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A set of polynucleotides, methods and kits for the detection of Bacillus anthracis

A technology of Bacillus anthracis and polynucleotides, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, and can solve problems such as lack of comparability, lack of uniform value determination methods, and great differences in test results.

Active Publication Date: 2018-06-26
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 Bacillus anthracis are still blank
In the actual real-time fluorescent PCR of Bacillus anthracis, 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. The accuracy is poor, resulting in great differences in test results between laboratories and lack of comparability

Method used

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  • A set of polynucleotides, methods and kits for the detection of Bacillus anthracis
  • A set of polynucleotides, methods and kits for the detection of Bacillus anthracis
  • A set of polynucleotides, methods and kits for the detection of Bacillus anthracis

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preparation example Construction

[0117] The present invention also provides a set of methods for preparing the above-mentioned plasmid standard molecule of Bacillus anthracis, comprising the following steps:

[0118] ① Artificially synthesizing the specific sequence of the Bacillus anthracis, the sequence is shown as SEQ ID NO: 1 and / or 3;

[0119] ② Cloning the sequence of Bacillus anthracis obtained in step ① to a cloning vector to obtain a plasmid standard molecule of Bacillus anthracis.

[0120] The artificial synthesis method described in step ① is preferably: the sequence is obtained by whole gene synthesis or PCR primer amplification.

[0121] The plasmid standard molecular construction method of the present invention preferably comprises the following steps:

[0122] ① Query the macromolecular toxin gene PA gene and / or macromolecular capsular gene capA gene of Bacillus anthracis in Genbank of NCBI (National Center for Biotechnology Information);

[0123] ②Analyze the above sequence, select a suitabl...

Embodiment 1

[0150] Example 1 Construction of Plasmid Standard Molecules

[0151] Experimental reagents and experimental equipment:

[0152] 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.

[0153] The experimental method includes the following steps:

[0154] 1. Search the Genotoxin PA gene sequence of Bacillus anthracis in GenBank;

[0155] 2. Analyze the above sequence, select a suitable sequence and a suitable restriction site, the genotoxin PA gene sequence is 2227bp in length, and BamHI restriction sites are added at both ends;

[0156] 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, splicin...

Embodiment 2

[0175] Example 2 Homogeneity Test of Plasmid Standard Molecules PA and capA

[0176] 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 a polynucleotide, a method and a kit for detecting Bacillus anthraci, and particularly discloses the polynucleotide which can be used as a standard molecule for real-time fluorescent PCR (polymerase chain reaction) detection of the Bacillus anthraci as well as DNA constructs PA and capA. By the aid of the plasmid standard molecule, the problem of lack of standard substances for real-time fluorescent PCR detection of the Bacillus anthraci is solved, the comparability of the detection result of a real-time fluorescent PCR method of the Bacillus anthraci is guaranteed, and a reliable quality control method is provided for detection with the real-time fluorescent PCR method of the Bacillus anthraci.

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 the detection of Bacillus anthracis. Background technique [0002] Bacillus anthraci belongs to the genus Bacillus. Bacillus anthracis can cause anthrax in sheep, cattle, horses and other animals and humans. Bacillus anthrax was once used as one of the lethal war agents by the imperialists. Normally, herdsmen, farmers, fur and slaughter workers are susceptible to infection, so it has attracted the attention of relevant departments in many countries around the world. [0003] At present, the detection methods of Bacillus anthracis are divided into two categories, one is the traditional culture and its improvement method, which depends on biochemical and morphological characteristics, the detection cycle is longer, and the operability is poor; the other is the polymerase chain reaction (polymerase...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/31C12N15/63
CPCC12Q1/689C12Q2600/166
Inventor 李妍刘刚许丽梁文闻艳丽李兰英徐勤任淑贞
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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