Detection of bacteria
First Claim
1. A method for detecting at least one type of bacterial contamination in a physiological sample comprising human cells said method comprising the steps of i) extracting bacterial DNA from said physiological sample, ii) amplifying said bacterial DNA by real-time PCR using an oligonucleotide primer pair, said oligonucleotide primer pair consisting of an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO:
- 1 and an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO;
2, thereby producing amplified bacterial DNA, and hybridizing said amplified bacterial DNA with at least two oligonucleotide hybridization probes, wherein one of said at least two oligonucleotide hybridization probes comprises at least 10 consecutive bases of SEQ ID NO;
5 and is marked with fluorescein at the 3′
terminus, and at least one of said at least two oligonucleotide hybridization probes is selected from the group consisting of SEQ ID NO;
6-SEQ ID NO;
11 and is marked with a fluorescent dye at the 5′
terminus and iii) evaluating the amplified bacterial DNA from step ii) by means of fusion curve analysis, thus detecting if present at least one type of bacterial contamination in said physiological sample; and
wherein said method further comprises detecting at least one internal control, wherein nucleic acid for said at least one internal control is added to the bacterial DNA after said extracting step (i), and said nucleic acid for said at least one internal control is co-amplified with the bacterial DNA in step (ii); and
wherein said nucleic acid of said at least one internal control is is co-amplified in step (ii) with an oligonucleotide primer pair, said oligonucleotide primer pair comprising an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO;
3 and an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO;
4.
1 Assignment
0 Petitions
Accused Products
Abstract
The invention relates to a method for detecting bacterial contaminations preferably in physiological samples as well as sequences of synthetic oligonucleotides used therefor. The method comprises the steps of i) extracting the nucleic acid, particularly bacterial DNA, ii) amplification by means of primers and detection by means of oligonucleotides, particularly fluorescence-marked oligonucleotides as hybridization probes, containing a sequence that is selected from among a group encompassing SEQ ID NO:5 to SEQ ID NO:35, preferably in real-time PCR, and iii) evaluation by means of fusion curve analysis.
23 Citations
14 Claims
-
1. A method for detecting at least one type of bacterial contamination in a physiological sample comprising human cells said method comprising the steps of i) extracting bacterial DNA from said physiological sample, ii) amplifying said bacterial DNA by real-time PCR using an oligonucleotide primer pair, said oligonucleotide primer pair consisting of an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO:
- 1 and an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO;
2, thereby producing amplified bacterial DNA, and hybridizing said amplified bacterial DNA with at least two oligonucleotide hybridization probes, wherein one of said at least two oligonucleotide hybridization probes comprises at least 10 consecutive bases of SEQ ID NO;
5 and is marked with fluorescein at the 3′
terminus, and at least one of said at least two oligonucleotide hybridization probes is selected from the group consisting of SEQ ID NO;
6-SEQ ID NO;
11 and is marked with a fluorescent dye at the 5′
terminus and iii) evaluating the amplified bacterial DNA from step ii) by means of fusion curve analysis, thus detecting if present at least one type of bacterial contamination in said physiological sample; and
wherein said method further comprises detecting at least one internal control, wherein nucleic acid for said at least one internal control is added to the bacterial DNA after said extracting step (i), and said nucleic acid for said at least one internal control is co-amplified with the bacterial DNA in step (ii); and
wherein said nucleic acid of said at least one internal control is is co-amplified in step (ii) with an oligonucleotide primer pair, said oligonucleotide primer pair comprising an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO;
3 and an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO;
4. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
- 1 and an oligonucleotide comprising at least 10 consecutive bases of SEQ ID NO;
Specification