Detection, identification and differentiation of eubacterial taxa using a hybridization assay

a technology of eubacterial taxa and hybridization assay, which is applied in the field of detection, identification and differentiation of eubacterial taxa using a hybridization assay, can solve the problems of difficult control of this organism in the hospital, and achieve the effect of rapid and reliable hybridization

Inactive Publication Date: 2006-06-01
INNOGENETICS NV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019] Another object of the present invention is a rapid and reliable hybridization method for detection and / or identification of Staphylococcus species, in particular of Staphylococcus aureus.

Problems solved by technology

Its propensity to establish prolonged carriage among hospitalized patients and increasing resistance to antibiotics makes control of this organism within the hospital very difficult.

Method used

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  • Detection, identification and differentiation of eubacterial taxa using a hybridization assay

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of the Samples to be Tested

[0208] 1 / . DNA from Pure Cultures

[0209] For extracting the DNA from pure cultures, different purification methods can be used: [0210] Lysis with lysostaphin (5 μg / μl) for 1 h at 37° C. and purification with the QIAamp blood DNA isolation kit (Qiagen) [0211] The method of Pitcher et al. (1989) [0212] The MagNAPure LC DNA isolation Kit m (Bacteria, Fungi) on the MagNAPure instrument. Bacterial cells grown O / N on LB plates or slants were suspended in 100 to 1000 μl TE pH8 for storage at −20° C. 2 μl to 20 μl was used for extraction according to the manufacturer's recommendations. [0213] QIAamp DNA mini kit (catalog no. 51306-QIAGEN). The culture was pre-treated enzymatically using lysozyme and lysostaphin.

[0214] 2 / . DNA from Positive Blood Culture Bottles

[0215] Blood samples were inoculated in aerobe blood culture bottles (BacT / ALERT FA) and incubated in a BacT / Alert 3D system (Organon Teknika) at 37° C. until positive. Positivity was monitore...

example 2

LightCycler (LC) Protocol

[0218] Following the instructions of the manufacturer of the kit LC-FastStart DNA Master Hybridization Probes (cat. No 3 003 248 or No 2 239 272): [0219] any sample material suitable for PCR in terms of purity, concentration, and absence of inhibitors can be used; [0220] the primers should be at a final concentration of 0,3 to 1 μM each; [0221] the HybProbes at a final concentration of 0,2 μM each, or double; [0222] the concentration of MgCl2 should be optimized, and may vary from 1 to 5 mM; [0223] and a negative control should be ran.

[0224] The amplification and melting conditions are described herein after. The LC software version 3.5 was used. The quantification settings were F2 / back F1 (samples). For the baseline adjustment the arithmetic mode was used. The crossing point (Ct) calculation was based on the second derivative maximum. The calculation method for the melting peak was polynomial. The peak area was used to calculate the Tm.

[0225] Amplificati...

example 3

Results on Purified DNA, Inclusivity and Cross Reactivity Tests

[0226] 1 / Inclusivity

[0227] All S. aureus isolates examined (n=63) were successfully amplified (Ct range 17.25-33.51) and gave one uniform melting peak with a mean Tm of 53.13° C.±0.52° C. whatever the geographical or specimen origin.

[0228] It has to be noted that the subspecies S. aureus subs. anaerobius fell together with the S. aureus species.

[0229] All isolates received as S. epidermidis (n=48) and S. haemolyticus (n=16) could be detected using the melting curve. Quantification curves usually were not observed. The average Tm value for S. epidermidis isolates was 44.55° C.±0.21° C. and for S. haemolyticus isolates it was 44.96° C.±0.24° C. There were no differences observed among isolates from different geographical or specimen origin.

[0230] All isolates received as S. aureus or S. haemolyticus reacted as expected. However, deviating results were obtained for five S. epidermidis isolates.

[0231] Two of these, one...

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Abstract

The present invention relates to a method for the specific detection and / or identification of Staphylococcus species, in particular Staphylococcus aureus, using new nucleic acid sequences derived from the ITS (Internal Transcribed Spacer) region. The present invention relates also to said new nucleic acid sequences derived from the ITS region, between the 16S and 23S ribosomal ribonucleic acid (rRNA) or rRNA genes, to be used for the specific detection and / or identification of Staphylococcus species, in particular of S. aureus, in a biological sample. It relates also to nucleic acid primers to be used for the amplification of said spacer region of Staphylococcus species in a sample.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for the specific detection and / or identification of Staphylococcus species, in particular Staphylococcus aureus, using new nucleic acid sequences derived from the ITS (Internal Transcribed Spacer) region. [0002] The present invention relates also to said new nucleic acid sequences derived from the ITS region, between the 16S and 23S ribosomal ribonucleic acid (rRNA) or rRNA genes, to be used for the specific detection and / or identification of Staphylococcus species, in particular of S. aureus, in a biological sample. [0003] It relates also to nucleic acid primers to be used for the amplification of said spacer region of Staphylococcus species in a sample. BACKGROUND OF TH INVENTION [0004] The genus Staphylococcus includes currently 32 described species and 15 subspecies. From the human clinical point of view, S. aureus is the most important, but some coagulase-negative species are emerging pathogens especially i...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04
CPCC12Q1/689
Inventor CLAEYS, SOFIEJANNES, GEERTHABERHAUSEN, GERDEMRICH, THOMASVERDOODT, LIA
Owner INNOGENETICS NV
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