WO2006050275A2 - Procede de detection rapide specifique par espece d'agents pathogenes - Google Patents

Procede de detection rapide specifique par espece d'agents pathogenes Download PDF

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WO2006050275A2
WO2006050275A2 PCT/US2005/039292 US2005039292W WO2006050275A2 WO 2006050275 A2 WO2006050275 A2 WO 2006050275A2 US 2005039292 W US2005039292 W US 2005039292W WO 2006050275 A2 WO2006050275 A2 WO 2006050275A2
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probe
specimen
probes
detector
pathogen
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PCT/US2005/039292
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WO2006050275A3 (fr
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Joseph C. Liao
David A. Haake
Bernard M. Churchill
Marc A. Suchard
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The Regents Of The University Of California
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Publication of WO2006050275A2 publication Critical patent/WO2006050275A2/fr
Publication of WO2006050275A3 publication Critical patent/WO2006050275A3/fr
Priority to US11/743,071 priority Critical patent/US7763426B2/en
Priority to US12/788,101 priority patent/US20110027782A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present invention relates generally to materials and methods for rapid species- specific detection of bacterial and other pathogens in specimens.
  • Urinary tract infection is the most common urological disease in the United States and the second most common bacterial infection of any organ system. UTIs are a major cause of patient morbidity and health-care expenditure for all age groups. UTIs account for approximately 7 million office visits, more than 1 million visits to emergency departments, and approximately 100,000 hospitalizations each year. The estimated annual cost to the United States health-care system is approximately $1.6 billion. In patients who are at risk for complicated UTIs (e.g. obstructive uropathy, immunocompromised state, neurogenic bladder, congenital urinary tract anomalies, and indwelling foreign bodies), delay in diagnosis and initiation of appropriate medical intervention can lead to life threatening systemic infections or permanently reduced renal function.
  • complicated UTIs e.g. obstructive uropathy, immunocompromised state, neurogenic bladder, congenital urinary tract anomalies, and indwelling foreign bodies
  • Urine specimens are the most common type of body fluid submitted for culture to clinical microbiology laboratories. Significant resources at these clinical laboratories are devoted to the time- consuming processing of urine specimens, although the majority of these specimens are negative or yield insignificant quantities of bacteria. A rapid test that could identify the uropathogens, or confirm the presence or absence of clinically significant bacteria with high sensitivity and specificity, would significantly reduce the workload of clinical microbiology laboratories.
  • Molecular biological techniques based on DNA hybridization are increasingly utilized in clinical diagnostic testing, and are especially useful in the identification of infectious agents that cannot be cultured.
  • Hybridization of oligonucleotides to the unique molecular sequence of an organism's DNA or RNA is highly sensitive for pathogen-specific identification, surpassing culturing methods that depend on morphological and biochemical characteristics.
  • More rapid real-time polymerase chain reaction (PCR) quantification involving amplification of target DNA or RNA currently requires technically demanding specimen processing procedures.
  • microscale devices are particularly compatible to detect and manipulate biological molecules of interest, such as nucleic acids and proteins, with nanoscale precision.
  • electrochemical sensors offer sensitivity, selectivity, portability and relative low cost for nucleic acids detection.
  • the basic electrochemical sensor design is comprised of a nucleic acid layer coupled with electrochemical transducers to detect sequence-specific hybridization events.
  • the invention disclosed herein addresses these needs and others by providing methods and materials for rapid, species-specific detection of pathogens.
  • the invention provides oligonucleotide probes that can be used separately or in combination for the detection and distinction of bacterial pathogens commonly found in urological and other patient specimens.
  • the invention provides methods for using such probes to rapidly assay for pathogens.
  • the invention provides a universal lysis buffer that enables lysis of both gram-negative and gram-positive bacteria, and permits immediate assay of lysed specimens (e.g., urine) without requiring a bacterial purification step, further accelerating the assay.
  • the invention provides a method for detecting the presence of a pathogen in a specimen.
  • the method comprises contacting a lysate of the specimen with a capture probe immobilized on a substrate.
  • the capture probe comprises a first oligonucleotide that specifically hybridizes with a first nucleic acid sequence of the pathogen to be detected.
  • the lysate has been brought into contact -with a detector probe that comprises a detectably labeled oligonucleotide that specifically hybridizes with a second nucleic acid sequence of the pathogen.
  • the lysate can be brought into contact with the detector probe simultaneously with, prior to, or subsequent to, the contacting of the lysate with the capture probe. Detection of the second detectable label complexed with the substrate is indicative of the presence of the corresponding pathogen.
  • the first and second nucleic acid sequences of the pathogen are adjacent to each other, such that no gap remains between the capture probe and the detector probe upon hybridization with the target nucleic acid sequences of the pathogen. In another embodiment, a gap between the first and second nucleic acid sequences is not greater than about 6 base pairs.
  • the lysate can be prepared by contacting the specimen with a universal lysis buffer capable of lysing both gram-negative and gram-positive bacteria.
  • a representative universal lysis buffer contains Triton X-100, KH 2 PO 4 , EDTA and lysozyme.
  • the lysate can be prepared by contacting the specimen with a first lysis buffer comprising a non-denaturing detergent (e.g., Triton X-IOO) and lysozyme, or a second lysis buffer comprising NaOH.
  • the lysing comprises contacting the specimen with both buffers in series, e.g. with the second lysis buffer, either before or after contacting the specimen with the first lysis buffer.
  • the contacting of the specimen with the buffer(s) typically occurs at room temperature.
  • the specimen is in contact with the lysis buffer for a total of about 10 minutes.
  • the contact with each buffer is typically about 5 minutes.
  • the oligonucleotide probes are typically less than 60 base pairs in length, preferably 10- 50 base pairs in length, and in most embodiments, the oligonucleotide probes are 20-40 base pairs in length. In one embodiment, the probe is about 40 base pairs in length.
  • the detectable labels for use with the invention can be selected from many such labels known in die art.
  • the label comprises a reporter enzyme, such as horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • the HRP is conjugated to an antibody or other binding partner and serves as a secondary label that binds to a primary label on the detector probe.
  • the primary label on the detector probe is fluorescein and the secondary label is HRP conjugated to an anti-fluorescein antibody.
  • the detectable label can be at the 3' and/ or 5' end of the detector probe. In some embodiments, the detectable label is at the 3' end of the detector probe.
  • the pathogen is typically a microorganism that can be found in bodily fluids.
  • the pathogen is a uropathogen.
  • Uropathogens include bacterial and fungal pathogens.
  • the pathogen is a bacterial pathogen, such as Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterococcus spp., Klebsiella pneumoniae, - Enterobacter aerogenes, Enterobacter clocae.
  • the specimen can be any specimen believed to contain or suspected of containing a pathogen, such as a bodily fluid.
  • Representative bodily fluids include blood and blood products, saliva, semen, prostatic secretions, cerebrospinal fluids and urine.
  • the specimen is urine.
  • the invention additionally provides an assay kit for use in carrying out the method of the invention.
  • the kit comprises one or more of the probes described herein, and, optionally, a container or substrate therefor.
  • the kit comprises a substrate to which one or more capture probes of the invention are bound or otherwise immobilized.
  • the kit further comprises a container and one or more detector probes corresponding to the capture probes.
  • the substrate is an electrochemical sensor array.
  • FIG. 1A-1D Components and performance of the electrochemical sensor.
  • the 16- sensor array (2.5 x 7.5 cm) was microfabricated with a thin, optical-grade layer of gold electrodes deposited on plastic (GeneFluidics Inc., Monterey Park, CA). Each sensor in the array contained three electrodes: a central working electrode, a circumferential reference electrode, and a short auxiliary electrode.
  • C Detection strategy: 1. Bacterial lysis to release 16s rRNA target (dashed line); 2. Hybridization of the target with the fluorescein— labeled detector probe; 3.
  • Hybridization of the target with the biotin— labeled capture probe 4. Binding of anti-fiuorescein antibody conjugated with horseradish peroxidase (HRP) to the target-probe sandwich; and 5. Generation of current by transfer of electrons to the electron transfer mediator, TMB.
  • HRP horseradish peroxidase
  • FIG. 2 Specificity of Entmbacteriaceae-spe ⁇ &c probe pair. Positive signals were seen for all Enterobacteriaceae spp. tested but not for Gram-positive uropathogens (Eo, Ef, Ss, Sa) or P. aeruginosa (Pa). See footnote of Table 2 (Example 1, below) for bacterial species abbreviations. Mean and standard deviation of experiments performed in duplicate are shown. NC refers to the negative control experiments performed with capture and detector probes but without bacterial lysate.
  • FIG. 3A-3B Direct, species-specific detection of uropathogens in representative clinical urine specimens using the electrochemical sensor array.
  • Current output for each of the probe pairs in the array are shown in nanoamperes.
  • the mean current output of duplicate sensors is shown above each bar, error bars represent the standard deviation.
  • the probe pair designation is shown below each bar, their species-specificity is given in Table 2.
  • the urinalysis and microbiological characteristics of each specimen are shown to the right of the bar graph. Background signal level was determined by averaging the log 10 results of the NC sensors and the sensors with the four lowest species-specific probe pairs (from among EC, PM, KE, PA, and EF). As described in the text, significant signals were 0.30 log units (5 standard deviations) above background.
  • E. coliva E. coliva.
  • FIG. 4. 1 UTI Chip' Signal Interpretation Algorithm. The three-step algorithm used to interpret the results of electrochemical sensor experiments on 78 specimens that met inclusion criteria are shown. Positive signals were those with a mean log of greater than 0.30 log units (5 standard deviations) over background. "UNI” and “EB” are eubacterial and probe signals, respectively. "Bkgnd” is the background signal, as defined in the text of Example 1. "MaxSpSp” refers to the maximum species- specific signal. Clinical microbiology results are given in shaded boxes. Two-letter species abbreviations are given in the footnote of Table 2 (Example 1). NSG indicates "No Significant Growth”. NG indicates "No Growth”. Correct results are in black letters (darkest font), false-positive results are in medium dark letters (1 Sm; 2 NG), false- negative results are in red letters (gray font).
  • FIG. 5 Model of the electrochemical redox reporter complex.
  • Components of the complex include the horseradish peroxidase (HRP)-Fab conjugate bound to the fluorescein (3' small circle)-modified detector probe (5' small circle).
  • the fluorescein- modified detector probe and rRNA target (dashed line) are anchored to the sensor surface by the biotin-modified capture probe.
  • the relative sizes of the electrochemical redox reporter complex components are provided indicating that the fluorescein of a 3'- modified detector probe would be ⁇ 258A from the sensor surface.
  • FIG. 6 The effect of lysis conditions on electrochemical signal intensity. ⁇ nterococcus organisms were treated with various lysis methods followed by direct electrochemical detection of 16S rRNA in the crude bacterial lysates. Under each condition, 10 5 ⁇ nterococcus cells were treated at room temperature for a total of 10 min.
  • the lysis methods are: 1) NaOH for 10 min; 2) Triton X-IOO for 5 min, then NaOH for 5 min; 3) Triton X-IOO with lysozyme for 10 min; 4) NaOH for 5 min, then Triton X-IOO with lysozyme for 5 min; 5) Triton X-IOO with lysozyme for 5 min, then NaOH for 5 min.
  • Background current output was measured using negative control (NC) sensors to which no cell lysates were applied. Current output was measured in duplicate for each lysis condition. Signal output was measured in nano-amperes (nA).
  • FIG. 7 The effect of oligonucleotide length on electrochemical signal intensity.
  • Single stranded oligonucleotides ('Test Probes') ranging from 20-60 bps in length modified at the 5'- and 3'-ends with biotin and fluorescein, respectively, were tested to examine the effect of probe length on signal intensity. Hybridization was not required in these experiments because, as shown in the inset, the double-labeled probes served as a bridge between fluorescein and biotin on the electrochemical sensor surface.
  • the highest signal output was obtained using the 40 bp probe, yielding a lower limit of detection at a concentration of 10 pM. Background current output was measured using negative control (NC) sensors to which no cell lysates were applied. Signal output was measured in nano-amperes (nA). Asterisks indicate lower limits of detection that differ significantly from background (two-tailed t test for paired samples, P ⁇ .05).
  • FIG. 8A-8B Effects of gap between the probe hybridization sites on signal intensity, probe configuration, and probe modification on signal intensity.
  • Fig. 8A same capture probe was paired with different detector probes to examine the effects of removing the short sequence gap between the probe hybridization sites and location of fluorescein modification (5' or 3').
  • Enterococcm cells were lysed by sequential treatment with the combination of Triton X-IOO and lysozyme followed by NaOH.
  • Fig. 8B shows the results from 5-fold serial dilutions of Enterococcus cells using the detector and capture configuration which confer the highest signal (Fig. 8A, bar 4) and the 'original' EF detector probe ( Figure 8A, bar 1).
  • FIG. 9 Effects of hybridization with a mixture of two detector probes on electrochemical sensor specificity.
  • Detector probes specific for Enterococcus or Escherichia coli 16S rRNA were examined alone and in combination using electrochemical sensors functionalized with an Enterococcus-s ⁇ & ⁇ B.c capture probe. There was no significant decrease in signal intensity for detection of Enterococcus rRNA when hybridization was performed with a mixture of Enterococcus- and E. «>/z-specific detector probes. There was no increase in nonspecific detection of E. coli rRNA for hybridization with a mixture of detector probes. Similar results were obtained with other 2-, 3-, and 5-detector probe mixtures. Background current output was measured using negative control (NC) sensors to which no cell lysates were applied. Signal output was measured in nano-amperes (nA).
  • NC negative control
  • FIG. 10 Effects of hybridization with a mixture of seven detector probes on electrochemical sensor specificity.
  • a single electrochemical sensor array immobilized with 7 different capture probes is shown schematically in the inset. 10 6 Enterococcus cells were lysed and released rRNA hybridized with a mixture of seven different detector probes before application on the sensor array. Background current output was measured using negative control (NC) sensors to which no cell lysates were applied. Signal output was measured in nano-amperes (nA). The results demonstrate species-specific detection of Enterococcus using a mixture of seven detector probes. Asterisks indicate results that differ significantly from background (two-tailed t test for paired samples, P ⁇ .05). DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is based on the discovery and development of materials and methods for a rapid detection assay that permits species-specific detection of pathogens in clinical specimens.
  • the invention provides an electrochemical sensor array and oligonucleotide probes specific for clinically relevant pathogen species as well as methods for rapid and efficient use of same.
  • an "oligonucleotide probe” is an oligonucleotide having a nucleotide sequence sufficiently complementary to its target nucleic acid sequence to be able to form a detectable hybrid probe:target duplex under high stringency hybridization conditions.
  • An oligonucleotide probe is an isolated chemical species and may include additional nucleotides outside of the targeted region as long as such nucleotides do not prevent hybridization under high stringency hybridization conditions.
  • Non- complementary sequences such as promoter sequences, restriction endonuclease recognition sites, or sequences that confer a desired secondary or tertiary structure such as a catalytic active site can be used to facilitate detection using the invented probes.
  • An oligonucleotide probe optionally may be labeled with a detectable marker such as a radioisotope, a fluorescent moiety, a chemiluminescent moiety, an enzyme or a ligand, which can be used to detect or confirm probe hybridization to its target sequence.
  • a detectable marker such as a radioisotope, a fluorescent moiety, a chemiluminescent moiety, an enzyme or a ligand, which can be used to detect or confirm probe hybridization to its target sequence.
  • Probe specificity refers to the ability of a probe to distinguish between target and non- target sequences.
  • nucleic acid refers to a deoxyribo- nucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally-occurring nucleotides.
  • a “detectable marker” or “label” is a molecule attached to, or synthesized as part of a nucleic acid probe. This molecule should be uniquely detectable and will allow the probe to be detected as a result. These detectable moieties are often radioisotopes, chemiluminescent molecules, enzymes, haptens, or even unique oligonucleotide sequences.
  • hybrid or a “duplex” is a complex formed between two single- stranded nucleic acid sequences by Watson-Crick base pairings or non-canonical base pairings between the complementary bases.
  • hybridization is the process by which two complementary strands of nucleic acid combine to form a double-stranded structure (“hybrid” or “duplex”).
  • Stringency is used to describe the temperature and solvent composition existing during hybridization and the subsequent processing steps. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency conditions are chosen to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid. Exemplary stringency conditions are described hereinbelow.
  • complementarity is a property conferred by the base sequence of a single strand of DNA or RNA which may form a hybrid or double-stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs on the respective strands.
  • Adenine (A) ordinarily complements thymine (T) or Uracil (U), while guanine (G) ordinarily complements cytosine (C).
  • adjacent in the context of nucleotide sequences and oligonucleotides, means immediately next to one another (end to end), such that two adjacent molecules do not overlap with one another and there is no gap between them.
  • two oligonucleotide probes hybridized to adjacent regions of a target nucleic acid molecule have no nucleotides of the target sequence (unpaired with either of the two probes) between them.
  • the phrases “consist essentially of or “consisting essentially of” mean that the oligonucleotide has a nucleotide sequence substantially similar to a specified nucleotide sequence. Any additions or deletions are non-material variations of the specified nucleotide sequence which do not prevent the oligonucleotide from having its claimed property, such as being able to preferentially hybridize under high stringency hybridization conditions to its target nucleic acid over non-target nucleic acids.
  • substantially corresponding probes of the invention can vary from the referred-to sequence and still hybridize to the same target nucleic acid sequence. This variation from the nucleic acid may be stated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe and its target sequence. Probes of the present invention substantially correspond to a nucleic acid sequence if these percentages are from 100% to 80% or from 0 base mismatches in a 10 nucleotide target sequence to 2 bases mismatched in a 10 nucleotide target sequence. In preferred embodiments, the percentage is from 100% to 85%. In more preferred embodiments, this percentage is from 90% to 100%; in other preferred embodiments, this percentage is from 95% to 100%.
  • nucleic acids having a sufficient amount of contiguous complementary nucleotides to form, under high stringency hybridization conditions, a hybrid that is stable for detection.
  • oligonucleotide probes can hybridize with their target nucleic acids to form stable probe:target hybrids (thereby indicating the presence of the target nucleic acids) without forming stable probe:non-target hybrids (that would indicate the presence of non-target nucleic acids from other organisms).
  • the probe hybridizes to target nucleic acid to a sufficiently greater extent than to non-target nucleic acid to enable one skilled in the art to accurately detect the presence of E. coli, P. mirabilis, P.
  • Preferential hybridization can be measured using techniques known in the art and described herein.
  • a "target nucleic acid sequence region" of a pathogen refers to a nucleic acid sequence present in the nucleic acid of an organism or a sequence complementary thereto, which is not present in the nucleic acids of other species.
  • Nucleic acids having nucleotide sequences complementary to a target sequence may be generated by target amplification techniques such as polymerase chain reaction (PCR) or transcription mediated amplification.
  • room temperature means about 20-25 0 C.
  • the invention provides oligonucleotide probes that are specific for E. coli, P. mirabilis, P. aeruginosa, Enterococcus spp., the K " lebsiella-Enterobacter group, and the Enterobacte ⁇ aceae group, as well as a universal bacterial detection probe. Direct detection of pathogens using these probes has been demonstrated in both inoculated urine and clinical urine samples from symptomatic patients.
  • the probes include capture probes and detector probes, described in greater detail below. Additional probes are listed in Table 5, in Example 2.
  • CTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCAT (SEQ ID NO: 6)
  • GTCCATCCATCAGCGACACCCGAAAGCGCCTTTCA (SEQ ID NO: 10)
  • Oligonucleotides may be prepared using any of a variety of techniques known in the art. Oligonucleotide probes of the invention include the sequences shown in Table 1, Table 5, and equivalent sequences that exhibit essentially the same ability to form a detectable hybrid probe:target duplex under high stringency hybridization conditions. Oligonucleotide probes typically range in size from 10 to 100 nucleotides in length. Preferred probes are 20-60 nucleotides in length, with 20-40 nucleotides being optimal for some conditions, as illustrated in the Examples below. A variety of detectable labels are known in the art, including but not limited to, enzymatic, fluorescent, and radioisotope labels.
  • highly stringent conditions or “high stringency conditions” are those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 0 C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 75O mM sodium chloride, 75 mM sodium citrate at 42 0 C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulf
  • Suitable “moderately stringent conditions” include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS. Any polynucleotide may be further modified to increase stability.
  • flanking sequences at the 5' and/or 3' ends Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/ or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.
  • flanking sequences at the 5' and/or 3' ends Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/ or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as
  • Nucleotide sequences can be joined to a variety of other nucleotide sequences using established recombinant DNA techniques.
