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A polynucleotide, method and kit for real-time fluorescent quantitative PCR detection of Vibrio cholerae

A technology of Vibrio cholerae and polynucleotide, applied in the field of bioengineering, can solve the problems of lack of uniform determination method, lack of comparability, poor determination accuracy of plasmid DNA molecules, etc.

Active Publication Date: 2018-12-21
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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 Vibrio cholerae are still blank
In the actual real-time fluorescent PCR of Vibrio cholerae, most of the units use plasmid DNA molecules designed and constructed by themselves as standard products, and the specific fragments of the target genes are different, and there is no uniform value determination method. The accuracy of the value is poor, resulting in great differences in the test results between laboratories and lack of comparability

Method used

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  • A polynucleotide, method and kit for real-time fluorescent quantitative PCR detection of Vibrio cholerae
  • A polynucleotide, method and kit for real-time fluorescent quantitative PCR detection of Vibrio cholerae
  • A polynucleotide, method and kit for real-time fluorescent quantitative PCR detection of Vibrio cholerae

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preparation example Construction

[0092] 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 Vibrio cholerae as described above, comprising the following steps:

[0093] ① Artificially synthesizing the virulence regulator toxR gene sequence of Vibrio cholerae, the virulence regulator toxR gene sequence is shown in SEQ ID NO: 1 in the sequence listing;

[0094] ② Cloning the virulence regulator toxR gene sequence of Vibrio cholerae obtained in step ① into a cloning vector to obtain a plasmid standard molecule of Vibrio cholerae.

[0095] The artificial synthesis method described in step ① is preferably: the sequence is obtained by whole gene synthesis or PCR primer amplification.

[0096] The plasmid standard molecular construction method of the present invention preferably comprises the following steps:

[0097] ① Query the virulence regulator toxR gene of Vibrio cholerae in Genbank o...

Embodiment 1

[0115] The construction of embodiment 1 plasmid standard molecule

[0116] Experimental reagents and experimental equipment:

[0117] 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.

[0118] The experimental method includes the following steps:

[0119] 1. Search the gene virulence regulator toxR gene sequence of Vibrio cholerae in GenBank,

[0120] 2. Analyze the above sequence, select a suitable sequence and a suitable restriction site, the length of the gene virulence regulator toxR gene sequence is 885bp, and BamHI restriction sites are added at both ends;

[0121] 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 synthes...

Embodiment 2

[0139] The homogeneity test of embodiment 2 plasmid standard molecule PLW07

[0140] 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 diffe...

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Abstract

The invention discloses polynucleotide, a method and a kit for real-time fluorescence quantitative PCR (Polymerase Chain Reaction) detection of vibrio cholerae, and particularly provides the polynucleotide, usable for vibrio cholerae PCR detection, and a primer pair matched with the polynucleotide. A polynucleotide sequence is prepared into a standard plasmid molecule PLW07 by using an appropriate framework plasmid, and real-time fluorescence PCR detection is carried out by virtue of the primer pair; the specificity and the sensitivity are extremely high, and the stability is high.

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 Vibrio cholerae and its construction method, quantitative method and application. Background technique [0002] Vibrio cholerae (Vibrio cholerae) is a pathogenic bacterium that can cause a severe infectious disease with diarrhea as the main symptom. Rapid and accurate identification of Vibrio cholerae plays an important role in the prevention and treatment of the disease. It has been found for a long time that Vibrio cholerae group 01 can cause the epidemic of cholera. Group 0139 cholera, as an emerging infectious disease, caused a cholera pandemic in India and Bangladesh in 1992. Since 1993, outbreaks caused by Vibrio cholerae of group 0139 have also occurred to varying degrees in many areas of my country. In recent years, the proportion of outbreaks of grou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12Q1/6851C12Q1/06C12N15/11C12N15/63C12N15/66C12R1/63
CPCY02A50/30
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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