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A set of polynucleotides, methods and kits for the detection of bacterial drug resistance gene ndm-1

A technology of drug-resistant genes and polynucleotides, applied in the field of bioengineering, can solve the problems of poor accuracy of plasmid DNA molecular determination, lack of unified determination methods, and great differences in detection results.

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

Since the development of plasmid DNA standard materials for NDM-1 drug-resistant gene PCR-related detection methods is still blank, in the actual NDM-1 drug-resistant gene PCR detection, most units use plasmid DNA molecules designed and constructed by themselves as standard products , the specific fragments of the target gene are different, and at the same time, there is a lack of a unified valuation method, and the accuracy of the plasmid DNA molecular valuation is poor, resulting in great differences in the test results between laboratories, lack of comparability, validity and reliability sex

Method used

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  • A set of polynucleotides, methods and kits for the detection of bacterial drug resistance gene ndm-1
  • A set of polynucleotides, methods and kits for the detection of bacterial drug resistance gene ndm-1
  • A set of polynucleotides, methods and kits for the detection of bacterial drug resistance gene ndm-1

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Experimental program
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preparation example Construction

[0083] The present invention also provides a set of preparation methods for the plasmid standard molecule detected by the bacterial drug resistance gene NDM-1 as described above, comprising the following steps:

[0084] ① Artificially synthesizing the specific sequence of the bacterial drug resistance gene NDM-1, the sequence is shown in SEQ ID NO: 1;

[0085] ② Step ① The sequence of the bacterial drug resistance gene NDM-1 obtained is cloned into a cloning vector to obtain a plasmid standard molecule for detection of the bacterial drug resistance gene NDM-1.

[0086] The artificial synthesis method described in step ① is preferably: the sequence is obtained by whole gene synthesis or PCR primer amplification.

[0087] The plasmid standard molecular construction method of the present invention preferably comprises the following steps:

[0088] ① Query bacterial drug resistance gene NDM-1 in Genbank of NCBI (National Center for Biotechnology Information);

[0089] ②Analyze t...

Embodiment 1

[0111] Example 1 Construction of Plasmid Standard Molecules

[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 the bacterial drug resistance gene NDM-1 sequence in GenBank;

[0116] 2. Analyze the above sequence, select a suitable sequence and a suitable restriction site, the length of the bacterial drug resistance gene NDM-1 sequence is 813bp, and BamHI restriction sites are added 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 the synthesis of single-stranded...

Embodiment 2

[0135] Example 2 Homogeneity Test of Plasmid Standard Molecule pXL08

[0136] 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 difference i...

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Abstract

The invention discloses polynucleotide, a method and a kit for detection of a bacterial drug-resistant gene, namely, a New Delhi metallo-blactamase-1 (NDM-1) gene. Specifically, the invention discloses polynucleotide and a DNA construction material pXL08 capable of being used as real-time fluorescent PCR detection standard molecules for the bacterial drug-resistant gene NDM-1. According to the invention, due to plasmid standard molecules, the difficult problem of lack of standard substances in the real-time fluorescent PCR detection of the bacterial drug-resistant gene NDM-1 is solved, the comparability of a real-time fluorescent PCR detection result of the bacterial drug-resistant gene NDM-1 is ensured, and a reliable quality control method is provided for the real-time fluorescent PCR detection of the bacterial drug-resistant gene NDM-1.

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 bacterial drug resistance gene - New Delhi metallo-beta-lactamase-1 gene (NDM-1). Background technique [0002] In recent years, with the widespread use of antibiotics, a variety of bacteria have developed drug resistance to varying degrees. In September 2010, scholars from India, the United Kingdom, Sweden, Pakistan and Australia jointly reported a bacterium that can resist almost all antibiotics, called "super bacteria", which can produce a new metallo-β-lactamase, Its hydrolytic activity is much higher than that of existing metallo-β-lactamases. Since the first case of infection was found in New Delhi, India, the enzyme was called New Delhi metallo-blactamase-1 (New Delhi metallo-blactamase-1, NDM-1). NDM-1 has a strong hydrolysis effect and can resist almost all antibiotics current...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12N15/11C12N15/63
CPCC12Q1/689C12Q2600/166
Inventor 许丽刘刚梁文李妍闻艳丽李兰英徐勤
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH