Method for quickly detecting microbial dna/rna, kit therefor and the use of said method

• US 20050079490A1
• Filed: 12/27/2000
• Published: 04/14/2005
• Est. Priority Date: 12/23/1999
• Status: Active Grant

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Roche Diagnostics Corporation (Roche Holding AG)
• Inventors: Stuber, Frank, Hoeft, Andreas
• Granted Patent: US 7,169,555 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for identifying microbial DNA/RNA comprising providing a sample containing microbial DNA/RNA; enriching DNA/RNA in the sample by a PCR method wherein the DNA/RNA that is identified comprise copies which are amplified by the PCR method; preliminarily classifying at least one microorganism by identifying the microbial DNA/RNA by a melting curve; adding at least one labeled oligonucleotide whereby temperature-dependent hybridization takes place and identifying the DNA/RNA on the basis of a physical property of the resultant DNA/RNA oligonucleotide complex, such as the temperature-dependence of the hybridization.

Claims

1. A method for identifying microbial DNA/RNA, characterized in that

a sample containing RNA/DNA is provided;

DNA/RNA in the sample is enriched

at least one labeled oligonucleotide is added and temperature-dependent hybridization takes place

the DNA/RNA is identified on the basis of the physical properties of the DNA/RNA oligonucleotide complex, such as the temperature-dependence of the hybridization.

2. The method for identifying microbial DNA/RNA as claimed in claim 1, characterized in that:

the sample containing microbial DNA/RNA is enriched by means of a PCR method; and

the DNA/RNA copies which have been amplified by the PCR are identified.

3. The method as claimed in claim 2, characterized in that the PCR method is a real-time PCR method and the quantity of microbial DNA/RNA in the initial sample is thereby determined.

4. The method as claimed in claim 2, characterized in that a preliminary classification of the at least one microorganism is carried out for identifying the microbial DNA/RNA by way of the melting curve.

5. The method as claimed in claim 2, characterized in that a reverse dot blot, using suitable DNA/RNA probes, or a DNA chip/DNA microarray is employed for identifying the microbial DNA/RNA.

6. The method as claimed in one of the preceding claims, characterized in that, before being employed for amplifying the microbial DNA/RNA, the polymerases which are used in the PCR are decontaminated, for example by restriction enzyme treatment or by means of methoxypsoralen/UV irradiation.

7. The method as claimed in one of the preceding claims, characterized in that the sample to be investigated is body fluid, smears or tissue.

8. The method as claimed in one of the preceding claims, characterized in that the oligonucleotides [lacuna] with FRET-suitable dye-labeled probes and the Lower Primer exhibits a dye which is capable of being transferred and which is suitable for FRET, with the probes and primers being designed such that they bind to adjacent regions of a sequence to be amplified.

9. A kit for rapidly identifying microbial DNA/RNA in samples, characterized by decontaminated polymerase and a solution containing a standard DNA/RNA concentration of a known (template) DNA/RNA.

11. The kit as claimed in claim 9, characterized in that it furthermore contains RNA or DNA probes which may be labeled.

10. The kit as claimed in claim 10, characterized in that it furthermore contains at least one optionally labeled consensus primer pair, in particular having a length of up to about 23 base pairs.

12. The use of the method as claimed in one of the claims for rapidly identifying microbial genomic DNA or RNA.

13. The use as claimed in claim 12 for diagnosing bacterial infections, such as sepsis, or fungal infections, or in foodstuff monitoring, water monitoring or applications in veterinary medicine.

14. A program for identifying DNA/RNA in an automated machine during a PCR in the presence of at least one labeled DNA/RNA probe which, on binding to the DNA/RNA, produces an optical signal which is typical for the binding, comprising the following steps:

introducing the specific DNA/RNA probe,

obtaining and storing experimental values of signals which are specific for the binding of the DNA/RNA probe to DNA/RNA segments in dependence on the temperature during a PCR method;

calculating the first derivative of the optical signal I with respect to the temperature, i.e. dI/dT, and

storing the curve form (melting curve) of the fluorescence signal; and

comparing the dI/dT curve form for the specific probe with a stored table of values in which typical values for particular microorganisms are filed; and

selecting the curve form which is most similar to the experimental values in a selected region; and

outputing information with regard to at least one microorganism species which has been determined on the basis of the similarity of the measured and stored curve forms.