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Polynucleotide for staphylococcus aureus detection, method and kit

A polynucleotide and staphylococcal technology, applied in the field of polynucleotides, methods and kits, can solve the problems of poor accuracy of plasmid DNA molecule determination, lack of comparability, and great differences in test results

Active Publication Date: 2015-11-18
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 genetic testing, but plasmid DNA standard materials for the real-time fluorescent PCR detection method of Staphylococcus aureus are still blank
In the actual real-time fluorescent PCR of Staphylococcus aureus, 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, and there is no uniform value determination method. The accuracy of the fixed value is poor, resulting in great differences in the test results between laboratories and lack of comparability

Method used

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  • Polynucleotide for staphylococcus aureus detection, method and kit
  • Polynucleotide for staphylococcus aureus detection, method and kit
  • Polynucleotide for staphylococcus aureus detection, method and kit

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preparation example Construction

[0092] The present invention also provides a set of methods for preparing the above-mentioned plasmid standard molecule of Staphylococcus aureus, comprising the following steps:

[0093] ① Artificially synthesizing the specific sequence of the Staphylococcus aureus, the sequence is shown in SEQ ID NO: 1;

[0094] ② Cloning the sequence of Staphylococcus aureus obtained in step ① to a cloning vector to obtain a plasmid standard molecule of Staphylococcus aureus.

[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 thermostable nuclease gene nuc gene and / or thermostable nuclease gene nuc of Staphylococcus aureus in Genbank of NCBI (National Center for Biotechnology Information);

[0098] ②Analyze the above sequence, select a suitabl...

Embodiment 1

[0111] The construction of embodiment 1 plasmid standard molecule

[0112] Experimental reagents and experimental equipment:

[0113] 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.

[0114] The experimental method includes the following steps:

[0115] 1. Search for the sequence of the thermostable nuclease nuc gene of Staphylococcus aureus in GenBank;

[0116] 2. Analyze the above sequence, select a suitable sequence and a suitable restriction site, the sequence length of gene thermostable nuclease nuc gene is 534bp, add EcoRI and BamHI restriction sites at both ends;

[0117] 3. Send the processed sequence to Treasure Bioengineering (Dalian) Co., Ltd., which will be responsible for the artificial synthesis of the whole gene, including t...

Embodiment 2

[0136] The homogeneity test of embodiment 2 plasmid standard molecule LY03

[0137] 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 differ...

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Abstract

The invention discloses polynucleotide for staphylococcus aureus detection, a method and a kit and particularly discloses the polynucleotide of standard molecules and capable of being used for real-time fluorescence PCR detection of staphylococcus aureus and a DNA construct LY03. According to the polynucleotide for staphylococcus aureus detection, the method and the kit, plasmid standard molecules solve the problem that standard matter lacks in real-time fluorescence PCR detection of the staphylococcus aureus, the result comparability of the real-time fluorescence PCR method detection of staphylococcus aureus is guaranteed, and a reliable quality control method is provided for real-time fluorescence PCR method detection of staphylococcus aureus.

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 detecting Staphylococcus aureus. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is an important human pathogenic bacteria, belonging to the genus Staphylococcus (Staphylococcus). As for the presence of Staphylococcus aureus in quick-frozen food, the Ministry of Health announced the national food safety standard "Quick-frozen Noodle and Rice Products" on November 24, 2011, allowing the presence of Staphylococcus aureus in limited quantities. [0003] At present, the detection methods of Staphylococcus aureus are divided into two categories, one is the traditional culture and its improved methods, which rely on biochemical and morphological characteristics, the detection cycle is longer, and the operability is poor; the other is the polymerase chain reaction ( Polymeras...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12N15/10
CPCC12Q1/689C12Q2600/166
Inventor 刘刚李妍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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