Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-03-01
2004-03-02
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091210, C536S024300, C536S024320, C536S024330, C536S025320
Reexamination Certificate
active
06699670
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the filed of clinical diagnoses. In particular it relates to diagnoses of bacterial infections generically and specifically.
BACKGROUND OF THE INVENTION
Currently, the standard method for diagnosing the presence of bacterial pathogens in clinical samples relies on culture techniques. However, active research is underway using new molecular methods to decrease detection time and increase assay sensitivity. Polymerase chain reaction (PCR) has emerged as the molecular method of choice in achieving these objectives. The utility of PCR and other molecular methods is evidenced by the recent guidelines issued by the 1999 National Committee for Clinical Laboratory Standards, encouraging the use of such methods in clinical laboratories performing bacterial identification assays (11).
To detect the presence of any bacterial pathogen in a clinical sample, primers annealing to regions of DNA conserved across a wide range of bacterial genomes have been employed. The design of such universal primers has often focused on the 16S rRNA gene (14). The presence of multiple copies of this gene within the bacterial genome facilitates its amplification by PCR. Further, sufficient sequence variability allows phylogenetic information to be attained for the purposes of microbial identification. However, up to the present, assays that provide for both universal detection and speciation require a second post-PCR processing step, which can be technically cumbersome and lengthen the time to reporting of results.
Contamination has plagued universal PCR-based bacterial detection systems. High sequence conservation of the DNA region chosen for PCR primer annealing coupled with the immense amplification power of PCR, results in the amplification of exceedingly minor bacterial contamination leading to false positives. Attempts to decontaminate PCR materials have involved nearly all known methods to destroy DNA including UV irradiation, 8-MOP treatment, and incubation with various enzymes such as DNase, restriction enzymes, or both in combination (2,3). Thus far, none of these methods have been shown to be entirely effective or reproducible.
Assessment of bacterial contamination can most reliably be made using real-time detection methods to characterize PCR amplification. Briefly, real-time PCR amplifications are reported by the cycle number at which PCR product accumulates significantly over baseline, as detected by interaction with fluorogenic probes (C
T
) (7). Aside from saving time and labor, this technique has been shown to be more objective and consistent than the traditional methods of amplification detection and template quantification involving gel electrophoresis (13). With this more precise technique however, Corless et al. found that most decontamination methods decreased PCR sensitivity. The implication of this finding was that the decontamination effect of the aforementioned methodologies could at least in part be explained by a reduction of the sensitivity of the PCR amplification system.
The quantitative capacity of real-time PCR has thus redefined the standards by which a decontamination method is measured. Not only would a particular method be required to yield negative results for controls under the more precise probe-based real-time system, but also, the method must be shown to preserve the sensitivity of the PCR assay. There is a continuing need in the art for methods which preserve sensitivity and eliminate false positive results in detecting bacterial infections.
BRIEF SUMMARY OF THE INVENTION
One embodiment of the invention provides a method for detecting and determining species source of eubacterial DNA in a sample. Template DNA in a sample is amplified using a real-time polymerase chain reaction (PCR). The PCR employs primers and at least two fluorogenic probes. The primers amplify a segment of a
S. aureus
16S rRNA gene comprising a conserved region and a first divergent region if a
S. aureus
16 rRNA gene is present in a PCR reaction. The conserved region comprises at least 18 contiguous nucleotides which are at least 80% identical among at least 10 eubacterial species. The first divergent region comprises at least 10 contiguous nucleotides and differs by at least 3 nucleotides from a second divergent region found in
Bradyrhizobium japonicum
16S rRNA gene. Each of the fluorogenic probes comprises a reporter dye and a quencher dye. A first of the two fluorogenic probes hybridizes to the conserved region and the second of the two fluorogenic probes hybridizes to a third divergent region of a first species of eubacteria. The reporter dyes of the first and the second probes have non-overlapping emission spectra. Fluorescence emissions of the reporter dyes are monitored. Presence of eubacteria in the sample is determined if emissions characteristic of the reporter dye of the first probe are detected. Presence of the first species of eubacteria in the sample is determined if emissions characteristic of the reporter dye on the second probe are detected.
Another embodiment of the invention provides another method for detecting and determining species source of eubacterial DNA in a sample. A real-time PCR reaction mixture is filtered to remove double stranded DNA contaminants having a length of ≧125 bp, forming a filtrate. The PCR reaction mixture comprises primers and at least two fluorogenic probes. The primers amplify a segment of a
S. aureus
16S rRNA gene comprising a conserved region and a first divergent region if a
S. aureus
16 rRNA gene is present in a PCR reaction. The conserved region comprises at least 18 contiguous nucleotides which are at least 80% identical among at least 10 eubacterial species. The first divergent region comprises at least 10 contiguous nucleotides and differs by at least 3 nucleotides from a second divergent region of
Bradyrhizobium japonicum
16S rRNA gene. Each of the probes comprises a reporter dye and a quencher dye. A first of the two fluorogenic probes hybridizes to the conserved region and the second of the two fluorogenic probes hybridizes to a third divergent region of a first eubacterial species. The reporter dyes of the first and the second probes have non-overlapping emission spectra. A sample comprising template DNA is added to the filtrate. Template DNA in the filtrate is amplified. Fluorescence emissions of the reporter dyes are monitored. Presence of eubacteria in the sample is determined if emissions characteristic of the reporter dye of the first probe are detected. Presence of the first species of eubacteria in the sample is determined if emissions characteristic of the reporter dye on the second probe are detected.
Another aspect of the invention is a pair of polymerase chain reaction primers for amplifying a segment of a 16S rRNA gene of eubacteria comprising a conserved region and a divergent region. The pair comprises primers p890F and p1033R (SEQ ID NO: 1 and 2, respectively.)
A further embodiment of the invention is a kit for detecting bacteremia. One component of the kit is a pair of primers that amplify a segment of a
S. aureus
16S rRNA gene if a
S. aureus
16 rRNA gene is present in a PCR reaction. The segment comprises a conserved region and a first divergent region. The conserved region is common to at least 10 species of eubacteria. The first divergent region comprises at least 10 contiguous nucleotides and differs by at least 3 nucleotides from a second divergent region of 16S rRNA gene from
Bradyrhizobium japonicum
. Another component of the kit is at least two fluorogenic probes. Each probe comprises a reporter dye and a quencher dye. A first of the two fluorogenic probes hybridizes to the conserved region. A second of the two fluorogenic probes hybridizes to a third divergent region in a first species of eubacteria. The third divergent region comprises at least 10 contiguous nucleotides and differs by at least 3 nucleotides from the second divergent region. The reporter dyes of the first and the second probes have non-overlapping emission spectra.
The present i
Kelen Gabor David
Lin Shin
Rothman Richard Eric
Yang Samuel
Banner & Witcoff , Ltd.
Horlick Kenneth R.
The Johns Hopkins University
Wilder Cynthia
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