EP1759000A1 - Polynucleotides pour la detection d'escherichia coli - Google Patents

Polynucleotides pour la detection d'escherichia coli

Info

Publication number
EP1759000A1
EP1759000A1 EP05742611A EP05742611A EP1759000A1 EP 1759000 A1 EP1759000 A1 EP 1759000A1 EP 05742611 A EP05742611 A EP 05742611A EP 05742611 A EP05742611 A EP 05742611A EP 1759000 A1 EP1759000 A1 EP 1759000A1
Authority
EP
European Patent Office
Prior art keywords
sequence
seq
consecutive nucleotides
polynucleotide
set forth
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05742611A
Other languages
German (de)
English (en)
Other versions
EP1759000A4 (fr
Inventor
Eliane Ubalijoro
Daniel Plante
Nancy Bourassa
Hélène DUMAS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Warnex Research Inc
Original Assignee
Warnex Research Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Warnex Research Inc filed Critical Warnex Research Inc
Publication of EP1759000A1 publication Critical patent/EP1759000A1/fr
Publication of EP1759000A4 publication Critical patent/EP1759000A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention pertains to the field of detection of microbial contaminants and, in particular, the invention relates to the detection of contamination by Escherichia coli.
  • Escherichia coli strains are widely distributed within the intestinal tracts of humans and warm-blooded animals. Most E. coli strains are considered harmless members of the microflora of the intestinal tract, however, four particular groups of E. coli strains, referred to as the enterovirulent Escherichia coli group, are pathogenic and cause gastrointestinal illnesses. This bacterium is commonly associated with contamination of food and water arising from fecal contaminations and E. coli is commonly used as an indicator of fecal contamination for diagnostic purposes. [Conway, PL (1995) Microbial ecology of the human large intestine. In: Human colonic bacteria: role in nutrition, physiology, and pathology, GR Gibson and GT Macfarlane, eds. pp.1-24.
  • a number of detection technologies require the culturing of bacterial samples for time periods of up to eight days. However, in that time, the product being tested must be placed in circulation for purchase and consumption. Therefore, a system that can rapidly identify the presence of E. coli in food samples is desirable.
  • nucleic acid hybridization An additional technology utilized for detection of bacterial contamination, is nucleic acid hybridization.
  • the target sequence of interest is typically amplified and then hybridized to an oligonucleotide probe which possesses a complementary nucleic acid sequence to that of the target molecule.
  • the probe can be modified so that detection of the hybridization product may occur, for examples, the probe can be labelled with a radioisotope or fluorescent moiety.
  • E. coli nucleic acid sequences for detection of this bacterium has been described. Many of the described detection methods, however, are specific for certain strains of E. coli, such as 0157, or are not specific for E. coli. For example, U.S. Patent No. 5,654,417 describes DNA fragments useful for detecting E. coli
  • O157:H7 strains and U.S. Patent No. 6,365,723 describes genomic sequences, which can be used as diagnostic probes. These sequences are present in E.coli O157:H7 but absent from E. coli K12.
  • Desmarchelier et al. J. Clin. Microbiol. (1998) 36:1801-1804) describe a PCR-based method for detecting E. coli 0157 that involves amplification of a region of the O-antigen synthesis genes followed by gel electrophoresis and Southern blot analysis to confirm the identify of the amplified fragment. The method was capable of identifying two serotypes of E. coli 0157; the O157:H7 and O157:H- serotypes.
  • a particularly useful modification of the above technology provides for the concurrent amplification and detection of the target sequence (i.e. in "real time") through the use of specially adapted oligonucleotide probes.
  • probes include molecular beacon probes (Tyagi et al., (1996) Nature Biotechnol. 14:303- 308), TaqMan ® probes (U.S. Patent Nos. 5,691,146 and 5,876,930) and Scorpion probes (Whitcombe et al., (1999) Nature Biotechnol. 17:804-807).
  • TaqMan ® probes to detect Escherichia coli in water samples is described by Frahm and Obst inJ. Microbiol. Methods (2003) 52:123-131.
  • Molecular beacons represent a powerful tool for the rapid detection of specific nucleotide sequences and are capable of detecting the presence of a complementary nucleotide sequence even in homogenous solutions.
  • Molecular beacons can be described as hairpin stem-and-loop oligonucleotide sequences, in which the loop portion of the molecule represents a probe sequence, which is complementary to a predetermined sequence in a target nucleotide.
  • One arm of the beacon sequence is attached to a fluorescent moiety, while the other arm of the beacon is attached to a non-fluorescent quencher.
  • the stem portion of the stem-and-loop sequence holds the two arms of the beacon in close proximity. Under these circumstances, the fluorescent moiety is quenched.
  • the probe When the beacon encounters a nucleic acid sequence complementary to its probe sequence, the probe hybridizes to the nucleic acid sequence, forming a stable complex and, as a result, the arms of the probe are separated and the fluorophore emits light. Thus, the emission of light is indicative of the presence of the specific nucleic acid sequence.
  • Individual molecular beacons are highly specific for the DNA sequences they are complementary to.
  • a molecular beacon probe designed to specifically detect the E. coli O157:H7 serotype has been described (Fortin et al, (2001) Analytical Biochem. 289:281-288).
  • the probe was designed to hybridise to an amplified target sequence from the r ⁇ E O- antigen synthesis gene of E.coli O157:H7 that is either 496 base pair (bp) or 146 bp in length, depending on the primers used.
  • the probe was also able to detect E. coli O157:NM and O157:H- serotypes, but was not intended to detect other strains of E. coli.
  • the prpR gene from several bacteria, including E. coli and Salmonella typhimurium has been characterized.
  • the sequence of theprpR gene has extensive homology with numerous members of the sigma-54-dependent family of transcriptional activators.
  • the PrpR protein is believed to be a transcriptional activator for theprp operon, which encodes enzymes involved in propionate catabolism [Blattner, FR, et al., (1997) Science 277:1453-1474; HorswiU, AR, and Escalante-Semerena, JC (1997) Journal of Bacteriology. 179:928-940; Shingler, V (1996) Molecular Microbiology. 19:409-416].
  • An object of the present invention is to provide polynucleotides for the detection of Escherichia coli.
  • a combination of polynucleotides for amplification and detection of E. coli nucleic acid sequences selected from the group of: (a) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ TD NOs:2-l 1; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:2-l 1 and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 12, or the complement thereof; (b) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs
  • a method of detecting E. coli in a sample comprising the steps of: (i) contacting a sample suspected of containing, or known to contain, E.coli with a first pair of polynucleotide primers capable of amplifying a first E. coli target sequence within the prpR gene, or a second pair of polynucleotide primers capable of amplifying a second E.
  • a method of detecting E. coli in a sample comprising the steps of: (i) contacting a sample suspected of containing, or known to contain, one or more E. coli target nucleotide sequences with a combination of polynucleotides of the invention under conditions that permit amplification of said target sequence(s), and (ii) detecting any amplified target sequence(s), wherein detection of an amplified target sequence indicates the presence of E.coli species in the sample.
  • kits for the detection of E. coli comprising a combination of polynucleotides selected from the group of: (a) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of S ⁇ Q JD NOs:2-l 1 ; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of S ⁇ Q ID NOs:2- 11 and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in S ⁇ Q ID NO: 12, or the complement thereof; (b) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of S ⁇ Q ID NOs:26-35; a second polynucleotide primer comprising at least 7 consecutive nucleotides of
  • a pair of polynucleotide primers for amplification of a portion of an E. coliprpR gene comprising: (a) a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of S ⁇ Q JD NOs:2-l 1; and (b) a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of S ⁇ Q ID NOs:2-l 1.
  • a pair of polynucleotide primers for amplification of a portion of an E. coli genome comprising the 3' region of the ykgM gene
  • said pair of polynucleotide primers comprising: (a) a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of S ⁇ Q JD NOs:26-35; and (b) a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of S ⁇ Q JD NOs:26-35.
  • an isolated E. coli specific polynucleotide consisting essentially of: (a) the sequence as set forth in S ⁇ Q JD NO: 12 or S ⁇ Q ID NO:36, or a fragment of said sequence, or (b) a sequence that is the complement of (a).
  • a polynucleotide primer of between 7 and 100 nucleotides in length for the amplification of a portion of an E. coliprpR gene, said polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in S ⁇ Q ID NO: 12, or the complement thereof.
  • a polynucleotide primer of between 7 and 100 nucleotides in length for the amplification of a portion of anE. coli genome comprising the 3' region of One ykgM gene, said polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 36, or the complement thereof.
  • a polynucleotide probe of between 7 and 100 nucleotides in length for detection of E. coli, said polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 12 or 36, or the complement thereof.
  • Figure 1 presents a multiple sequence alignment showing conserved regions of a portion of the prpR gene from various E. coli strains [SEQ ID NOs:2-ll].
