Genetic typing chip of 10 common pathogenic legionella and detection kit

A gene chip and Legionella technology, applied in the field of gene chips and detection kits containing the chip, can solve the problems of low resolution, inability to distinguish species, inability to distinguish Escherichia coli and Shigella, etc., and achieve repeatability Strong, accurate and easy-to-use effects

Active Publication Date: 2013-06-19
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 abili...

Method used

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  • Genetic typing chip of 10 common pathogenic legionella and detection kit
  • Genetic typing chip of 10 common pathogenic legionella and detection kit
  • Genetic typing chip of 10 common pathogenic legionella and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Design and preparation of probes

[0087] 1. Sequence acquisition:

[0088] (1) Obtaining the ITS sequence of Legionella longbeach: the ITS sequence of Legionella longbeach was downloaded from the GenBank public database;

[0089] (2) Legionella pneumophila, Legionella anserii, Legionella bozemannii, Legionella Dumov, Legionella feifei, Legionella golemannii, Legionella jordan river, Legionella mirini, Legionella mei The ITS sequence of was deciphered by our laboratory;

[0090] 2. Example of probe design:

[0091] Probes for Legionella anseri: Import the ITS sequence of Legionella anseri into Glustal X software, select a representative sequence and perform Blastn comparison in the public data NCBI to determine whether it can be used as a specific target and the position of the specific target. Import the sequences into OligoArray 2.0 software. Run the program to design probes online.

[0092] 3. Probe synthesis: Extend the 5' end of the probe sequence in...

Embodiment 2

[0099] Example 2 Design and preparation of primers

[0100] 1. Example of primer design:

[0101] Because the 16s and 23s sequences are conserved among different Legionella species, we can ensure that all Legionella can be amplified by designing universal primers at the end of 16s and the beginning of 23s. Taking upstream primers as an example, first put the 16s sequences of different bacteria into MEGA software for analysis, and select the most versatile fragments to import into Primer Primer 5.0 software, set the length to 70bp-10bp, and G+C% value to 40%-60 %, Hairpin: NONE, Dimer: NONE, False Priming: NONE, Cross Dimer: NONE.

[0102] The design method of the downstream primer is the same as that of the above-mentioned probe primer, and the design parameters used are also the same.

[0103] 2. Primer synthesis: commission the primer sequences in Table 2 to be synthesized (PAGE purified) by Probe Synthesis Co., Ltd.

[0104] SEQ ID serial number Sequence (5...

Embodiment 3

[0106] Specific identification of a genotyping array for the detection of important pathogenic Legionella species

[0107] The specificity experiment of gene chip of the present invention:

[0108] 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.

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Abstract

The invention provides a genetic typing chip and a detection kit of 10 common pathogenic legionella (legionella pneumophila, legionellaanisa, legionella bozemanii, legionella dumoffii, legionella fairfieldensis, legionella gormanii, legionellajordanis, legionella longbeachae, legionella maceachernii, legionella maceachernii and the like). The genetic chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier. The oligonucleotide probe comprises a DNA (deoxyribonucleic acid) fragment or a complementary DNA fragment selected on a 16s-23s intermediate zone sequence (ITS). Genomic DNA of a sample to be detected is amplified and labeled by a designed primer and is hybridized by the genetic chip, and legionella in different types can be detected according to hybridizing signals. The genetic chip can detect common pathogenic legionella and is simple and convenient to operate, high in accuracy and strong in repeatability, and guide medical administration more accurately.

Description

technical field [0001] The invention relates to a gene chip and a detection kit containing the chip, in particular to Legionella pneumophila, Legionella anseri, Legionella bozimannii, Legionella dumov, Legionella feifei, Gormans Gene chips and detection kits for 10 species of Legionella, including Legionella, Legionella Jordan River, Legionella longbeach, Legionella micheni, and Legionella mei. 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C40B40/06
CPCY02A50/30
Inventor 王磊曹勃阳刘向前王敏冯露
Owner NANKAI UNIV
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