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Polynucleotide, method and kit for detecting salmonella bacteria

A Salmonella, polynucleotide technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of lack of uniform determination method, lack of comparability, poor determination accuracy of plasmid DNA molecules, etc.

Active Publication Date: 2016-02-03
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, plasmid DNA molecules have been more and more in-depth research and application as standard materials for gene detection, but the plasmid DNA standard materials for the real-time fluorescent PCR detection method of Salmonella are still blank.
In the actual real-time fluorescent PCR of Salmonella, 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. At the same time, there is a lack of unified value determination methods, and the value of the plasmid DNA molecules is accurate. The degree of difference is poor, resulting in great differences in test results between laboratories, lack of comparability

Method used

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  • Polynucleotide, method and kit for detecting salmonella bacteria
  • Polynucleotide, method and kit for detecting salmonella bacteria
  • Polynucleotide, method and kit for detecting salmonella bacteria

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preparation example Construction

[0115] The present invention also provides a set of methods for preparing the plasmid standard molecule of Salmonella as above, comprising the following steps:

[0116] ① Artificially synthesizing the specific sequence of the Salmonella, the sequence is shown in SEQ ID NO: 1 and / or 3;

[0117] ② Cloning the sequence of Salmonella obtained in step ① to a cloning vector to obtain a plasmid standard molecule of Salmonella.

[0118] The artificial synthesis method described in step ① is preferably: the sequence is obtained by whole gene synthesis or PCR primer amplification.

[0119] The plasmid standard molecular construction method of the present invention preferably comprises the following steps:

[0120] ① Query the internalization gene InlA gene and / or transcriptional activation regulatory protein gene prfA gene of Salmonella in Genbank of NCBI (National Center for Biotechnology Information);

[0121] ②Analyze the above sequence, select a suitable sequence and a suitable re...

Embodiment 1

[0134] The construction of embodiment 1 plasmid standard molecule

[0135] Experimental reagents and experimental equipment:

[0136] 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.

[0137] The experimental method includes the following steps:

[0138] 1. Search the invA gene sequence of Salmonella in GenBank;

[0139] 2. Analyze the above sequence, select a suitable sequence and a suitable restriction site, the length of the invA gene sequence of Salmonella is 2058bp, and add KpnI restriction sites at both ends;

[0140] 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 OligoDNA, splicing of DNA...

Embodiment 2

[0159] Example 2 Homogeneity Inspection of Plasmid Standard Molecules pXL10 and pXL11

[0160]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 lit...

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Abstract

The invention discloses polynucleotide, a method and a kit for detecting salmonella bacteria. Specifically, the invention discloses polynucleotide and DNA constructs pXL10 and pXL11, which can be used as standard molecules for real-time fluorescent PCR (polymerase chain reaction) detection of the salmonella bacteria. The plasmid standard molecules disclosed by the invention solve the difficulty that the real-time fluorescent PCR detection of the salmonella bacteria lacks standard substances, thereby ensuring the comparability of a result of a real-time fluorescent PCR method detection of the salmonella bacteria, and providing a reliable quality control method for the real-time fluorescent PCR method detection of the salmonella bacteria.

Description

technical field [0001] The invention relates to a set of plasmid molecules in the technical field of bioengineering, in particular to a set of polynucleotides, methods and kits for Salmonella detection. Background technique [0002] With the progress of society and the improvement of people's living standards, food safety has become a global focus. Food poisoning incidents caused by various food-borne pathogenic microorganisms have attracted more and more attention from experts and governments. Timely and effective detection and identification of pathogenic bacteria has become the key to the prevention and treatment of foodborne diseases. Salmonella is an important food-borne pathogen, and food poisoning incidents caused by Salmonella are frequently reported in countries all over the world. In February 2007, the "peanut butter incident" in the United States, and the "tomato incident" in the United States from 2005 to 2006 were all caused by Salmonella. In the Principality ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/31C12N15/63
CPCC12Q1/689C12Q2600/166Y02A50/30
Inventor 许丽刘刚梁文李妍闻艳丽李兰英徐勤
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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