  • the sequences depicted represent the non-coding strand of the gene. Shaded blocks highlight the following regions: bases 469 to 490: forward primer SEQ ID NO: 14; bases 519 to 541: binding site for a molecular beacon probe [SEQ JD NO:16]; bases 571 to 588: binding site for reverse primer [SEQ ID NO:15];
  • Figure 2 presents the arrangement of PCR primers and a molecular beacon probe on the prpR gene sequence in one embodiment of the invention. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature on the PCR product generated with primers SEQ ID NOs:14 & 15;
  • Figure 3 presents the secondary structure of a molecular beacon probe in accordance with one embodiment of the invention [SEQ ID NO: 16];
  • Figure 4 presents a multiple sequence alignment of a portion of the ykgM gene and the region of the genome adjacent to the 3' end of the ykgM gene from several isolates of E. coli [SEQ JD NOs:26-35], The sequences depicted represent the non-coding strand. Shaded blocks highlight the following regions: bases 212 to 232 : forward primer
  • Figure 6 presents the secondary structure of a molecular beacon probe in accordance with one embodiment of the invention [SEQ ID NO:40];
  • Figure 7 presents (A) the sequence of a portion of an E. coliprpR gene [SEQ JD NO:l] comprising the prpR consensus sequence identified in one embodiment of the invention, (B) the sequence of the prpR conserved region (consensus sequence) [SEQ ID NO:12].
  • Figure 8 presents (A) the sequence (non-coding strand) of an E. coli ykgM gene [S ⁇ Q TD NO:25] and (B) the sequence of the E. coli ykgM consensus sequence [S ⁇ Q ID NO:36], (C) the sequence of a highly conserved region [S ⁇ Q JD NO:37] identified within the consensus sequence.
  • the present invention is based on the identification of highly conserved regions
  • consensus sequences constitute suitable target sequences for the design of primers and probes capable of specifically amplifying and detecting nucleic acids sequences from one or more E. coli strains in a test sample.
  • the present invention thus provides for primer and probe sequences capable of amplifying and/or detecting all or part of an E. coli consensus sequence that are suitable for use in detecting the presence of various E. coli strains in a sample, such as a clinical sample, microbiological pure culture, or a sample related to food, environmental or pharmaceutical quality control processes.
  • the present invention contemplates methods of detecting E. coli strains in a sample using primers and/or probes targeting a single consensus sequence, as well as methods using combinations of primer and/or probe sets, wherein each set targets the same or different consensus sequences. In one embodiment, the use of such combinations increases the specificity of detection.
  • the invention provides diagnostic assays that can be carried out in real time and addresses the need for rapid detection of E. coli in a variety of biological samples.
  • the primers and probes provided by the present invention are capable of distinguishing E. coli target sequences from Shigella sequences, i.e. specifically amplify and/or detect E. co/f sequences but not Shigella sequences.
  • the primers and probes of the invention demonstrate a specificity for E. coli nucleic acid sequences of at least 95%, as defined herein. In one embodiment, the primers and probes of the invention demonstrate a specificity for E. coli nucleic acid sequences of at least 97%. In another embodiment, the primers and probes of the invention demonstrate a specificity for E. coli nucleic acid sequences of at least 98%.. In further embodiments, the primers and probes of the invention demonstrate a specificity for E. coli nucleic acid sequences of at least 99%, and at least 99.5%.
  • combinations of primer and probe sets that target the same or different E. coli consensus sequences are also contemplated for the detection of E. coli. Such combinations can demonstrate greater specificity than one, or both, of the sets of primers/probes alone.
  • a combination of primer and/or probe sets that target different consensus sequences and which demonstrate a specificity for E. coli of at least 98%.
  • a combination of primer and/or probe sets that target different consensus sequences and which demonstrate a specificity forE. coli of at least 99%.
  • the primers and probes demonstrate a sensitivity in detecting E. coli strains of at least 90%. In another embodiment, the primers and probes demonstrate a sensitivity of at least 91%. In further embodiments, the primers and probes demonstrate a sensitivity of at least 92%.
  • the present invention provides for a combination of primer and probe sets that target different consensus sequences and which demonstrate a sensitivity of at least 95%.
  • a combination of primer and probe sets that target different consensus sequences and which demonstrate a sensitivity of at least 96% there are provided combinations of primer and probe sets that target different consensus sequences and which demonstrate a sensitivity of at least 97%, and at least 98%.
  • oligonucleotide and “polynucleotide” as used interchangeably in the present application refer to a polymer of greater than one nucleotide in length of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), hybrid RNA/DNA, modified RNA or DNA, or RNA or DNA mimetics.
  • the polynucleotides may be single- or double-stranded.
  • the terms include polynucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as polynucleotides having non-naturally-occurring portions which function similarly.
  • backbone backbone linkages
  • Such modified or substituted polynucleotides are well-known in the art and for the purposes of the present invention, are referred to as "analogues.”
  • primer and “polynucleotide primer,” as used herein, refer to a short, single-stranded polynucleotide capable of hybridizing to a complementary sequence in a nucleic acid sample.
  • a primer serves as an initiation point for template- dependent nucleic acid synthesis. Nucleotides are added to a primer by a nucleic acid polymerase in 1 accordance with the sequence of the template nucleic acid strand.
  • a “primer pair” or “primer set” refers to a set of primers including a 5' upstream primer that hybridizes with the 5' end of the sequence to be amplified and a 3 ' downstream primer that hybridizes with the complementary 3 ' end of the sequence to be amplified.
  • forward primer refers to a primer which anneals to the 5 ' end of the sequence to be amplified.
  • reverse primer refers to a primer which anneals to the complementary 3 ' end of the sequence to be amplified.
  • probe and “polynucleotide probe,” as used herein, refer to a polynucleotide used for detecting the presence of a specific nucleotide sequence in a sample. Probes specifically hybridize to a target nucleotide sequence, or the complementary sequence thereof, and can be single- or double-stranded.
  • hybridize refers to the ability of a polynucleotide to bind detectably and specifically to a target nucleotide sequence.
  • Polynucleotides specifically hybridize to target nucleotide sequences under hybridization and wash conditions that minimize appreciable amounts of detectable binding to non-specific nucleic acids.
  • High stringency conditions can be used to achieve specific hybridization conditions as is known in the art.
  • hybridization and washing are performed at high stringency according to conventional hybridization procedures and employing one or more washing step in a solution comprising 1-3 x SSC, 0.1-1% SDS at 50-70°C for 5-30 minutes.
  • specificity refers to the ability of a primer or primer pair to amplify, or a probe to detect, nucleic acid sequences from E. coli but not other bacterial species. "% specificity" is defined by a negative validation test as described herein whereby the primers and/or probe are tested against a panel of at least 100 bacterial species other than E. coli. Thus, for example, a pair of primers that does not amplify any nucleic acid sequences from the panel of bacterial species would be defined as demonstrating 100% specificity and a pair of primers that amplified a nucleic acid sequence from one bacterial species in a panel of 100 species would be defined as demonstrating 99% specificity.
  • sensitivity refers to the ability of a primer or primer pair to amplify, or a probe to detect, nucleic acid sequences from a range of E.coli strains.
  • "% sensitivity" is defined by a positive validation test as described herein whereby the primers and/or probe are tested against a panel of at least 100 E. coli strains.
  • a pair of primers that amplifies nucleic acid sequences from all E. coli strains in the panel would be defined as demonstrating 100% sensitivity and a pair of primers that amplified nucleic acid sequences from ninety E. coli strains in a panel of 100 strains would be defined as demonstrating 90% sensitivity.
  • corresponding to refers to a polynucleotide sequence that is identical to all or a portion of a reference polynucleotide sequence.
  • complementary to is used herein to indicate that the a polynucleotide sequence is identical to all or a portion of the complementary strand of a reference polynucleotide sequence.
  • the nucleotide sequence "TATAC” corresponds to a reference sequence "TATAC” and is complementary to a reference sequence "GTATA.”
  • hairpin or “hairpin loop” refer to a single strand of DNA or RNA, the ends of which comprise complementary sequences, whereby the ends anneal together to form a "stem” and the region between the ends is not annealed and forms a "loop.”
  • Some probes, such as molecular beacons have such "hairpin” structure when not hybridized to a target sequence.
  • the loop is a single-stranded structure containing sequences complementary to the target sequence, whereas the stem self-hybridises to form a double-stranded region and is typically unrelated to the target sequence, however, nucleotides that are both complementary to the target sequence and that can self-hybridise can also be included in the stem region.
  • target sequence or “target nucleotide sequence,” as used herein, refer to a particular nucleic acid sequence in a test sample to which a primer and/or probe is intended to specifically hybridize.
  • a “target sequence” is typically longer than the primer or probe sequence and thus can contain multiple “primer target sequences” and “probe target sequences.”
  • a target sequence may be single- or double-stranded.
  • primer target sequence refers to a nucleic acid sequence in a test sample to which a primer is intended to specifically hybridize.
  • probe target sequence refers to a nucleic acid sequence in a test sample to which a probe is intended to specifically hybridize.
  • the term "about” refers to a +/-10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • prpR consensus sequence This sequence is referred to herein as the prpR consensus sequence.
  • An exemplary multiple sequence alignment of portions of the non-coding strand of the prpR gene is shown in Figure 1.
  • One skilled in the art will appreciate that similar alignments can be conducted using shorter sequences and or the coding strand of the gene, such as the region shown in Figure 7A [SEQ ID NO:l].
  • the present invention provides isolated E. coli specific polynucleotides consisting of the consensus sequence as set forth in SEQ ID NO: 12 (shown in Figure 7), or in SEQ ID NO:36 (shown in Figure 8), or the complement of these sequences, that can be used as target sequences for the design of primers and or probes for the specific detection of E. coli.
