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Plasmid standard molecular adaptable to sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection

A technology of Enterobacter sakazakii and standard molecules, which is applied in the field of bioengineering, can solve the problems of lack of positive standard products and configuration of positive standard products, and achieve the effect of ensuring comparability, good traceability and strong uniformity

Inactive Publication Date: 2014-01-08
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a real-time fluorescent PCR detection method suitable for Enterobacter sakazakii in order to overcome the lack of positive standard and positive standard configuration in the existing Enterobacter sakazakii real-time fluorescent PCR detection method. Plasmid standard molecule and construction method, quantitative method and application of the plasmid standard molecule

Method used

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  • Plasmid standard molecular adaptable to sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection
  • Plasmid standard molecular adaptable to sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection
  • Plasmid standard molecular adaptable to sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection

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Effect test

Embodiment 1

[0054] The construction of embodiment 1 plasmid standard molecule

[0055] Experimental reagents and experimental equipment:

[0056] A large number of plasmid extraction kits (OMEGA), and other biochemical reagents are imported or domestic analytically pure biochemical reagents; experimental equipment includes centrifuges, constant temperature water baths, constant temperature culture shakers, pipette guns, etc.

[0057] The experimental method includes the following steps:

[0058] 1. Search the 16S rDNA gene of Enterobacter sakazakii in GenBank,

[0059] 2. Analyzing the above sequence, selecting a suitable sequence and a suitable enzyme cutting site. The length of the M16S rDNA gene sequence is 1493bp, with HindⅢ restriction sites added at both ends;

[0060] 3. Send the processed sequence to Treasure Bioengineering (Dalian) Co., Ltd., which will be responsible for the artificial synthesis of the whole gene, including the synthesis of single-stranded Oligo DNA, splicing...

Embodiment 2

[0078] The homogeneity test of embodiment 2 plasmid standard molecule pXL05

[0079] 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. Homogeneity evaluation must be carried out during the development (production) of reference materials to prove that they have good homogeneity. The value of plasmid standard molecules with good uniformity will not be affected by factors such as aliquoting, and there is little dif...

Embodiment 3

[0100] The stability investigation of embodiment 3 plasmid standard molecules

[0101] Stability refers to the ability of the characteristic values ​​of the standard substance to remain within the specified range under specific time intervals and storage conditions. Stability is a basic property of standard substances, which is used to describe the characteristics of standard substances changing with time, that is, to describe the time distribution characteristics of standard substances. Stability assessment must be performed during the development of reference materials. Stability assessment not only evaluates the measurement uncertainties related to material stability, but also identifies suitable storage and transport conditions. Plasmid standard molecules with good stability, their characteristic values ​​will not change over time under appropriate storage and transportation conditions, and the test results will not be affected by their instability, which ensures the reli...

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Abstract

The invention discloses a plasmid standard molecular adaptable to sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection, a preparation method thereof, a quantitation method thereof and application. The plasmid standard molecular comprises a 16S rDNA gene sequence of the sakazakii. The 16S rDNA gene sequence is shown as SEQ ID NO: 1 in sequence lists. The plasmid standard molecular is quantitated by the ultraviolet light spectrophotometry method and a high resolution inductively coupled plasma emission mass spectrometry (HR-ICP-MS), and good traceability is guaranteed. By the aid of the plasmid standard molecular, the problem of the lack of standard material during the sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection, comparability of results of the sakazakii real-time fluorescent quantitation PCR detection, and a reliable quality control method is provided for the sakazakii real-time fluorescent quantitation PCR detection.

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 fluorescence quantitative PCR detection of Enterobacter sakazakii and its construction method, quantitative method and application. Background technique [0002] Enterobacter sakazakii (Enterobacter sakazakii) is a pathogenic bacterium newly discovered in dairy products that has attracted widespread attention in recent years. Since 1961, cases of neonatal meningitis, bacteremia and necrotizing enterocolitis caused by this bacteria have been reported one after another around the world, and Enterobacter sakazakii has attracted attention as an important food-borne pathogen. Many research reports have shown that infant formula milk powder is the main infection channel of meningitis, sepsis and necrotizing enterocolitis in infants and premature infants, and the overall mortality rate is as high as 80%. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/31C12Q1/68C12Q1/06G01N21/64
CPCY02A50/30
Inventor 许丽刘刚李兰英梁文李妍徐勤闻艳丽
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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