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Primer for amplifying specific regions of common pathogenic type Legionella gyrB genes and application thereof

A technology of Legionella and pathogenic type, applied in the field of Legionella Mikdide, can solve the problems of high price, time-consuming and laborious, unfavorable rapid diagnosis and so on

Active Publication Date: 2011-08-31
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because Legionella is not easy to isolate and culture, and there are many types
The use of bacteriological culture method to identify it requires expensive special medium, and the growth of bacteria is relatively slow, and it takes more than 3-7 days to produce results, which is not conducive to rapid clinical diagnosis. At the same time, laboratory personnel are required to have relatively high technology. Time-consuming

Method used

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  • Primer for amplifying specific regions of common pathogenic type Legionella gyrB genes and application thereof
  • Primer for amplifying specific regions of common pathogenic type Legionella gyrB genes and application thereof
  • Primer for amplifying specific regions of common pathogenic type Legionella gyrB genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 : Genome Extraction

[0080] In BCYE plate medium at 37°C, 5% CO 2 Culture Legionella Mickel-Daider, Legionella bozemannii and Legionella longbeach respectively overnight, scrape the colonies with 50mM Tris-HCl (pH8.0), and collect the bacteria by centrifugation. The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg / ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul 20mg / ml proteinase K, 15ul 10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg / ml RNase, and incubate at 65°C for 30 minutes. Add an equal volume of phenol to extract the mixture, take the supernatant, and then extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether. Extract to remove residual phenol. The supernatant was used to precipitate DNA with 2 times volume of ethanol, and the DNA wa...

Embodiment 2

[0081] Example 2 : Amplification of the gyrB gene from Legionella meek-daid, Legionella bozemannii, Legionella longbeach by PCR

[0082] gyrB was amplified by PCR using the genomes of Legionella mikk-Dade, Legionella bozemannii and Legionella longbeach as templates. In the present invention, degenerate primers are designed according to the conserved region of gyrB, and the sequence of amplifying its gyrB degenerate primers is: upstream primer (5'-WCVGGTYTGCAYCAYATG'), downstream primer (5'-GTCTGBGAKGARAAYTTVGG'); The PCR reaction program is as follows: pre-denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 1 minute, thus performing 30 cycles; finally, extending at 72°C for 5 minutes to obtain PCR The size and specificity of PCR products were detected by 0.8% agarose gel electrophoresis. Combine 2 tubes of PCR products, and use the UNIQ-10 Column DNA Gel Recovery Kit from Shanghai Sangon B...

Embodiment 3

[0083] Example 3 : Sequencing and splicing of gyrB gene

[0084] The gel-cut and purified fragments were bidirectionally sequenced with an ABI377 automatic DNA sequencer, and the Staden Package was used for sequence splicing to obtain the sequence of the entire gyrB gene (SEQ ID NO: 1-3).

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Abstract

The invention relates to a primer for respectively amplifying specific regions of gyrB genes (DNAgyrase B subunit gene, hereinafter referred to as gyrB) in Legionella micdadei, Legionella bozemanii and Legionella longbeachae, but also provides a PCR (Polymerase Chain Reaction) reagent kit comprising the primer for amplifying the specific regions of gyrB genes in Legionella micdadei, Legionella bozemanii and Legionella longbeachae. The PCR reagent kit for detecting Legionella micdadei, Legionella bozemanii and Legionella longbeachae is simple, convenient and quick and has good specificity and high sensitivity, can be applied to the fields of supervision and detection of water bodies and clinic samples, detection of pathogenic bacteria in drinking water, bacteriology classification, epidemiology survey and the like and has profound and lasting social benefit and great economic benefit.

Description

technical field [0001] The present invention relates to specific nucleotides of gyrB gene (DNA gyrase B subunitgene, hereinafter referred to as gyrB) in Legionella micdadei; Legionella bozemanii; Legionella longbeachae , especially relate to oligonucleotides specific to gyrB in Legionella mikk-Dade, Legionella bozemannii and Legionella longbeach and applications thereof. Background technique [0002] Legionella is a Gram-negative bacillus and is an opportunistic pathogen widely present in soil and water environments in nature. Legionella can cause Legionnaires' disease, which is a bacterial infectious disease with symptoms of respiratory tract infection and systemic poisoning symptoms. Since Legionnaires' disease was first discovered in the United States in 1976, the disease has shown two characteristics of worldwide distribution and high fatality rate. There are 42 species and 64 serotypes of Legionella, at least 22 of which are related to human diseases, and the most clo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 王磊周光朋曹勃阳窦岩刘衍伟
Owner NANKAI UNIV
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