  • target sequences suitable for the specific detection of E. coli comprise at least 60% of the sequence set forth in S ⁇ Q ID NO: 12 or 36, or the complement thereof.
  • the target sequences comprise at least 75% of the sequence set forth in SEQ JD NO:12 or 36, or the complement thereof.
  • the target sequences comprise at least 80% of the sequence set forth in SEQ ID NO: 12 or 36, or the complement thereof.
  • Target sequences comprising at least 85%, 90%, 95% and 98% of the sequence set forth in SEQ JD NO: 12 or 36, or the complement thereof, are also contemplated.
  • portions of the consensus sequences can be expressed in terms of consecutive nucleotides of the sequence set forth in SEQ ID NO: 12 or 36.
  • target sequences comprising portions of the consensus sequences that include at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 and at least 105 consecutive nucleotides of the sequence set forth in SEQ ID NO.T2 or 36, or the complement thereof, are contemplated.
  • at least 65 consecutive nucleotides it is meant that the target sequence may comprise any number of consecutive nucleotides between 65 and the full length of the sequence set forth in SEQ JD NO:12 or 36 (i.e.
  • this range includes portions of the consensus sequence that comprise at least 66, at least 67, at least 68, at least 69, etc, consecutive nucleotides of the sequence set forth in SEQ ID NO:12 or 36.
  • the highly conserved region of the prpR consensus sequence thus identified is 23 nucleotides in length and has a sequence corresponding to SEQ ID NO: 13 (as shown below and in Figure 7C).
  • the highly conserved region of the ykgM consensus sequence is 28 nucleotides in length and has a sequence corresponding to SEQ ID NO:37 (as shown below and in Figure 8C).
  • one embodiment of the present invention provides for target sequences that comprise all or a portion of a sequence corresponding to SEQ ID NO: 13 or 37, or the complement thereof.
  • the target sequences may include additional nucleotide sequences that are found upstream and/or downstream of the consensus sequence in the E. coli genome.
  • the assays provided by the present invention typically include an amplification step, it may be desirable to select an overall length for the target sequence such that the assay can be conducted fairly rapidly.
  • the target sequence typically has an overall length of less than about 500 nucleotides.
  • the target sequence has an overall length of less than about 400 nucleotides.
  • the target sequence has an overall length of less than about 350 nucleotides.
  • the target sequence has an overall length of less than or equal to about 300, about 250, about 200, and about 150 nucleotides.
  • the present invention provides for polynucleotides for the amplification and/or detection of E. coli nucleic acids in a sample.
  • the polynucleotide primers and probes of the invention comprise a sequence that corresponds to or is complementary to the portion of the E. coliprpR gene shown in any one of SEQ ID NOs:2-l 1 or the region of the E. coli genome encompassing the 3 ' end of the ykgM gene as shown in any one of SEQ JD NOs:26-35 (referred to herein as the ykgM 3' region) that are capable of specifically hybridizing to E. coli nucleic acids.
  • the polynucleotides of the invention comprise a sequence that corresponds to or is complementary to a portion of the E. coliprpR gene sequence as set forth in SEQ JD NO: 1 or the regions of the ykgM 3 ' region as set forth in SEQ ID NOs:26-35.
  • the polynucleotides of the present invention are generally between about 7 and about 100 nucleotides in length.
  • the optimal, length for a selected polynucleotide will vary depending on its intended application (i.e. primer, probe or combined primer/probe) and on whether any additional features, such as tags, self-complementary "stems" and labels (as described below), are to be incorporated.
  • the polynucleotides are between about 10 and about 100 nucleotides in length.
  • the polynucleotides are between about 12 and about 100 nucleotides in length.
  • the polynucleotides are between about 12 and about 50 nucleotides and between about 12 and about 40 nucleotides in length.
  • the entire length of the polynucleotide primer or probe does not need to correspond to or be complementary to its target sequence within the E. coli prpR gene or ykgM 3 ' region in order to specifically hybridize thereto.
  • the polynucleotide primers and probes may comprise nucleotides at the 5 ' and/or 3 ' termini that are not complementary to the target sequence.
  • Such non-complementary nucleotides may provide additional functionality to the primer/probe, for example, they may provide a restriction enzyme recognition sequence or a "tag" that facilitates detection, isolation or purification.
  • the additional nucleotides may provide a self-complementary sequence that allows the primer/probe to adopt a hairpin configuration. Such configurations are necessary for certain probes, for example, molecular beacon and Scorpion probes.
  • one or more position within the polynucleotide can be degenerate, i.e. can be filled by one of two or more alternate nucleotides.
  • certain positions in a gene can vary in the nucleotide that is present at that position depending on the strain of bacteria that the gene originated from.
  • position 487 of the alignment shown in Figure 1 can contain a thymine (“T") nucleotide or a cytosine (“C”) nucleotide depending on which strain of E. coli the prpR gene originates from.
  • T thymine
  • C cytosine
  • a "degenerate" primer or probe can be designed to target this sequence that contains either a T or a C at the position corresponding to position 487 in the alignment.
  • Degenerate primers or probes are typically prepared by synthesising a "pool" of polynucleotide primers or probes that contains approximately equal amounts of, for example, a polynucleotide containing a T at the degenerate position and a polynucleotide containing a C at the degenerate position.
  • the polynucleotide primers and probes of the invention comprise a sequence of at least 7 consecutive nucleotides that correspond to or are complementary to a portion of the E.
  • any one of S ⁇ Q ID NOs:2-l 1 or 26-35 the optimal length of the sequence corresponding or complementary to the target E. coli sequences will be dependent on the specific application for the polynucleotide, for example, whether it is to be used as a primer or a probe and, if the latter, the type of probe. Optimal lengths can be readily determined by the skilled artisan.
  • the polynucleotides comprise at least 10 consecutive nucleotides corresponding or complementary to a portion of the E. coli sequences shown in any one of S ⁇ Q ID NOs:2-l 1 or 26-35. In another embodiment, the polynucleotides comprise at least 12 consecutive nucleotides corresponding or complementary to a portion of the E. coli sequences shown in any one of S ⁇ Q JD NOs:2-l 1 or 26-35. In a further embodiment, the polynucleotides comprise at least 15 consecutive nucleotides corresponding or complementary to a portion of the E. coli sequences shown in any , one of S ⁇ Q JD NOs:2-ll or 26-35.
  • Polynucleotides comprising at least 18, at least 20, at least 22, at least 24, at least 26, at least 27 and at least 28 consecutive nucleotides corresponding or complementary to a portion of the E. coli sequences shown in any one of S ⁇ Q JD NOs:2-l 1 or 26-35 are also contemplated.
  • polynucleotides of the invention are set forth in Table 1. Further non-limiting examples for the polynucleotides of the invention include polynucleotides that comprise at least 7 consecutive nucleotides of any one of S ⁇ Q ID NOs:14, 15, 17, 19, 20, 22, 24, 38, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63.
  • the polynucleotide primers of the present invention comprise a sequence that corresponds to or is complementary to a portion of the E. coli sequences shown in any one of SEQ JD NOs:2-l 1 or 26-35.
  • the primers are capable of amplifying a target nucleotide sequence comprising all or a portion of the 120 nucleotide consensus sequence as shown in SEQ ID NO: 12, or comprising all or a portion of the 144 nucleotide consensus sequence as shown in SEQ ID NO:36.
  • the present invention provides for primer pairs capable of amplifying an E. coli target nucleotide sequence, wherein the target sequence is less than about 500 nucleotides in length and comprises at least 65 , consecutive nucleotides of SEQ ID NO: 12 or 36, or the complement thereof.
  • pairs of primers can be selected to comprise a forward primer corresponding to a portion of the E. coliprpR gene sequence upstream of or within the region of the gene corresponding to SEQ ID NO: 12 and a reverse primer that it is complementary to a portion of the E. coliprpR gene sequence downstream of or within the region of the gene corresponding to SEQ ID NO: 12.
  • pairs of primers can be selected to comprise a forward primer corresponding to a portion of the E. coli ykgM ' 3' ' region upstream of or within the region corresponding to SEQ JD NO:36 and a reverse primer that it is complementary to a portion of the E. coli ykgM 3 ' region downstream of or within the region corresponding to SEQ ID NO:36.
  • the primers comprise at least 7 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs:2-l 1 or 26-35. In one embodiment, the primers comprise at least 7 consecutive nucleotides of SEQ ID NOs:2-l 1 or 26-35. In one embodiment, the primers comprise at least 7 consecutive nucleotides of SEQ ID NOs:2-l 1 or 26-35. In one embodiment, the primers comprise at least 7 consecutive nucleotides of SEQ ID NOs:2-l 1 or 26-35. In one embodiment, the primers comprise at least 7 consecutive nucleotides of SEQ ID NOs:2-l 1 or 26-35. In one embodiment, the primers comprise at least 7 consecutive nucleotides of SEQ ID NOs:2-l 1 or 26-35. In one embodiment, the primers comprise at least 7 consecutive nucleotides of SEQ ID NOs:2-l 1 or 26-35. In one embodiment, the primers comprise at least 7 consecutive nucleotides of SEQ ID NOs:2-l
  • the primers comprise at least 7 consecutive nucleotides of the sequence set forth in SEQ ID NO:12 or 36.
  • primer pairs can be readily determined by a worker skilled in the art.
  • primers are selected that specifically hybridize to the appropriate region of the E. coli genome, as described above.
  • primers are selected that contain minimal sequence repeats and that demonstrate a low potential of forming dimers, cross dimers, or hairpin structures and of cross priming.
  • Such properties can be determined by methods known in the art, for example, using the computer modelling program OLIGO ® Primer Analysis Software (distributed by National Biosciences, Inc., Madison, MN).
  • Non-limiting examples of suitable primer sequences include sequnces that comprise SEQ ID NO: 14, 15, 20, 38 or 39 shown in Table 1, as well as primers comprising at least 7 consecutive nucleotides of any one of SEQ JD NOs:14, 15, 17, 19, 20, 22, 24, 38, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 or 63.
  • the probe polynucleotides of the invention are designed to correspond to or be complementary to a portion of the consensus sequences shown in SEQ JD NO: 12 and 36.
  • the probe polynucleotides therefore, comprise at least 7 consecutive nucleotides of the sequence set forth in SEQ JD NO: 12 or 36, or the complement thereof.
  • highly conserved regions were identified within the E.coli consensus sequences.
  • the present invention provides for probe polynucleotides comprising at least 7 consecutive nucleotides of the sequence set forth in SEQ ID NO: 13 or 37, or the complement thereof.
  • Non-limiting examples of suitable probe sequences include sequences that comprise SEQ JD NO:17, 19, 22, 24, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 or 63 shown in Table 1, as well as probes comprising at least 7 consecutive nucleotides of any one of SEQ ID NOs:14, 15, 17, 19, 20, 22, 24, 38, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 or 63.
  • the probes comprises at least 7 consecutive nucleotides of any one of SEQ ID NOs: 17, 19, 22, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 or 63.
  • the probe may be a hybridization probe, the binding of which to a target nucleotide sequence can be detected using a general DNA binding dye such as ethidium bromide, SYBR ® Green, SYBR ® Gold and the like.
  • the probe can incorporate one or more detectable labels. Detectable labels are molecules or moieties a property or characteristic of which can be detected directly or indirectly and are chosen such that the ability of the probe to hybridize with its target sequence is not affected.
  • Labels suitable for use with the probes of the present invention include those that can be directly detected, such as radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles, and the like.
  • directly detectable labels may require additional components, such as substrates, triggering reagents, light, and the like to enable detection of the label.
  • the present invention also contemplates the use of labels that are detected indirectly.
  • Indirectly detectable labels are typically specific binding members used in conjunction with a "conjugate” that is attached or coupled to a directly detectable label. Coupling chemistries for synthesising such conjugates are well-known in the art and are designed such that the specific binding property of the specific binding member and the detectable property of the label remain intact.
  • “specific binding member” and “conjugate” refer to the two members of a binding pair, i.e. two different molecules, where the specific binding member binds specifically to the probe, and the "conjugate” specifically binds to the specific binding member. Binding between the two members of the pair is typically chemical or physical in nature.
  • binding pairs include, but are not limited to, antigens and antibodies; avidin/streptavidin and biotin; haptens and antibodies specific for haptens; complementary nucleotide sequences; enzyme cofactors / substrates and enzymes; and the like.
  • the probe is labelled with a fluorophore.
  • the probe may additionally incorporate a quencher for the fluorophore.
  • Fluorescently labelled probes can be particularly useful for the real-time detection of target nucleotide sequences in a test sample.
  • Examples of probes that are labelled with both a fluorophore and a quencher that are contemplated by the present invention include, but are not limited to, molecular beacon probes and TaqMan ® probes. Such probes are well known in the art (see for example, U.S. Patent Nos.
  • a molecular beacon probe is a hairpin shaped oligonucleotide sequence, which undergoes a conformational change when it hybridizes to a perfectly complementary target sequence.
  • the secondary structure of a typical molecular beacon probe includes a loop sequence, which is capable of hybridizing to a target sequence and a pair of arm (or "stem") sequences.
  • One arm is attached to a fluorophore, while the other arm is attached to a quencher.
  • the arm sequences are complementary to each other so as to enable the arms to hybridize together to form a molecular duplex and the beacon adopts a hairpin conformation in which the fluorophore and quencher are in close proximity and interact such that emission of fluorescence is prevented.
  • Hybridization between the loop sequence and the target sequence forces the molecular beacon probe to undergo a conformational change in which arm sequences are forced apart and the fluorophore is physically separated from the quencher. As a result, the fluorescence of the fluorophore is restored. The fluorescence generated can be monitored and related to the presence of the target nucleotide sequence. If no target sequence is present in the sample, no fluorescence will be observed.
