Nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof

A technology of Legionella pneumophila and nucleotides, which is used in the determination/inspection of microorganisms, resistance to vector-borne diseases, DNA/RNA fragments, etc. , time-consuming and other problems, to achieve the effect of broad market application prospects, fast speed, and simplified operation methods

Inactive Publication Date: 2012-01-11
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In addition, there are many types of Legionella, and conventional bacterial culture and serum identification and typing are time-consuming, heavy workload, and low-throughput, which cannot meet the needs of epidemiological investigations and rapid analysis of sudden pathogenic events; Type method Because MAbs typing requires special technology, expensive instruments and equipment can only be used in specific laboratories

Method used

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  • Nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof
  • Nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof
  • Nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 : Genome Extraction

[0035] In BCYE plate medium at 37°C, 5% CO 2 Cultivate Legionella pneumophila type 1 strain overnight, scrape colonies with 50mM Tris-HCl (pH8.0), and collect bacteria by centrifugation. The cells were resuspended with 250ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, and 10ul 10mg / ml lysozyme was added to continue 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 20 minutes. 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 chloroform:isoamyl alcohol (49:1). 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 finally the DNA was resuspended in 30ul TE. Genomic DNA was detected by 0.4% agarose gel electrophoresis.

Embodiment 2

[0036] Example 2 : Screening of specific primers

[0037] Design primers for the specific region of the wzt gene of Legionella pneumophila type 1; use this pair of primers (upstream primer is 5'-GTACTACTACAACCGCAGCAGA-3', see sequence SEQ ID NO: 1; downstream primer is 5'-CGAGTGATATCCATGACTGAAC-3', See sequence SEQ ID NO: 2). Take 1 standard strain of Legionella pneumophila type 1, 12 standard strains of other serotypes of Legionella pneumophila, 7 other strains of Legionella, 1 clinical isolate of Legionella pneumophila type 1, and 7 strains of Legionella Other serotype clinical isolates and 6 closely related bacteria were used as templates for PCR. The system was 0.4ul of 5uM primers, 2.5ul of 10×buffer, 0.25ul of 10mM dNTP, 0.2ul of 5U / ul Taq polymerase and 3ul of the sample template to be tested. 0.2ml thin-walled PCR tube, and finally with ddH 2 O make up to 25ul. All primers gave positive results in Legionella pneumophila type 1, and did not get any PCR product band...

Embodiment 3

[0038] Example 3 : PCR detection kit

[0039] 1. Preparation of materials and equipment required for testing experiments

[0040] 1. Kit composition:

[0041] 1) dNTP (10mM) 30ul;

[0042] 2) 10×buffer (10×enzyme-specific reaction buffer) 50ul;

[0043] 3) Taq polymerase (5U / ul) 5ul;

[0044] 4) Primer mixture (5uM) 10ul;

[0045] 5) Positive control substance (KP) 10ul;

[0046] 6) Negative control substance (KN) 10ul;

[0047] 7)ddH 2 O 5ml.

[0048] Each kit can be used to detect 10 samples.

[0049] Among them, 10×buffer, dNTP, and Taq polymerase were provided by Treasure Bioengineering (Dalian) Co., Ltd.; the primer mixture was a self-designed sequence provided to Shanghai Yingjun Biotechnology Co., Ltd. for synthesis; positive control substance, negative control substance and ddH 2 O is prepared by ourselves.

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Abstract

The invention relates to a nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof. The nucleotide is a nucleotide shown as SEQ ID NO: 1 and/or a nucleotide shown as SEQ ID NO: 2 and is complementary with the nucleotides. The nucleotides can be used for preparing a PCR (Polymerase Chain Reaction) kit for detecting Legionella pneumophila serogroup 1, a gene chip or a micro-array. The nucleotide specific to the wzt of the Legionella pneumophila serogroup 1 provided by the invention as well as the PCR kit, the gene chip or the micro-array including the nucleotide have the advantages of strong practical applicability, simple preparation method of the PCR kit, short detection period, high speed, strong maneuverability, easiness for industrial production, low detection cost, high accuracy and high sensitivity.

Description

technical field [0001] The present invention relates to a nucleotide and its application, in particular to a wzt (ABC transporter of LPS O-antigen for Legionella pneumophila serogroup 1), ABC transporter of LPS O-antigen, hereinafter Referred to as wzt) specific nucleotides and their applications. Background technique [0002] Legionella (legionella) is a Gram-negative bacillus that widely exists in water bodies and soils in natural and artificial environments. Febrile pulmonary disease - Legionnaires' disease. 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. The growth and reproduction of Legionella is closely related to environmental factors. The cooling water, condensate water and hot spring water of the cooling tower of the central air conditioner are the suitable living environment for Legionella in the outside world. When the Legionella in the en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 曹勃阳周光朋朱之燕王磊冯露
Owner NANKAI UNIV
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