  • a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids.
  • Vectors of particular interest include probe generation vectors.
  • a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.
  • the invention provides a method for species-specific detection of pathogens in clinical and other specimens.
  • the pathogens to be detected include those which infect human or animal subjects.
  • Representative pathogens for detection include bacterial pathogens, such as those listed in Tables 1 and 5, and those responsible for fungal infection in body fluids (e.g., Candida albicans and Candida glabrat ⁇ ), as well as others known to those skilled in the art.
  • the method generally comprises contacting one or more probes of the invention with a specimen. For example, one can perform the method using one, two, three, four, five, six or all of the probes described herein, and/ or using one or more of these probes in combination with other probes known in the art.
  • the method can be carried out quickly, with a minimum of specimen preparation prior to assay.
  • the specimen is lysed with a lysis buffer prior to contact with the probes of the invention.
  • the lysis buffer is sufficient to release nucleic acid molecules from the pathogen to be detected, such that the target regions of the nucleic acid molecules of the pathogen are able to hybridize with the probes.
  • the lysis buffer(s) can be selected in accordance with the target pathogen(s).
  • the method is performed using an electrochemical sensor.
  • an electrochemical sensor suitable for use with the invention is described in the U.S. patent application assigned publication number 20020123048.
  • the sensor array can be an integral component of a point-of-care system for molecular detection of pathogens in body fluids.
  • Those skilled in the art will appreciate the ease with which the particular method described in detail herein can be adapted for use with other materials, such as an automated sample preparation cartridge or optical sensors, as well as other conventional detection methods, employing the probes described herein.
  • the method comprises contacting a lysate of the specimen with a capture probe immobilized on a substrate.
  • the capture probe comprises a first oligonucleotide that specifically hybridizes with a first nucleic acid sequence of the pathogen to be detected.
  • the lysate has been brought into contact with a detector probe that comprises a detectably labeled oligonucleotide that specifically hybridizes with a second nucleic acid sequence of the pathogen.
  • the lysate can be brought into contact with the detector probe simultaneously with, prior to, or subsequent to, the contacting of the lysate with the capture probe. Detection of the second detectable label complexed with the substrate is indicative of the presence of the corresponding pathogen.
  • the first and second nucleic acid sequences of the pathogen are adjacent to each other, such that no gap remains between the capture probe and the detector probe upon hybridization with the target nucleic acid sequences of the pathogen.
  • a gap between the first and second nucleic acid sequences is not greater than about 6 base pairs.
  • the lysis preparation comprises the universal lysis buffer containing 1% Triton X-IOO, 0.1 M KH 2 PO 4 , 2 niM EDTA and 1 mg/ml lysozyme.
  • Use of the universal lysis buffer obviates the need to use separate lysis buffer for gram-positive and gram-negative bacteria.
  • the time-consuming steps of bacterial RNA and/ or DNA purification are not necessary, permitting direct application of a lysed urine sample to the capture probes, improving speed and efficiency of the assay.
  • the method can be performed by first lysing a specimen of interest to release nucleic acid molecules of the pathogen.
  • the lysate can be prepared by contacting the specimen with a first lysis buffer comprising a non-denaturing detergent (e.g., Triton X-IOO) and lysozyme, or a second lysis buffer comprising NaOH.
  • a non-denaturing detergent e.g., Triton X-IOO
  • the lysing comprises contacting the specimen with both buffets in series, e.g. with the second lysis buffer, either before or after contacting the specimen with the first lysis buffer.
  • the contacting of the specimen with the buffer(s) typically occurs at room temperature.
  • the specimen is in contact with the lysis buffer for a total of about 10 minutes. Where a first and second lysis buffer is used, the contact with each buffer is typically about 5 minutes.
  • the time and temperature under which the contact with lysis buffer occurs can be varied (e.g. higher temperatures will accelerate the lysis) and also optimized for a particular specimen, target pathogen and other assay conditions.
  • the method comprises contacting a specimen with one or more detector probes of the invention under conditions permitting hybridization of target nucleic acid molecules of pathogens (e.g., bacteria) present in the specimen with the detector probes, resulting in hybridized target nucleic acid molecules.
  • pathogens e.g., bacteria
  • One or more hybridized target probes are brought into contact with one or more capture probes, under conditions permitting hybridization of capture probes with target nucleic acid molecules. Accordingly, the target nucleic acid ultimately hybridizes with both capture probe(s) and detector probe(s).
  • detector probe hybridizes with the target nucleic acid first, after which the hybridized material is brought into contact with an immobilized capture probe. Following a wash, the dectectortargetcapture combination is immobilized on a surface to which the capture probe has been bound. Detection of probe bound to target nucleic acid is indicative of presence of pathogen.
  • the method comprises detection of current associated with binding of probe to target.
  • the capture probe is labeled with biotin and immobilized onto a surface treated with streptavidi ⁇ .
  • the detector probe in this example is tagged with fluorescein, providing an antigen to which a horse radish peroxidase-labeled antibody binds.
  • This peroxidase in the presence of its substrate (typically, hydrogen peroxide and tetramethylbenzidine), catalyzes a well-characterized redox reaction and generates a measurable electroreduction current under a fixed voltage potential, thereby providing an electrochemical signal to detect presence of the target nucleic acid.
  • substrate typically, hydrogen peroxide and tetramethylbenzidine
  • kits are also within the scope of the invention.
  • Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method.
  • the containers can comprise a probe that is or can be detectably labeled.
  • the kit can also include a container comprising a reporter-means, such as a biotin-binding protein, e.g., avidin or streptavidin, bound to a detectable label, e.g., an enzymatic, fluorescent, or radioisotope label.
  • the kit comprises a container and one or more detector probes disclosed herein.
  • the kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a label can be provided on the container to indicate that the composition is used for a specific application, and can also indicate directions for use. Directions and or other information can also be included on an insert which is included with the kit
  • the kit further comprises a substrate to which one or more capture probes are immobilized.
  • the substrate comprises an electrochemical sensor array. Capture probes specific for different species of bacteria can be positioned on different sensors of the array.
  • An assay kit comprises all the materials to be used in performance of the assay with the exception of the specimen or sample to be assayed.
  • a typical assay kit comprises an electrochemical sensor array to which a plurality of capture probes have been immobilized and a plurality of corresponding detector probes.
  • the plurality of probes includes probes specific for each of the bacterial pathogens disclosed herein.
  • the kit includes one or more control probes and/or a universal probe.
  • Example 1 Rapid Species-Specific Detection of Uropathogens Using an Electrochemical Sensor Array
  • the sensor array can be an integral component of a point-of-care system for molecular detection of pathogens in body fluids.
  • Uropathogen isolates and clinical utine specimens were obtained from the UCLA Clinical Microbiology Laboratory with approval from the UCLA Institutional Review Board and appropriate Health
  • Isolates were received in vials containing Brucella broth with 15% glycerol (BBL, Maryland) and were stored at -70°C. Overnight bacteria cultures were freshly inoculated into Luria Broth (LB) grown to logarithmic phase as measured by OD 600 . Concentrations in the logarithmic phase specimens were determined by serial plating, typically yielding 10 7 -10 8 bacteria/ml. The uropathogens grown in LB were stored as frozen pellets at — 70 0 C until time of experimentation. Appropriate dilutions were made when determining the target specificity of the probes such that the different uropathogen numbers were within one order of magnitude of each other.
  • Probe design Species- and group-specific capture and detector probe pairs were designed using a bioinformatics-based approach that compared 16S rDNA sequences obtained from the NCBI database (Bethesda, MD) and from uropathogen isolates with estimated hybridization accessibility of 16S rRNA target sequences (14). In addition to species- and group-specific probe pairs, a universal probe pair was designed to hybridize with all bacterial 16S rDNA sequences. Both capture and detector probes were 27-35 bps in length, their hybridization sites typically separated by a gap of 6 bps. Capture and detector probes were synthesized with 5' biotin and 5' fluorescein modifications, respectively (MWG, High Point, NC).
  • Electrochemical sensor array Electrochemical sensor arrays were provided by GeneFluidics (Monterey Park, CA). As shown in Figure IA, each sensor in the 16-sensor array consists of a central working electrode, surrounded by a reference electrode and an auxiliary electrode. The single layer electrode design populated with alkanethiolate self- assembled monolayer (SAM) surface modifications was described previously (16) with modifications in the electrode configuration and fabrication process. Sensor arrays used in the current study were batch fabricated by deposition of a 50 ntn gold layer onto a plastic substrate. 40 ⁇ l of 0.1 KiMK 3 Fe(CN) 6 (potassium hexacyanoferrate, Sigma, St.
  • each working electrode was incubated with 2.5 ⁇ l of NHS /EDC (100 mM JV-hydroxysuccinimide, 400 mM JV-3-dimethylaminopropyl-N- ethylcarbodiimide) for 10 min.
  • Activated sensors were incubated in biotin (5 mg/ml in 50 mM sodium acetate) (Pierce, Rockford, IL) for 10 min.
  • Biotinylated sensors were incubated in 4 ⁇ l of 0.5 U/ml of streptavidin in RNase-free H 2 O (Cat. No. 821739, MP Biomedicals, Aurora, OH) for 10 min.
  • Stteptavidin-coated sensors were incubated with biotinylated capture probes (4 ⁇ l, 1 ⁇ M in 1 M phosphate buffer, pH 7.4) for 30 min.
  • IM NaOH was preceded by resuspension of the bacterial pellet in 10 ⁇ l of 0.1% Triton X-IOO, 2 mM EDTA, and 1 mg/ml lysozyme (Sigma) in 2OmM Tris-HCl, pH 8.0, and incubation at room temperature for 5 min. 50 ⁇ l of the detector probe (0.25 ⁇ M) in 2.5% bovine serum albumin (Sigma, St. Louis, MO) 1 M phosphate buffer, pH 7.4, were added to the bacterial lysate and incubated for 10 min at 65°C to allow target-probe hybridization.
  • Clinical validity study design Clinical urine specimens were received from routine urine cultures collected from inpatients and outpatients and submitted to the UCLA Clinical Microbiology Laboratory. Routine plating on tripticase soy agar with 5% sheep blood was performed on each specimen for phenotypic identification and colony counting while an aliquot of each specimen was held at 4°C overnight. On the day after plating, specimens were selected for inclusion in the study on the basis of a rapid indole test for the purpose of including uropathogens other than Escherichia coliv ' a. approximately one-half of the specimens. The other half of the specimens were divided between E. coli- containing specimens and specimens determined to have "no significant growth" or "no growth” (see definitions below).
  • a receiver operating characteristic (ROC) curve analysis (29, 51) was performed to determine the optimal threshold for a positive result to maximize weighted accuracy, where weighted accuracy was defined as (5*sensitivity + specificity) /6 to account for the greater diagnostic importance of minimizing false negative results than of minimizing false positive results.
  • Sensor results were determined in a three-step algorithm. First, the average of the log 10 UNI results was compared with background to determine whether the specimen contained bacteria. Second, for specimens predicted to contain bacteria, the identity of the bacteria in the specimen was determined by comparing the average log 10 result of the highest species-specific signal (from among the EC, PM, KE, PA, and EF sensors) with background. Third, if no species-specific signal was positive, then the average log 10 result of the EB probe pair was compared with background to determine if the bacteria present in the specimen were members of the Enterobacte ⁇ aceae family.
  • ROC receiver operating characteristic
  • probes for the 'UTI Chip' The electrochemical sensor was used to determine which probe pairs had the greatest sensitivity and specificity for binding to 16S rRNA in lysates of uropathogens. Capture and detector probes were designed to hybridize to species- and group-specific regions of the 16S rRNA molecule and that are accessible to hybridization with oligonucleotide probes, as determined by prior flow cytometric analysis (14).
  • Table 2 summarizes of the observed specificity of the probe pairs using the electrochemical sensor array. Significant sequence similarities between Klebsiella and Enterobacter spp. 16S sequences precluded design of species-specific probes for these organisms.
  • the Enterococcus probe pair (EF) was specific for both E.faecalis and E. faedum.
  • the universal probe pair (UNI) detects all of the uropathogens tested.
  • Figure 2 shows detection by the EB probe pair of all members of the Enterobacteriaceae family tested, but not equal numbers of P. aeruginosa, Staphylococcus or Enterococcus spp. As shown in Table 2 and Figure 2, both the UNI and EB probes detect less common uropathogens such as Citrobacter and Morganella spp., for which the species-specific probes are not yet available.
  • Cf Citrobacter freundii
  • Ea Enterobacter aerogenes
  • Ec Escherichia coli
  • Ef E. faedum
  • El E. cloacae
  • Eo Enterococcus faecalis
  • Pm Proteus mirabilis
  • Kp Klebsiella pneumoniae
  • Pa Pseudomonas aeruginosa
  • Mm Morganella morganii
  • Sa Staphylococcus aureus
  • Ss S. saprophytic .
  • Species-specific detection of uropathogens in clinical urine specimens were tested using the UTI Chip and 7 probe pairs with specificities relevant to the detection of the most prevalent uropathogens.
  • Clinical urine specimens represented the most significant challenge to the electrochemical sensor detection strategy because they frequently also contained high concentrations of host proteins, white blood cells, red blood cells, cellular debris in addition to wide variations in pH.
  • the UTI Chip produced accurate results with clinical urine specimens representing a broad range of uropathogen species and concentrations, as well as broad range of urine specimen parameters related to the host inflammatory response to UTI.
  • Figure 3A illustrates that a high signal-to-noise ratio was maintained in the case of a clinical urine specimen from a patient with an E. colt UTI in which a high numbers of white blood cells were present and the pH of the urine was 5.5.
