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Identification of pathogens

A pathogen and identification method technology, which is applied in the field of identification of pathogens infected by body fluids, to achieve the effects of reducing recovery care, accelerating treatment, and high sensitivity

Inactive Publication Date: 2009-08-05
奥地利研究中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, however, these routine diagnostic methods last at least 24 hours due to the need to enrich for microorganisms

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0095] Example 1: Sample-reference strain

[0096] All reference strains tested in this study were obtained from the American Type Culture Collection (ATCC) or the "Deutsche Sammlung für Mikroorganismen und Zellkultur" (DSMZ). In addition to reference strains, probe specificity and sensitivity were tested with clinical isolates that had been identified by classical microbiological methods. For long-term storage, store all bacterial strains in 50% glycerol stock solutions at -80°C. For most experiments a pure culture was used of a number of bacteria / ml obtained by culturing each microorganism in Caso broth overnight at 37°C and finally adjusting the concentration of microorganisms / ml using Mc Farland standard # 0.5. Microarray testing of the following bacteria: Escherichia coli (ATCC 35218, EC5, EC17, 81617, 68933, 68307), Enterobacter aerogenes (DSMZ 30053, 12676), Enterobacter cloacae (26385, 79232, 93840, 12720, 74892 ), Klebsiella pneumoniae (25809, 85813, 26385, 13253), ...

Embodiment 2

[0097] Embodiment 2: the design of oligonucleotide probe

[0098] Probe design and analysis was performed with the ARB software package (Ludwig et al., 2004). Ribosomal DNA (rDNA) sequences of selected pathogenic bacteria and yeasts were downloaded from GenBank at the NCBI homepage (www.ncbi.nlm.ni-h.gov) and uploaded to the ARB software package to create sequences comprising more than 27,000 16S The rDNA sequence also has a database of more than 7,000 18S rDNA sequences to detect possible mismatches with eukaryotic sequences.

[0099] After aligning the new sequence to a pre-existing database, the dendrogram is calculated using the neighbor joining method (see figure 1 ).

[0100]Use of probe design functions (including variable parameter settings such as probe length (20 bases), maximum non-group hits, G+C content, melting temperature, and minimum hairpin loop) , according to the results of the ARB software, probes for the species and the selected genus were designed.

...

Embodiment 3

[0121] Embodiment 3: the preparation of microarray

[0122] Oligonucleotide probes were from VBC Genomics (Austria). Five thymine residues were added at the 5' end of each oligonucleotide as a spacer. To ensure covalent attachment to reactive aldehyde groups on the microarray surface (CSS-100 Silylated Slides, Cel Associates, USA), the probes were 5' amino modified. Probes were printed onto silanized glass slides at different concentrations (50 μM, 20 μM and 10 μM in 3x SSC and 1.5 M betaine monohydrate) by a contact arrayer (Omnigrid, GeneMachines) while conditioning the air. Humidity is between 55 and 60%.

[0123] Six copies of each probe were printed per microarray. Spotting was performed with a SMP3 needle (TeleChem, USA) to obtain a spot size of 100 μm in diameter.

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Abstract

Disclosed is a method for identification of microbial pathogens in a body fluid sample comprising the following steps: a) providing a body fluid sample; b) lysing the microbial pathogens and performing a nucleic acid amplification reaction on the microbial DNA encoding 16S or 18S rRNA wherein or whereafter the amplified nucleic acids are labelled; c) contacting the labelled amplified nucleic acids of step b) with a microarray comprising on defined areas on the microarray's surface immobilised probes for microbial DNA encoding 16S or 18S rRNA from microbial pathogens; d) detecting the binding of one or more species of the labelled amplified nucleic acids to a probe by detecting a labelled amplified nucleic acid being specifically bound to the microarray; and e) identifying a microbial pathogen in the body fluid sample by correlating the detected binding of the labelled amplified nucleic acids with the defined areas of the immobilised probes for microbial DNA encoding 16S or 18S rRNA from microbial pathogens.

Description

technical field [0001] The present invention relates to the identification of pathogens of bodily fluid infections. Background technique [0002] Despite advances in the diagnosis and early treatment of bloodborne infections, mortality remains high. The traditional method of identifying microorganisms is a blood culture-based method that requires cultivation of microorganisms with subsequent morphological and physiological characterization (Peters et al., 2004). [0003] The frequency of human pathogens is periodically monitored by various scientists and several clinical research programs. show that more than 95% of all bloodstream infections are caused by only 15 different genera. Staphylococcus sp. and Escherichia sp. account for more than 50% of infections. Diversity studies vary only slightly across countries and laboratories. While overall pathogen infection rates were stable over time, in particular Pseudomonas aeruginosa infection increased significantly and repre...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G16B25/00
CPCC12Q1/6837C12Q1/6895G06F19/20C12Q1/689C12Q2600/16G16B25/00C12Q1/6844G16B25/10G16B25/30
Inventor H·维辛格梅尔R·皮希勒L·博罗赛C·内哈默尔
Owner 奥地利研究中心有限公司
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