Polynucleotide, method and kit for detection of bacterial drug-resistant gene NDM-1

A technology of NDM-1 and drug-resistant genes, applied in the field of bioengineering, can solve the problems of lack of unified value determination methods, great differences in detection results, and poor accuracy of plasmid DNA molecular value determination.

Active Publication Date: 2016-03-16
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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  • Polynucleotide, method and kit for detection of bacterial drug-resistant gene NDM-1
  • Polynucleotide, method and kit for detection of bacterial drug-resistant gene NDM-1
  • Polynucleotide, method and kit for detection of bacterial drug-resistant gene NDM-1

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preparation example Construction

[0083] The present invention also provides a method for preparing a set of plasmid standard molecules for detection of bacterial drug resistance gene NDM-1 as described above, including the following steps:

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

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

[0086] Wherein, the artificial synthesis method described in step ① is preferably: whole gene synthesis or PCR primer amplification method to obtain the sequence.

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

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

[0089] ②Analyze the above seq...

Embodiment 1

[0111] Example 1 Construction of plasmid standard molecules

[0112] Experimental reagents and experimental instruments:

[0113] Plasmid mass extraction kit (OMEGA), other biochemical reagents are imported aliquots or domestically produced analytical pure biochemical reagents; experimental instruments include centrifuges, constant temperature water baths, constant temperature culture shakers, pipettes, etc.

[0114] The experimental method includes the following steps:

[0115] 1. Search for the sequence of the bacterial drug resistance gene NDM-1 in GenBank;

[0116] 2. Analyze the above sequence, select the appropriate sequence and the appropriate restriction site, the length of the bacterial drug resistance gene NDM-1 sequence is 813bp, with BamHI restriction sites added at both ends;

[0117] 3. Send the processed sequence to Bao Bioengineering (Dalian) Co., Ltd., who will be responsible for the artificial synthesis service of the whole gene, including the synthesis of single-strand...

Embodiment 2

[0135] Example 2 Uniformity inspection of plasmid standard molecule pXL08

[0136] Uniformity is the consistency state of the structure or composition that characterizes a set or multiple characteristics of a substance. By measuring samples of specified sizes taken from different packaging units (such as bottles, bags, etc.) or taken from different positions of the same packaging unit, and the measurement results fall within the specified uncertainty range, it can be considered that the reference material has a specified characteristic value Is uniform. Uniformity is the basic attribute of a reference material, which is used to describe the spatial distribution of the characteristics of a reference material. The uniformity evaluation must be carried out during the development (production) of the reference material to prove its good uniformity. The uniformity of the plasmid standard molecule will not be affected by factors such as aliquoting, and there is little difference in th...

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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 present invention relates to a set of plasmid molecules in the field of bioengineering technology, in particular to a set of polynucleotides, methods and kits for detecting bacterial drug resistance gene-New Delhi metal beta-lactamase-1 gene (NDM-1). Background technique [0002] In recent years, with the widespread use of antibiotics, many kinds of bacteria have shown different degrees of drug resistance. In September 2010, scholars from India, the United Kingdom, Sweden, Pakistan and Australia jointly reported a bacteria that can resist almost all antibiotics, called "super bacteria", which can produce a new metal β-lactamase. Its hydrolysis activity is much higher than the existing metal β-lactamase. Since the first case of infection was found in New Delhi, India, the enzyme is called New Delhimetallo-blactamase-1 (NewDelhimetallo-blactamase-1, NDM-1). NDM-1 has a powerful hydrolysis effect and can resist almost all antibiotics currently used, whic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/63
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
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