  • the high amperometric signals using the UNI, EB, and EC sensors indicated that the streptavidin coating on the sensor surface remained intact and the target-probe hybridization step of the electrochemical sensor protocol was not inhibited by host cells or changes in urinary pH.
  • the low background signals with the remaining sensors indicated that the SAM layer was also unaffected by exposure to urine.
  • Figure 3B illustrates result obtained in a urine specimen from a patient with a K.
  • Clinical validity study involving blinded clinical urine specimens A total of 89 blinded clinical urine specimens were received from the clinical microbiology laboratory. Eleven urine specimens were found to contain more than one organism and were excluded from further analysis. The remaining 78 specimens that were analyzed included 58 with bacteria speciated by the clinical microbiology laboratory, 8 specimens classified by the clinical microbiology laboratory as "no significant growth” and 12 specimens that were "no growth”.
  • the 58 positive specimens contained a broad diversity of uropathogens: 26 contained E. coli, 3 contained P. mirabilis, 8 contained K. pneumoniae, 1 contained E. cloacae, 5 contained E. aerogenes, 1 contained P. aeruginosa, 2 contained C. freundii, 1 contained C. koseri, 1 contained S. marcescens, 8 contained Enterococcus spp., and 2 contained S. saprophyticus.
  • Chip' was lower for Gram-positive organisms; three specimens containing Enterococd and one specimen containing S. saprophytics were falsely negative using the UNI probe pair.
  • the resulting biosensor demonstrated a high signal-to-noise ratio and low variance between duplicate sensors that is maintained despite contact with bacteria obtained directly from clinical urine specimens.
  • the sensor technique utilized here is an electrochemical sandwich assay in which target 16S rRNA is bound by both a capture and detector probe (16). The capture probe anchors the target to the sensor and the detector probe provides a means for recognizing target bound on the sensor surface.
  • This sandwich strategy has been successfully employed in several types of electrochemical sensors (5, 7, 46, 50).
  • the detector probe is linked directly or indirectly to HRP for amperometric detection of redox current (5) ⁇ (7, 50).
  • An exception to this approach is an electrochemical sandwich assay involving a ferrocene-modified detector probe (46).
  • the ferrocene moieties mediate electron transfer to the gold electrode via a phenylacetylene molecular wire embedded in the electrode's SAM.
  • the SAM reduces background current by insulating the working electrode when a potential difference is applied between the working and reference electrodes (3).
  • the sensor When our electrochemical sensor results are to be read, the sensor is placed in a potentiostat and a voltage of — 20OmV is applied between the working and auxiliary electrodes, resulting in polarization of the working electrode with negative charges.
  • HRP substrates such as TMB, then serve to transfer electrons from the electrode surface to HRP across the SAM (11, 16, 27).
  • Electrochemical sensors directly detect nucleic acid targets by hybridization, so that sensitivity and specificity problems associated with nucleic acid amplification in the presence of biological inhibitors are avoided. Accuracy of the 'UTI Chip' was demonstrated for samples with significant amounts of somatic cells, urinary protein, and ranges of pH. In contrast, PCR detection assays for urine specimens are subject to false negative results due to DNA polymerase inhibitors, which may not be removed even after a nucleic acid purification step (24, 25). Application of PCR assays to complex mixtures of nucleic acids can produce biased target amplification resulting in problems with specificity (36, 44).
  • 16S rRNA was chosen as the sensor target because it exists in high copy number in bacterial cells and is an essential component of ribosomes. 16S rDNA sequences of the relevant species of bacteria are well characterized and contain regions of diversity and conservation that are useful for molecular diagnostic purposes (37). Similar to probes used for 16S rRNA- based fluorescence in situ hybridization assays, the oligonucleotide probes that were developed for use with the electrochemical sensor array hybridize with species-specific and surface-accessible regions of the 16S rRNA target molecule.
  • the panel of probes described in the clinical feasibility study was able to detect and identify a broad range of Gram-negative uropathogens.
  • the absence of a positive signal from the UNI probe effectively rules out a Gram-negative bacterial UTI.
  • Our detection system had reduced sensitivity for Gram-positive uropathogens such as Enterococct/s species and S. saprophytics.
  • the most likely explanation for this problem is resistance of the Gram-positive cell wall to the alkaline lysis method used in our study. Development of alternative lysis methods that would be applicable to all potential uropathogens is an area of active investigation in our laboratory.
  • the optical-grade surface characteristics of the gold electrodes in our electrochemical sensor array allowed for formation of pinhole-free SAMs.
  • Highly insulating SAMs improve sensitivity by reducing sensor background and increasing the signal-to-noise ratio.
  • Sensitivity was also improved by integrating liquid-phase detector probe/target hybridization for maximum signal detection efficiency and solid-phase probe/sensor immobilization for maximum target capture efficiency (16).
  • the standard diagnostic criterion for UTI is greater than 10 5 cfu/ml from clean-catch voided urine sample (23), although actual concentrations of uropathogens in clinical urine specimens are frequently higher.
  • a robust uropathogen diagnostic system should be able to detect and quantify bacteria over a wide spectrum of bacterial concentrations and urine parameters.
  • coli contain between 5 x 10 3 to 2 x 10 4 copies of 16S rRNA per cell (31). Therefore, we estimate that the rRNA detection limit of the sensor is within femtomolar (3 x 10 "16 ) range, which compares favorably to other electrochemical DNA sensors (8). This level of sensitivity is achieved using raw bacterial lysates from actual body fluids, and represents an important advance compared to previous studies.
  • Example 2 Determinants of signal intensity for bacterial pathogen detection using an electrochemical DNA biosensor array
  • This example describes the determinants of electrochemical signal intensity using a sensor assay that involves hybridization of target rRNA to a fluorescein-modified detector probe and a biotin-modified capture probe anchored to streptavidin on the sensor surface. Signal is generated by an oxidation-reduction current produced by the action of horseradish peroxidase (HRP) conjugated to an anti-fluorescein monoclonal Fab bound to the detector probe.
  • HRP horseradish peroxidase
  • a 12-fold increase in electrochemical signal intensity for detection of Enterococcal 16S rRNA was achieved using a two-step approach involving initial treatment with Triton X-100 and lysozyme followed by alkaline lysis. This universal lysis system was shown to be effective for both Gram-positive and Gram- negative organisms. The location of fluoiecein modification was found to be a determinant of signal intensity, indicating that the distance from the sensor surface at which the HRP-Fab conjugate binds to fluorescein is important. Signal intensity was consistently higher for 3'-modified than for 5'-modified detector probes, effectively moving fluorescein away from the sensor surface.
  • Electrochemical DNA biosensors contain a recognition layer consisting of single- stranded oligonucleotides commonly known as capture probes.
  • the mechanism of detection used for the electrochemical sensors in this study involves a redox reporter molecule that binds to a second oligonucleotide referred to as a detector probe. Binding of the capture and detector probes to the nucleic acid target functions as a three- component 'sandwich' assay to generate an electronic readout via the reporter molecule.
  • Whole cell bacterial lysates are mixed with fluorescein-labeled detector probes for the initial target-probe hybridization in liquid phase.
