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Specific primers and kit for detecting various serotype pathogenic bacteria of legionella pneumophilia

A specific technology for Legionella pneumophila, applied in the field of multiplex PCR rapid detection, can solve the problems of unstandardized methods, poor stability of monoclonal antibody typing, and easy to be affected by environmental factors, etc., achieving simple method and promising market application wide, saving manpower and material resources

Active Publication Date: 2011-08-03
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, many molecular typing methods are used in the research of Legionella typing, but most of the methods are not standardized. The disadvantage of traditional serological typing identification is that it is easily affected by environmental factors and may exist between other bacteria species. Cross-reaction occurs due to the common antigen, and the monoclonal antibody typing developed from this also has the defect of poor stability

Method used

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  • Specific primers and kit for detecting various serotype pathogenic bacteria of legionella pneumophilia
  • Specific primers and kit for detecting various serotype pathogenic bacteria of legionella pneumophilia
  • Specific primers and kit for detecting various serotype pathogenic bacteria of legionella pneumophilia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 : Genome Extraction

[0042] 1) Take a little bacterial solution from the strain preservation tube and inoculate it on the Legionella BCYE growth plate; 2.5% CO 2 , cultured at 37°C for 5-7 days.

[0043] 2) Add 1mL of 50mM Tris-HCl (pH8.0) to the plate, scrape the bacteria with a sterilized coating rod, take an appropriate amount of bacteria solution into a 1.5mL centrifuge tube, centrifuge at 10000rpm for 5 minutes to accumulate bacteria, and remove the supernatant.

[0044] 3) Add 250 μL of 50 mM Tris-HCl (pH8.0) to resuspend, add 10 μL of 0.5M EDTA (pH8.0), and mix thoroughly.

[0045] 4) Add 15 μL of 20 mg / mL lysozyme, mix thoroughly, and incubate at 37°C for 20 minutes.

[0046] 5) Add 3 μL 20mg / mL proteinase K, and mix gently.

[0047] 6) Add 20 μL of 10% SDS, and bathe in water at 50° C. for 1 hour until the solution is clear.

[0048] 7) Add 2 μL of 25mg / mL RNAase, and bathe in water at 65°C for 20 minutes.

[0049] 8) Add an equal volume of ph...

Embodiment 2

[0054] Embodiment 2: the design of primer

[0055] The sequence downloaded from NCBI, combined with the sequence tested by the laboratory, designed primers for the specific regions of the wzt gene of Legionella pneumophila types 1 and 6, and the wzm gene of Legionella pneumophila types 4, 10 and 13. The primer sequences are as follows 1 shows:

[0056] Table 1 The specific primer sequences of Legionella pneumophila serotypes 1, 4, 6, 10 and 13

[0057]

Embodiment 3

[0058] Example 3 : Screening of specific primers

[0059] For the wzt gene of Legionella pneumophila type 1 and 6, the specific region design primers of the wzm gene of Legionella pneumophila 4, 10 and 13 types, the primer sequences are as shown in table 1 (SEQ ID NO: 1-SEQ ID NO: 10). Collected 1 standard strain of Legionella pneumophila type 1, 14 standard strains of other serotypes of Legionella pneumophila, 7 strains of other legionella genus, 1 clinical isolate of Legionella pneumophila type 1, 4 strains of Legionella The specificity of the primers was verified for clinical isolates belonging to other serotypes and 6 closely related bacteria. The strain numbers and sources are shown in Table 2 below.

[0060] Table 2 Standard strains for testing

[0061]

[0062]

[0063]

[0064] The clinical strains used in this patent are shown in Table 3 below:

[0065] Table 3 The biochemical test results of the tested clinical strains

[0066]

[0067] The PCR syst...

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Abstract

The invention relates to specific polymerase chain reaction (PCR) primers for detecting various serotype pathogenic bacteria of legionella pneumophilia, a method for quickly detecting by adopting a multi-PCR kit, and application. The kit comprises 10*PCR reaction liquid, MgCl2, dNTP, primers, and DNA polymerase; wherein the sequences of the primers is one or both of (1) and (2), wherein (1) wzt or wzm specific nucleotide sequences of legionella pneumophila serogroups 1, 4, 6, 10 and 13; and (2) complementary DNA sequences of DNA sequences selected from (1). The primers have high practicability for wzt specific nucleotide of common legionella pneumophila serogroups, and a PCR kit, a gene chip or a microarray comprising the nucleotide; and the PCR kit is easy and convenient to prepare, short in detection period, high in speed and accuracy, and strong in operability, detection cost is reduced, and the PCR kit is suitable for industrial production.

Description

technical field [0001] The present invention relates to a kind of method and application thereof of multiplex PCR fast detection, relate in particular to a kind of method that can be used to detect Legionella pneumophila serotype 1 type (Legionella pneumophila serogroup 1), 4 types (Legionella pneumophila serogroup 4), 6 types ( Legionella pneumophila serogroup 6), type 10 (Legionella pneumophila serogroup 10) and type 13 (Legionella pneumophila serogroup 13) five pathogenic bacteria PCR techniques and the sequences of PCR primers used. Background technique [0002] Legionella (Legionella) was first discovered in the "Philadelphia Legionnaires' Disease Incident" in 1976, hence the name. Legionella is a facultative intracellular parasite, which widely exists in nature and artificial water bodies and soil environments. Inhalation of Legionella-contaminated aerosols, drinking pollution or wound contact with contaminated water may lead to Legionella infection , the acute febril...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 王磊周光朋姚芳芳曹勃阳冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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