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Nucleotide specific to legionella pneumophila O12 type wzm and wecA genes and application of nucleotide

A technology of Legionella pneumophila and nucleotides, which is applied in recombinant DNA technology, microbial determination/inspection, resistance to vector-borne diseases, etc. Achieve the effect of wide market application prospect, high detection accuracy and simplified operation process

Active Publication Date: 2017-07-07
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the genotyping of 15 known serotypes of Legionella pneumophila, O12 serotype and O15 serotype are more difficult to identify than other serotypes. At present, there is no method that can quickly distinguish O12 and O15 serotypes report

Method used

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  • Nucleotide specific to legionella pneumophila O12 type wzm and wecA genes and application of nucleotide
  • Nucleotide specific to legionella pneumophila O12 type wzm and wecA genes and application of nucleotide
  • Nucleotide specific to legionella pneumophila O12 type wzm and wecA genes and application of nucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Genome Extraction

[0032] (1) In the ultra-clean workbench, draw a small amount of bacterial liquid from the preservation tube of the standard strain (see Table 2), and inoculate it onto the growth plate of Legionella BCYE (Qingdao Hi-tech Industrial Park Haibo Biotechnology Co., Ltd.), 37°C, 5.0% CO 2 Culture for 5-7 days.

[0033] (2) Add 2 mL of sterile water to the BCYE growth plate, gently scrape up the bacterial lawn with a sterile round glass rod, then suck the bacterial suspension into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 5 minutes, discard the supernatant , repeat the wash once.

[0034] (3) Add 250 μL of 50 mM Tris-HCl buffer (pH 8.0) to resuspend, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant.

[0035] (4) Add 250 μL of 50 mM Tris-HCl buffer (pH 8.0) to resuspend, add 10 μL of 0.45M EDTA (pH 8.0), fully suspend, and incubate at 37°C for 20 minutes.

[0036] (5) Add 10 μL of 20 mg / mL lysozyme and incubate at 37°C for ...

Embodiment 2

[0048] Primer design

[0049] Primer design is the key to the present invention. The O-antigen gene cluster sequence of Legionella pneumophila serotype O12 was self-tested by our laboratory. Through comparative analysis, the selected wxya and wecA As the specific target gene of this bacterium, it targets the O12 type of Legionella pneumophila wxya and wecA Design primers for the specific region of the gene; as shown in Table 1:

[0050] wxya The upstream primer of the gene is 5'- GGGGATATTCAACCGTTA -3', see sequence SEQ ID NO:1; the downstream primer is 5'-TAAATCCTATTACAAATATAGC -3', see sequence SEQ ID NO:2.

[0051] wecA The upstream primer of the gene is 5'-ATTATATCATCATCCCTTTT-3', see sequence SEQID NO:3; the downstream primer is 5'-CTGTGTCCAACTCAATAAT-3', see sequence SEQID NO:4;

[0052] Table 1 The specific primer sequences of Legionella pneumophila serotype O9

[0053]

Embodiment 3

[0055] Screening of specific primers

[0056] One standard strain of Legionella pneumophila type O12 and 14 standard strains of other serotypes of Legionella pneumophila were collected. The strain numbers and sources are shown in Table 2 below.

[0057] Table 2 Standard strains for testing

[0058]

[0059]

[0060] The PCR system is divided into two parts, the PCR system of each part is 0.4 μL of 10 μM primer, 2.5 μL of 10×buffer, 0.25 μL of 10mM dNTP, 0.2 μL of 5 U / μL Taq polymerase and 3 μL of the sample template to be tested to 0.2 mL thin-walled PCR tubes, and finally with ddH 2 O to make up to 25 μL.

[0061] Used primer SEQ ID NO:1 and / or SEQ ID NO:2 obtains positive result in the template of Legionella pneumophila O12 type and O15 type, does not obtain any PCR product band in other groups; SEQ ID NO:3 and / or Or SEQ ID NO: 4 has no PCR product band in the O15 type, but a positive result is obtained in the O12 type. So these oligonucleotide fragments are highly...

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Abstract

The invention relates to a nucleotide specific to legionella pneumophila O12 type wzm and wecA genes and an application of the nucleotide. The nucleotide is a nucleotide shown as in SEQ ID NO:1 and / or SEQ ID NO:2, a nucleotide shown as in SEQ ID NO:3 and / or SEQ ID NO:4, and a nucleotide complementary to the above nucleotides. The nucleotides are used for preparing a PCR (Polymerase Chain Reaction) kit, a gene chip or a microarray which are used for detecting the legionella pneumophila O12 type. The practicability of the nucleotide specific to the legionella pneumophila O12 type wzm and wecA genes and the practicability of the PCR kit, the gene chip and the microarray comprising the nucleotide are strong, the preparation method of the PCR kit is simple, the detection period is short, the detection is fast, the maneuverability is strong, the commercialized production is easy to realize, the detection cost is relatively low, and the accuracy and the sensitivity are high.

Description

technical field [0001] The present invention relates to a kind of nucleotide and its application, especially a kind of nucleotide for Legionella pneumophila serogroup O12 (Legionella pneumophila serogroup 12) wxya Gene (ABC transporter of LPS O-antigen, ABCtransporter of LPS O-antigen, hereinafter referred to as wxya Gene ) and wecA Gene (UDP-GleNAe: Und-PGleNAe-l-P transferase, involved in the biosynthesis of O antigen) specific nucleotides and their applications, through which specific nucleotides can accurately identify Legionella pneumophila O12 and Other serotypes of Legionella pneumophila were identified. Background technique [0002] Legionella gets its name from the "mystery disease" that broke out in July 1976, infecting 221 people and killing 34. The first outbreak was detected among people attending an American Legion meeting in Philadelphia for the U.S. Bicentennial celebration. Six months later, the causative agent was identified as a previously unknown ba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/689Y02A50/30
Inventor 王磊任薇杨双席道义曹博阳冯露
Owner NANKAI UNIV
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