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Primer for amplifying gene specific area of DNA gyrase B subunit gene (gyrB) in legionella pneumophilia and application thereof

A Legionella pneumophila and gene technology, applied in the field of nucleotides, can solve the problems of difficult isolation and culture of Legionella, high price, unfavorable rapid diagnosis, etc.

Inactive Publication Date: 2011-02-09
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 gene specific area of DNA gyrase B subunit gene (gyrB) in legionella pneumophilia and application thereof
  • Primer for amplifying gene specific area of DNA gyrase B subunit gene (gyrB) in legionella pneumophilia and application thereof
  • Primer for amplifying gene specific area of DNA gyrase B subunit gene (gyrB) in legionella pneumophilia and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 : Genome Extraction

[0081] In BCYE plate medium at 37°C, 5% CO 2 Cultivate Legionella pneumophila overnight, scrape the colony 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 was rolled out with glass wool and washed with 70% ethanol, and...

Embodiment 2

[0082] Example 2 : Amplification of the gyrB gene in Legionella pneumophila by PCR

[0083]gyrB was amplified by PCR using the genome of Legionella pneumophila as a template. Since the full length of the gyrB gene is 2418, which exceeds the length that can be read by a DNA sequencer at one time, the gyrB gene is divided into three segments for cloning and sequencing in the present invention. Three pairs of primers were designed according to the conserved region of gyrB: upstream primer (5′-TAGCATAATACGGCAAGAGGG-3′), downstream primer (5′-TGGCATACCCTTCGTTTT-3′); upstream primer (5′-ATGGCTATCAAGAAACTAT-3′), downstream primer Primer (5'-TCTTCTCTTGTACCGCTT-3'); upstream primer (5'-TAAAGCGTGGAAAGCAGG-3'), downstream primer (5'-ACAGGAAGTGAACTAAGGG-3'). 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, ext...

Embodiment 3

[0084] Example 3 : Construction of clones of gyrB gene

[0085] 1. Obtaining the connection product:

[0086] The PCR purified product was mixed with Promega's 3×10 -3 The pGEM-T-Easy vector was ligated at 16°C for 24 hours, with a total volume of 10 μl, which contained 1 μl of 10×buffer and 0.5 units of T4 DNA ligase to obtain the ligation product.

[0087] 2. Preparation of competent cells:

[0088] Competent cells Escherichia coli DH5α were prepared according to the method provided by Bio-Rad. Take a single colony of Escherichia coli DH5α in 5ml of LB medium, culture it at 180rpm for 10 hours, take 2ml of the culture and transfer it to 200ml of LB medium, shake vigorously at 37°C and 250rpm to about OD6000.5, then freeze The bath was cooled for 20 minutes and centrifuged at 4000 rpm for 15 minutes at 4°C. Drain the supernatant, blow off the bacteria with 200 ml of cold ice-precooled deionized sterilized water, and centrifuge at 4000 rpm for 15 minutes at 4°C. Blow of...

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Abstract

The invention relates to a primer for amplifying the gene specific area of a DNA gyrase B subunit gene (gyrB for short) in legionella pneumophilia and provides a polymerase chain reaction (PCR) kit which comprises the primer for amplifying the gene specific area of the gyrB in the legionella pneumophilia. The PCR kit is simple, convenient and rapid and has high specificity and sensitivity when used for detecting the legionella pneumophilia, can be applied to the fields such as the supervision and detection of water bodies and clinical samples, the detection of pathogenic bacteria in drinking water, bacteriology classification, the investigation of epidemiology and the like and has good social benefit and economic benefit.

Description

technical field [0001] The present invention relates to a specific nucleotide for gyrB gene (DNA gyrase B subunit gene, hereinafter referred to as gyrB) in Legionella pneumophila (Legionella pneumophila), in particular to oligokaryon specific for gyrB in Legionella pneumophila Glycolic acid and its application. 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 closely related to humans is Legionella pneumophila. Legionella...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10C12P19/34C12R1/01
Inventor 王磊曹勃阳周光朋杨磊
Owner TIANJIN BIOCHIP TECH CO LTD
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