Quantitative Assay for the Simultaneous Detection and Speciation of Bacterial Infections

a technology of simultaneous detection and speciation, applied in the field of clinical diagnosis, can solve the problems of false positives, amplification of exceedingly minor bacterial contamination, and long reporting tim

Inactive Publication Date: 2010-08-12
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
View PDF20 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]The present invention thus provides the art with methods and tools for rapidly determining both the presence of bacteria in a sample and the type of bacteria present in a single reaction.

Problems solved by technology

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.
Thus far, none of these methods have been shown to be entirely effective or reproducible.
With this more precise technique however, Corless et al. found that most decontamination methods decreased PCR sensitivity.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Quantitative Assay for the Simultaneous Detection and Speciation of Bacterial Infections
  • Quantitative Assay for the Simultaneous Detection and Speciation of Bacterial Infections
  • Quantitative Assay for the Simultaneous Detection and Speciation of Bacterial Infections

Examples

Experimental program
Comparison scheme
Effect test

example 1

Materials and Methods

Bacterial Species and DNA Isolation.

[0032]Fifteen common pathogenic microorganisms, all of which were eubacterial except one, Candida albicans, were obtained from the clinical laboratory (Division of Medical Microbiology, Johns Hopkins School of Medicine, Baltimore, Md.). The species and their ATCC strain are listed in Table 1. Microorganisms were grown in standard cultures, and DNA extracted using the QIAamp DNA kit (Qiagen Corp., Santa Clarita, Calif.).

[0033]With regard to generating standard curves for starting DNA template quantification, Staphylococcus aureus was grown in Luria-Bertani (LB) broth at 37° C. with continuous shaking to an optical density at 600 nm of 0.6. Equal aliquots were then plated to determine colony-forming units (CFU) and subjected to DNA extraction with the QIAamp DNA kit. The isolated DNA was quantified based on optical density at 260 nm and then serially diluted. Analogous procedures were performed for Staphylococcus epidermidis.

TAB...

example 2

Specificity of Universal TagMan PCR

[0042]The specificity of the primers and probes used for universal amplification of eubacterial 16S rRNA gene was first assessed with genomic DNA extracts from 14 different bacterial species together with one isolate from Candida albicans (Table 2). In each PCR assay, 5 ng of purified DNA were used. The assay's positivity was determined by examination of the amplification plot (CT vs ΔRn) generated by the Sequence Detection Software (FIG. 1). All 14 bacterial species were correctly amplified and detected with CT values in the range of 19.2 to 21.8. No amplification (CT>40) was detected when DNA isolated from C. albicans was used. The assay results were further verified by subjecting reaction products to gel electrophoresis, with visualization of bands of the expected size (162 bp) (data not shown).

TABLE 2Specificity of the Taqman assay usinguniversal primers and probes.Isolated MicroorganismsStrain (ATCC)Taqman PCR resultsStaphylococcus aureus29213...

example 3

Theoretical Detection Limit of Taqman PCR

[0043]The sensitivity of the TaqMan assay was determined by amplifying serial dilutions of eubacterial DNA. The minimal detection limit of the TaqMan system was defined as the amount of template DNA at which the relationship between CT and starting template DNA became nonlinear. Serial dilutions of S. aureus DNA (50 ng to 5 fg) were added to PCR reactions with universal primers (p890F+p1033R) and probe (UniProbe). The results are shown in Table 3. The standard curve in which CT values were plotted against starting template DNA is linear between 50 ng to 5 pg (FIG. 2). At DNA levels below 5 pg, this relationship became non-linear, and the CT's were similar to the CT of the no template control (NTC). This suggested the presence of contaminating eubacterial DNA in the NTC. The minimal detection limit of the assay was thus 5 pg of S. aureus DNA.

TABLE 3Sensitivity of the Taqman Assay with or without pre-filtration.CT for the following template DNA...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
optical densityaaaaaaaaaa
volumeaaaaaaaaaa
emission spectraaaaaaaaaaa
Login to view more

Abstract

An adaptation of the real-time PCR assay allows for highly sensitive detection of any eubacterial species with simultaneous speciation. The assay relies on a ‘multiprobe’ design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair, and it is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step can be used to effectively decontaminate or remove background DNA. The real-time system reliably identifies 14 common bacterial species with a detection limit of 50 fg.

Description

[0001]This application claims priority to and incorporates by reference provisional application Ser. No. 60 / 272,642 filed Mar. 1, 2001, the disclosure of which is expressly incorporated herein.[0002]The invention was made under funding from National Institutes of Health grant 3M01RR00052-39-5(S1). The U.S. government therefore retains certain rights in this invention.FIELD OF THE INVENTION[0003]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[0004]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 ev...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68B01L99/00C12Q1/686C12Q1/689
CPCC12Q1/686C12Q1/689C12Q2565/102C12Q2537/143
Inventor ROTHMAN, RICHARD E.YANG, SAMUELLIN, SHINKELEN, GABOR D.GAYDOS, CHARLOTTE A.
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap