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Genotyping chip for legionella pneumophila, and kit for detection of legionella pneumophila

A Legionella pneumophila and gene chip technology, applied in the field of gene chips and detection kits containing the chips, can solve the problems of inability to distinguish species, low resolution ability, inability to distinguish Escherichia coli and Shigella, etc.

Active Publication Date: 2013-06-26
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high conservation of 16S rRNA, this technology has low resolution ability, and cannot distinguish species, nor can it distinguish Escherichia coli and Shigella

Method used

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  • Genotyping chip for legionella pneumophila, and kit for detection of legionella pneumophila
  • Genotyping chip for legionella pneumophila, and kit for detection of legionella pneumophila
  • Genotyping chip for legionella pneumophila, and kit for detection of legionella pneumophila

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Probe design and preparation

[0093] The sequence gets:

[0094] (1) Legionella pneumophila O1 wx Acquisition of the gene: The gene of Legionella pneumophila O1 was downloaded from the GenBank public database wx gene sequence;

[0095] (2) Legionella pneumophila O4, O12, O13, O15 wxya The gene sequence was deciphered by our laboratory;

[0096] (3) Legionella pneumophila O2, O3, O5, O6, O7 wx The gene was deciphered by our laboratory;

[0097] (4) Legionella pneumophila O11 wec Gene A was deciphered by our laboratory;

[0098] (5) Acquisition of 16S rRNA gene sequence: The entire 16S rRNA gene sequence of Legionella pneumophila and its close relatives were downloaded from the GenBank public database.

[0099] 2. Example of probe design:

[0100] Probe for Legionella pneumophila O1: The Legionella pneumophila O1 wx The gene sequence was imported into the Glustal X software, and a representative sequence was selected for Blastn comparison...

Embodiment 2

[0109] Example 2 Primer design and preparation

[0110] 1. Example of primer design:

[0111] (1) All the Legionella pneumophila O1 wx Import the gene sequence into the Glustal X software, select a representative sequence and import it into the Primer Primer 5.0 software, set the length to 70bp-10bp, G+C% value 40%-60%, Hairpin: NONE, Dimer: NONE, False Priming: NONE, Cross Dimer: NONE. And look for a nucleotide sequence region suitable for general primer design, its characteristics basically meet the following conditions: a), the conserved region should include the Legionella pneumophila O1 wx ; b), this region should contain a variable region that is easy to design specific probes, ensuring that the difference in nucleotides between the probes is greater than 4 or more; c), this region is flanked by conserved regions to meet the design of primers; d), the amplified product of the designed primers should not be too large, otherwise the sensitivity of PCR will be af...

Embodiment 3

[0129] Specific identification and sensitivity detection of a genotyping chip for the detection of an important waterborne pathogen Legionella pneumophila

[0130] 1. The specificity experiment of the gene chip of the present invention:

[0131] Specificity means that a probe can only hybridize with the target gene of its corresponding bacterial species and cannot hybridize with gene fragments of other bacterial species. This requires us not only to carry out a large number of bioinformatics analysis, but the probes analyzed by bioinformatics will also have non-specific results or unstable hybridization signals. Therefore, probe-specific screening still needs a large number of experiments to verify. In particular, some probes used for high-throughput detection must be verified by hybridization with a large number of close and distant bacteria.

[0132] Using 49 strains of Legionella collected, including 39 standard strains, including standard strains of 15 serotypes of Leg...

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Abstract

The present invention provides a genotyping chip for legionella pneumophila, and a kit for detection of the legionella pneumophila. The chip and the kit are mainly provided according to the 11 serotypes of the legionella pneumophila, wherein the 11 serotypes comprise O1, O2, O3, O4, O5, O6, O7, O11, O12, O13 and O15. The gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise DNA fragments selected from wzm gene, wzt gene and wecA gene, or complementary DNA fragments of the wzm gene, the wzt gene and the wecA gene, wherein the wzm gene, the wzt gene and the wecA gene have significant biological evolution advantages. According to the present invention, the genomic DNA of the sample requiring detection is amplified by the designed primers; the resulting amplified genomic DNA is labeled; the resulting labeled genomic DNA is subjected to hybridization with the gene chip; according to the resulting hybridization signals, the different serotypes of the legionella pneumophila can be detected. With the gene chip of the present invention, the purpose of the detection of the serotypes of the legionella pneumophila can be achieved, the operation is convenient, the accuracy is high, the repeatability is strong, and the accurate medical medication guidance is provided.

Description

technical field [0001] The invention relates to a gene chip and a detection kit containing the chip, in particular to Legionella pneumophila O1, O2, O3, O4, O5, O6, O7, O11, O12, O13 and O15, which are important pathogens in water bodies A total of 11 serotype gene chips and detection kits. Background technique [0002] Water is a necessary condition for the existence of life. It enables us to multiply and thrive. Human life, production and entertainment cannot do without water. It is also a place and carrier for many pathogenic microorganisms to breed and spread. Once these pathogenic microorganisms enter the human body, they may cause illness or even death, seriously threatening human health. With the development of society and the improvement of living standards, people are more and more concerned about their own health issues, and the safety of various water bodies (including drinking water, rivers and lakes, swimming pools, etc.) has increasingly become a hot spot of c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C40B40/06C12R1/01
Inventor 王磊曹勃阳姚芳芳冯露周光朋
Owner NANKAI UNIV
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