  • the target-probe hybrids are deposited on the sensor surface for the second, solid phase hybridization with the capture probe.
  • the resulting capture-detector-target complex anchors the 16S rRNA to the sensor surface and provides for its detection (Fig. 5).
  • Coupling the reporter enzyme (anti- fluorescein monoclonal Fab fragment conjugated to horseradish peroxidase) to the detector probe generates a redox reaction at the sensor surface when the enzyme substrate is added.
  • Application of a fixed potential between the working and reference electrodes enables amperometric detection as the redox substrates are regenerated.
  • the amplitude of the electroreductioii current is related to the nucleic acid target concentration.
  • the sensitivity of electrochemical sensors is affected by the signal- to-noise ratio. Reduction of background noise is largely determined by the precision of the microfabrication process and deposition of a uniform alkanethioloate self-assembled monolayer.
  • This example examines a number of determinants of electrochemical signal intensity, namely, 1) Bacterial lysis and release of the 16S rRNA target molecules; 2) The distance of the redox reporter from the sensor surface; 3) The effect of a gap between the target hybridization regions of the capture and detector probes; and 4) Probe-probe and probe-target interactions during hybridization with mixtures of detector probes.
  • the detection system involves hybridization of a biotin-labeled oligonucleotide capture probe and a fluorescein-labeled oligonucleotide detector probe to a nucleotide target (16S rRNA in this case).
  • the biotin-label on the capture probe anchors the probe-target sandwich to the streptavidin self-assembled monolayer on the sensor surface.
  • the fluorescein-label on the detector probe is not used for optical detection, rather as a binding site for the anti-fluorescein monoclonal Fab — horseradish peroxidase (HRP) conjugate.
  • HRP horseradish peroxidase
  • Microfabricated electrochemical sensor arrays with an alkanethiolate self-assembled monolayer were obtained from GeneFluidics (Monterey Park, CA). SAM integrity was confirmed by cyclic voltammetry (CV) (1) using a 16-channel potentiostat (GeneFluidics). After CV characterization, sensor arrays were washed and dried. Washing steps were carried out by applying a stream of deionized H 2 O to the sensor surface for approximately 2-3 sec followed by 5 sec of drying under a stream of nitrogen.
  • Uropathogenic Enterococcusfecalis and Escherichia coli strains were obtained from the UCLA Uropathogen Strain Collection. Isolation of uropathogens from clinical urine specimens was approved by the UCLA Institutional Review Board. Isolates were inoculated into Brucella broth with 15% glycerol (BBL, Maryland) and were stored at -70 0 C. Bacteria were grown overnight in Luria Broth (LB) 5 inoculated into LB and grown to logarithmic phase as measured by OD 600 . Concentration of the logarithmic phase specimens was determined by serial plating, typically yielding 10 7 -10 8 bacteria/ml.
  • the detector probe/bacterial lysate mixture was incubated for 10 min at 65°C to allow hybridization of the detector probe to target rRNA.
  • 4 ⁇ l of the bacterial lysate/detector probe mixture was deposited on each of the working electrodes in the sensor array.
  • the sensor array was incubated for 10 min at 65 0 C in a humidified chamber. After washing and drying, 2.5 ⁇ l of 0.5 U/ml anti-fluorescein horseradish peroxidase (HRP) Fab conjugate (Roche, diluted in 0.5% casein in 100 mM sodium phosphate buffer, pH 7.4) were deposited on each of the working electrodes for 10-15 min.
  • HRP horseradish peroxidase
  • Oligonucleotide probe design Oligonucleotide probe design. Oligonucleotide probes were synthesized by MWG Biotech (High Point, NC). Capture probes are synthesized with 5' biotin. Detector probes with synthesized with 5'- and/ or 3'- fluorescein modifications. The fluorescein molecule was used as a binding site for the anti-fluorescein monoclonal Fab — horseradish peroxidase (HRP) conjugate redox reporter. Oligonucleotide probe pairs were designed to bind to species-specific regions of the 16S rRNA molecules of Escherichia coli and Enterococcus faecalis.
  • HRP horseradish peroxidase
  • CTGAA AGTGC TTTAC AACCC GAAGG CCTTC TTCAT (SEQ ID NO: 6)
  • CAAAG GTATT (SEQ ID NO: 21)
  • Capture probes were 5'- modified with biotin.
  • Detector probes were 5'- and/ or 3'- modified with fluorescein.
  • Test probes were 5'-modified with biotin and 3'-modified with fluorescein.
  • Probe sequence numbering based on E. coli 16S rDNA. Abbreviations for probe specificity: Enterococcus species (EF), Escherichia coli (EC), Proteus mirabilis (Pm), Pseudomonas aeruginosa (PA), the K/ebsiella-Enferobacter group (KE), the Enterobacteriaceae family (EB), and universal bacterial (UNI) probes.
  • EF Enterococcus species
  • EC Escherichia coli
  • Pm Proteus mirabilis
  • PA Pseudomonas aeruginosa
  • KE K/ebsiella-Enferobacter group
  • EB Enterobacteriace
  • this two-step process can be considered a universal lysis strategy for release of bacterial 16S rRNA.
  • Use of various concentrations of the denaturing detergent, sodium dodecyl sulfate, coupled with non-specific proteases (e.g. Proteinase K or Pronase) were not successful.
  • detector probes In the capture-detector-target complex, the fluorescein of 3'-modified detector probes would be farther away from the sensor surface than that of 5'-modified detector probes. Use of 3 '-fluorescein modified detector probes resulted in greater signal intensity than 5'- fluorescein modified detector probes for detection of Enterococcus 16S rRNA (Fig. 8). As shown in Table 6, the effect of the location of fluorescein modification was examined for a variety of detector probes and targets. Some of the detector probes (UNI, ENTBC, EC) were also modified with fluorescein at both the 5' and 3' positions.
  • FIG. 10 shows results obtained when a 7-detector-probe mixture was hybridized with 16S rRNA derived from 10 6 uropathogenic Enterococcus.
  • a 16-sensor array was prepared with immobilization of 7 capture probes on pairs of sensors in duplicate.
  • UNI capture probes were immobilized on the two negative control (NC) sensors to determine background signal levels using the 7-detector probe cocktail in the absence of target nucleic acids.
  • the positive signals (EF and UNI) can be easily distinguished from non-specific capture probes and background signal (NC).
  • Similar findings have been obtained when 16S rRNA derived from E. coli, P. mirabilis, P. aeruginosa, and K. pneumoniae was used as the target. These experiments indicate that signal intensity is not adversely affected by detector probe mixtures and that sensor specificity is a function of the immobilized capture probe.
  • This example demonstrates the determinants of signal intensity in a 40-minute DNA sandwich assay for direct molecular detection of uropathogens using a novel electrochemical sensor array.
  • This provides an integrated point-of-care diagnostic system (lab-on-a-chip) for urinary tract infections.
  • the sensor array would serve as the critical sensing component of an automated detection system when coupled with a microfluidics-based sample preparation module.
  • the studies described here demonstrate the dependence of the system on the strategy for bacterial 16S rRNA release, the distance between the capture and detector probe hybridization sites on the rRNA target, and the location of the fluorescein on the detector probe relative to the sensor surface.
  • a fundamental difference between Gram-positive and Gram-negative bacteria is the thicker peptidoglycan cell wall of Gram-positive organisms.
  • alkaline lysis is an effective lysis method for Gram-negative, but not Gram-positive, uropathogens.
  • the concentration of Triton X-100 (0.1%) was found to be a factor, since higher concentrations resulted in loss of surface tension of the crude lysate aliquot on the sensor surface and cross-contamination among adjacent sensors within the array.
  • the total lysis time of 10-minute is a significant improvement over prior reports of 'rapid' bacterial lysis, which may take up to 1 hour incubation time.
  • direct detection of bacterial 16S rRNA without the need for additional nucleic acid purification step was successfully achieved.
  • the mechanism of signal production (i.e. current output) by the electrochemical sensor used in these studies involves cycling of HRP redox reaction products driven by the applied voltage potential at the sensor surface (2, 4).
  • the HRP substrates may also have limited access into the enzyme active site if it is too close to the sensor surface and thus affecting the electron transfer between oxidized substrates and the electrodes.
  • the observations that a relatively short window of optimal distance between the HRP and the sensor surface illustrates the specificity of the detection strategy and that binding of detector probes to other regions of the 16S rRNA molecule would be unlikely contribute to signal intensity.
  • Figure 8A shows that for using EF probes of same length targeting the same region, removal of the i ⁇ terprobe gap distance ( Figure 8A, bar 1 and 2) resulted in 4-fold improvement of the signal output. Similar results are seen with removing the gap for EC probe hybridization site.
  • RNA targets with complex secondary structures such as 16S rRNA
  • binding of the first probe may result in unwinding of rRNA helix structures and stabilize binding of the second probe.
  • the thermodynamic advantages of such unwinding may be best realized with sequential binding of a flanking probe binding to a site immediately adjacent to the first probe.
  • Others have suggested the use of an unlabeled bridging oligonucleotide between capture and detector probes to improve electrochemical signals (3). RNA degradation could affect the success of the sandwich formation if the probe hybridization sites are not contiguous are situated far apart.
  • detector probes were added separately to the bacterial lysate for hybridization then deposited on the sensor surface containing the capture probe. For each capture probe, therefore, a separate detector probe is added. Since the capture probes are designed from different areas within the 16S rDNA, a 'universal' detector probe from a fixed region of the 16S rDNA is not possible since the distance between the capture probes and the universal detector probe is likely too large. We tested whether it would be possible to mix the detector probes as a cocktail.
  • Fig. 9 Representative results shown in Fig. 9 indicate that comparable positive signals are obtained with the 2-detector-probe cocktail with the appropriate target compared to using a single detector probe. Specificity of the capture probes was retained despite using detector probe combinations. The 2-detector probe mixture did not contribute to higher background signals. The use of probe mixtures would greatly simplify the detection protocol when using a multiple detector probe mixture for identification of uropathogens in a clinical urine specimen. The advantage of using a detector probe mixture is indicated in Fig. 10 in which a IJTI Chip' containing different capture probes is used to query a cultured specimen containing Enterococcus using the detector probe cocktail.
  • detector probe mixture would facilitate the design and fabrication of the microfluidics-based sample preparation module since a single reservoir and channel can be used as supposed to individual reservoir and channel for each detector probe.
  • This example examines several aspects of an electrochemical DNA biosensor system for uropathogen detection to identify the determinants of signal intensity.
  • a 'universal' lysis cocktail was developed, capable of releasing target nucleic acids from both Gram-positive and -negative uropathogens.
  • the effects of probe length, fluorescein modification position, and distance between capture and detector probe hybridization sites were examined.
  • the feasibility of a detector probe cocktail was demonstrated.
  • Our findings will improve the performance of electrochemical sensors for detection of bacterial pathogens in clinical specimens.
  • Simplified sample preparation will greatly reduce the design complexity of the microfluidics component when the sensor array is eventually integrated into an automated device. This provides for the development of a portable, point-of-care pathogen detection system that would revolutionize the diagnosis and management of infectious diseases.

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Abstract

L'invention concerne un procédé destiné à la détection d'un agent pathogène dans un échantillon. Ce procédé consiste à mettre un lysat de cet échantillon en contact avec une sonde de capture immobilisée sur un substrat. Ce lysat peut être préparé rapidement à partir d'un échantillon clinique, ce qui évite le recours à une culture d'échantillons ou à une épuration bactérienne avant l'analyse. La sonde de capture comprend un premier oligonucléotide qui s'hybride spécifiquement avec une première séquence nucléotidique de l'agent pathogène à détecter. Le lysat est mis en contact avec une sonde de détection comprenant un oligonucléotide marqué, décelable, qui s'hybride spécifiquement avec une seconde séquence nucléotidique de l'agent pathogène. La détection du second marqueur décelable combiné avec le substrat indique la présence de l'agent correspondant. L'invention concerne également des variations de ce procédé, permettant d'optimiser la détection. L'invention concerne également des sondes et des trousses destinées à être utilisées dans ce procédé.
PCT/US2005/039292 2004-11-01 2005-11-01 Procede de detection rapide specifique par espece d'agents pathogenes WO2006050275A2 (fr)

Priority Applications (2)

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US11/743,071 US7763426B2 (en) 2004-11-01 2007-05-01 Probes and methods for detection of Escheridia coli and antibiotic resistance
US12/788,101 US20110027782A1 (en) 2004-11-01 2010-05-26 Probes and methods for detection of pathogens and antibiotic resistance

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US62390304P 2004-11-01 2004-11-01
US60/623,903 2004-11-01

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103773842A (zh) * 2013-10-18 2014-05-07 南京农业大学 一种用于检测尿路感染病原菌的特异性纳米金dna探针
CN108474030A (zh) * 2015-11-27 2018-08-31 卡尤迪生物科技（北京）有限公司 核酸扩增方法和系统

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6348314B1 (en) * 1996-01-24 2002-02-19 Third Wave Technologies, Inc. Invasive cleavage of nucleic acids
US6391558B1 (en) * 1997-03-18 2002-05-21 Andcare, Inc. Electrochemical detection of nucleic acid sequences

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6348314B1 (en) * 1996-01-24 2002-02-19 Third Wave Technologies, Inc. Invasive cleavage of nucleic acids
US6391558B1 (en) * 1997-03-18 2002-05-21 Andcare, Inc. Electrochemical detection of nucleic acid sequences

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103773842A (zh) * 2013-10-18 2014-05-07 南京农业大学 一种用于检测尿路感染病原菌的特异性纳米金dna探针
CN108474030A (zh) * 2015-11-27 2018-08-31 卡尤迪生物科技（北京）有限公司 核酸扩增方法和系统

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