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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: 2013-03-06
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 : Extraction of the genome

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

[0043] 2) Add 1 mL of 50 mM Tris-HCl (pH 8.0) to the plate, scrape the bacteria with a sterilized coating rod, take an appropriate amount of bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm 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 well.

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

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

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

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

[0049] 8) Add an equal volume of phenol:...

Embodiment 2

[0054] Example 2: Design of primers

[0055] The sequences downloaded from NCBI, combined with laboratory self-test sequences, were designed for the specific regions of the wzt genes of Legionella pneumophila types 1 and 6, and the wzm genes of Legionella pneumophila types 4, 10 and 13. The primer sequences are as follows: 1 shows:

[0056] Table 1 Specific primer sequences for Legionella pneumophila serotypes 1, 4, 6, 10 and 13

[0057]

Embodiment 3

[0058] Example 3 : Screening of specific primers

[0059] Primers were designed for the specific regions of the wzt genes of Legionella pneumophila types 1 and 6 and the wzm genes of Legionella pneumophila types 4, 10 and 13. The primer sequences are 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 other strains of Legionella, 1 clinical isolate of Legionella pneumophila type 1, 4 Legionella The specificity of the primers was verified for clinical isolates of 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 Biochemical test results of tested clinical strains

[0066]

[0067] The PCR system is 0.3μl of 10uM prim...

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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 multiplex PCR rapid detection method and application thereof, in particular to a method that can be simultaneously used for detecting Legionella pneumophila serogroup 1, 4 (Legionellapneumophila serogroup 4), 6 ( The PCR technology of five pathogenic bacteria of Legionella pneumophila serogroup 6), 10 type (Legionella pneumophila serogroup 10) and 13 type (Legionella pneumophila serogroup13) and the sequences of PCR primers used. Background technique [0002] Legionella was first discovered in the "Philadelphia Legionella Incident" in 1976, from which it got its name. Legionella is a facultative intracellular parasite that widely exists in nature and artificial water and soil environments. Inhalation of Legionella-contaminated aerosols, drinking contamination or wound contact with polluted water, etc., may lead to Legionella infection. , Acute febrile pulmonary disease caused by Legionella - Legionnaires' disease is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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