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Assays and kits for serotyping pseudomonas aeruginosa and oligonucleotide sequences useful in such methods and kits

A technology of Pseudomonas aeruginosa and oligonucleotides, which is applied in the field of oligonucleotides and Pseudomonas aeruginosa serotype-specific antibodies, and can solve problems such as high difficulty and inability to achieve detection accuracy.

Inactive Publication Date: 2013-02-27
KENTA BIOTECH AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Simultaneous detection of several Pseudomonas aeruginosa serotypes (especially IATS-O1, IATS-O6, IATS-O11 and serogroup 2) in a single reaction is technically difficult due to the Primer pairs and probe pairs can interact with each other, often resulting in inability to achieve clinically required detection accuracy

Method used

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  • Assays and kits for serotyping pseudomonas aeruginosa and oligonucleotide sequences useful in such methods and kits
  • Assays and kits for serotyping pseudomonas aeruginosa and oligonucleotide sequences useful in such methods and kits
  • Assays and kits for serotyping pseudomonas aeruginosa and oligonucleotide sequences useful in such methods and kits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1: Microbiological samples

[0142] To improve the 4-valent Pseudomonas aeruginosa serotyping test, 16 reference strains and 94 clinical isolates were used. Reference strains were obtained from Institut Pasteur (Paris, France) or Belgium bacteria collection BCCM (Ghent, Belgium). Clinical isolates were obtained from university hospitals in Basel, Bern, Zurich, Switzerland or Jena, Germany, and from multi-national clinical studies on cystic fibrosis.

[0143] To compare a modified 4-valent Pseudomonas aeruginosa serotyping test with a commercial agglutination test, nearly five hundred respiratory Pseudomonas aeruginosa isolates collected from approximately 20 hospitals in the United States, Germany, Greece, and Belgium were used. things.

Embodiment 2

[0144] Embodiment 2: sample preparation

[0145] 2.1 Measurement of purified bacterial DNA

[0146] Each reference strain and clinical isolate was cultured overnight at 37°C in 5 ml of brain heart infusion medium BHI (from BD Bioscience). Bacterial DNA was isolated from bacterial cell pellets using a high-purity PCR template preparation kit (from Roche Diagnostics). DNA stocks of all DNA samples were prepared as 20 ng / μl standards prior to use in PCR. The DNA stock solution was serially diluted 1:10 with ultrapure water (from Roche Diagnostics) as a working solution. Take 5 μl of each DNA working solution as template solution.

[0147] 2.2 Measurements from bacterial cultures

[0148] Reference strains and clinical isolates were plated on BHI agar plates (from BD Bioscience). After overnight incubation at 37°C, one loop of bacteria was dissolved in 500 μl of ultrapure water (from Roche Diagnostics). Take 8 μl of each bacterial dilution as template solution.

[0149] 2.3...

Embodiment 3

[0151] Example 3: Primers and assay design

[0152] 3.1 Screening of target genes

[0153] Table 3: Target genes used to design serotype-specific primer / probe pairs

[0154]

[0155] 3.2 Primer and probe sequence design

[0156] Primer / probe oligonucleotides were designed with the software LightCycler Probe Design Software 2.0 (from Roche Applied Science). Based on the software design, serogroup 2-specific probes were optimized by obtaining spacing between probe pairs, and serotype O11-specific probes were improved by shortening and perturbing sensing probes.

[0157] Table 4: Quadrivalent Pseudomonas aeruginosa serotyping assay - primer and probe sequences

[0158] In order to specifically detect one serotype in a test sample, two sets of oligonucleotide sequences are used: 2 primers and 2 probes. To specifically detect, for example, the 4 most prevalent Pseudomonas aeruginosa serotypes mentioned above, multiple primers and probes were used in one experiment.

[0159]...

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Abstract

The present invention relates to assays, kits and oligonucleotides for the detection of Pseudomonas aeruginosa for a fast, sensitive and reliable detection of Pseudomonas aeruginosa in a species- and serotype-specific manner. In particular, the present invention provides an assay for the serotype-specific detection of Pseudomonas aeruginosa, a kit for the serotype-specific detection of Pseudomonas aeruginosa, as well as oligonucleotides useful in such assay or kit. The present invention further relates to the use of Pseudomonas aeruginosa serotype specific antibodies for serotype specific treatment of Pseudomonas aeruginosa infection in a patient detected for said specific Pseudomonas aeruginosa serotype with such an assay or kit.

Description

technical field [0001] The invention relates to a method for detecting the serotype specificity of Pseudomonas aeruginosa, a kit for the serotype specificity of Pseudomonas aeruginosa, and the oligonucleotide used in the method or the kit. The present invention further relates to Pseudomonas aeruginosa serotype-specific antibodies for use in the serotype-specific treatment of Pseudomonas aeruginosa infections in patients suffering from a disease caused by a particular Pseudomonas aeruginosa serotype. Background of the invention [0002] Pseudomonas aeruginosa is a ubiquitous Gram-negative environmental bacterium found in water and soil. It is a typical opportunistic pathogen that usually does not pose a threat to an immunocompetent host, which clears it through antibody regulation and phagocytosis. However, patients with cystic fibrosis and immunocompromised individuals—including burn patients, those who are intubated in the intensive care unit, cancer and AIDS patients, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/16C12Q1/689C12Q1/6844C12R2001/38
Inventor 托马斯·艾默里奇米歇尔·鲁道夫霍尔格·科赫
Owner KENTA BIOTECH AG
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