality of Escherichia coli qPCR detection The comparability of results between laboratories is expected to solve the contradiction between the rapid development of pathogenic nucleic acid detect...

Embodiment 1

[0038] Synthesis, fragment design and cloning of embodiment 1 plasmid

[0039] 1. Fragment Design

[0040] Sequence source Escherichia coli O157 uidA (GenBank: JQ068101.1), eaeA (Gen Bank: AJ579305.1), Stx1 (GenBank: FR875155.1), Stx2 (GenBank: AB030484.1), rfbE (GenBank: JQ907523.1 ), flicH7 (GenBank: LT717488.1). The nucleic acid sequence of a single gene can be downloaded from the NCBI website (https: / / www.ncbi.nlm.nihgov / nucleotide)), and the selected gene has clear representativeness and versatility through sequence comparison;

[0041] 2. Plasmid Construction

[0042] The six genes were concatenated together at a ratio of 1:1 (separated by the irrelevant gene fragment aagtcg), and cloned into pUC57 through the method of whole gene artificial synthesis (produced by Shanghai Gemma Biotechnology Engineering Co., Ltd.) To form a recombinant plasmid (the length of the chemically synthesized double-stranded DNA fragment is 5691bp, the length of the entire plasmid is digeste...

Embodiment 2

[0048] The mensuration of embodiment 2 homogeneity, fixed value and shelf life

[0049] 1. Judgment of pDNA homogeneity

[0050] Homogeneity is a basic property of reference materials, which is used to describe the spatial distribution characteristics of reference materials. The quality of plasmid DNA standard substances with good uniformity will not be affected by factors such as aliquoting, so as to ensure the reliability of the test results. In order to meet the requirements of the national standard material technical specification, the present invention uses the ultraviolet method (minimum sampling volume is 1 μ L) to analyze the uniformity between bottles and inside bottles of pNDA, and analyzes the data by F test.

[0051] 1) Sampling method for uniformity between bottles: In order to test the homogeneity between bottles, 10 tubes of pDNA were randomly selected, and the 1 μL sample extracted from each tube was repeatedly measured by UV for 3 times, and the average value...

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Abstract

The invention discloses an escherichia coli O157 nucleic acid detection plasmid DNA reference sample as well as a preparation method and application thereof, and belongs to the field of microbiological detection. The reference sample is formed by inserting fragments containing uidA, eaA, Stx1, Stx2, rfbE and flicH7 genes from different escherichia coli O157 strains into plasmids, the nucleotide sequences are shown as SEQ ID No.1, and the genes are connected in series according to a ratio of 1: 1. The genes are separated by irrelevant gene segments; and the reference substance contains six clear detection genes including udiA, rf bE, flicH7, Stx1, Stx2 and eaA, sequence sources are clear, the genes are stored in the plasmids in a single copy mode, and the reference sample can be used for qualitative detection, and can also be used as a reference substance with traceable quantity values for reagent performance identification and capability evaluation between laboratories.

Description

technical field [0001] The invention relates to the field of microbial detection, in particular to a plasmid DNA reference sample for detection of Escherichia coli O157 nucleic acid, a preparation method and an application thereof. Background technique [0002] Escherichia coli O157 causes widespread food poisoning worldwide, and the diagnosis of Escherichia coli O157 requires a variety of information, such as genotype, invasion, and virulence. However, the target genes required for many detections are not evenly distributed in a single strain. In practice, if it is necessary to detect multiple genes at the same time, or to evaluate the detection ability of multiple genes among different laboratories, it is necessary to use the genomes of multiple strains to prepare multiple standard curves for quantitative analysis. Given that biological assays are limited by the overall level of quality control and quantitative traceability, reference materials that can cover a wide range...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/10C12N15/11C12R1/19
CPCC12Q1/689C12Q2600/166
Inventor 陈瑶许龙岩袁慕云郝文波刘二龙
Owner SOUTHERN MEDICAL UNIVERSITY
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