  • This methodology as described further below, can also be used to quantify the amount of target nucleotide in a sample.
  • Figures 3 and 6 depict the secondary structure of exemplary hairpin loop molecular beacons having sequences corresponding to SEQ ID NO: 16 and 40, respectively.
  • Wavelength-shifting molecular beacon probes which incorporate two fluorophores, a "harvester fluorophore and an “emitter” fluorophore (see, Kramer, et al, (2000) Nature Biotechnology, 18:1191-1196) are also contemplated.
  • FRET fluorescence resonance energy transfer
  • TaqMan ® probes are dual-labelled fluorogenic nucleic acid probes that function on the same principles as molecular beacons.
  • TaqMan ® probes are composed of a polynucleotide that is complementary to a target sequence and is labelled at the 5' terminus with a fluorophore and at the 3' terminus with a quencher.
  • TaqMan ® probes like molecular beacons, are typically used as real-time probes in amplification reactions. In the free probe, the close proximity of the fluorophore and the quencher ensures that the fluorophore is internally quenched.
  • the probe is cleaved by the 5' nuclease activity of the polymerase and the fluorophore is released.
  • the released fluorophore can then fluoresce and produce a detectable signal.
  • Linear probes comprising a fluorophore and a high efficiency dark quencher, such as the Black Hole Quenchers (BHQTM; Biosearch Technologies, Inc., Novato, CA) are also contemplated.
  • BHQTM Black Hole Quenchers
  • the high quenching efficiency and lack of native fluorescence of the BHQTM dyes allows "random-coil" quenching to occur in linear probes labelled at one terminus with a fluorophore and at the other with a BHQTM dye thus ensuring that the fluorophore does not fluoresce when the probe is in solution.
  • the probe stretches out spatially separating the fluorophore and quencher and allowing the fluorophore to fluoresce.
  • the BHQTM dyes can also be used as the quencher moiety in molecular beacon or TaqMan ® probes.
  • two fluorescently labelled probes that amieal to adjacent regions of the target sequence can be used.
  • One of these probes a donor probe
  • a donor fluorophore such as fluorescein
  • the acceptor probe is labelled at the 5' end with an acceptor fluorophore, such as LC Red 640 or LC Red 705.
  • an acceptor fluorophore such as LC Red 640 or LC Red 705.
  • primers and probes are capable of functioning as both primer and probe in an amplification reaction.
  • combined primer/probe polynucleotides include, but are not limited to, Scorpion probes, duplex Scorpion probes, LuxTM primers and AmplifluorTM primers.
  • Scorpion probes consist of, from the 5' to 3' end, (i) a fluorophore, (ii) a specific probe sequence that is complementary to a portion of the target sequence and is held in a hairpin configuration by complementary stem loop sequences, (iii) a quencher, (iv) a PCR blocker (such as, hexethylene glycol) and (v) a primer sequence. After extension of the primer sequence in an amplification reaction, the probe folds back on itself so that the specific probe sequence can bind to its complement within the same DNA strand. This opens up the hairpin and the fluorophore can fluoresce.
  • Duplex Scorpion probes are a modification of Scorpion probes in which the fluorophore- coupled probe/primer containing the PCR blocker and the quencher-coupled sequence are provided as separate complementary polynucleotides. When the two polynucleotides are hybridized as a duplex molecule, the fluorophore is quenched. Upon dissociation of the duplex when the primer/probe binds the target sequence, the fluorophore and quencher become spatially separated and the fluorophore fluoresces.
  • the Amplifluor Universal Detection System also employs fluorophore/quencher combinations and is commercially available from Chemicon International (Temecula, CA).
  • LuxTM primers incorporate only a fluorophore and adopt a hairpin structure in solution that allows them to self-quench. Opening of the hairpin upon binding to a target sequence allows the fluorophore to fluoresce.
  • Suitable fluorophores and/or quenchers for use with the polynucleotides of the present invention are known in the art (see for example, Tyagi et al, Nature Biotechnol, 16:49-53 (1998); Marras et al, Genet. Anal: Biomolec. Eng., 14:151-156 (1999)). Many fluorophores and quenchers are available commercially, for example from Molecular Probes (Eugene, OR) or Biosearch Technologies, Inc. (Novato, CA).
  • fluorescein and fluorescein derivatives such as 6-carboxyfluoroscein (FAM), 5 '-tetrachlorofluorescein phosphoroamidite (TET), tetrachloro-6- carboxyfluoroscein, VIC and JOE, 5-(2'-aminoethyl) aminonaphthalene-1-sulphonic acid (EDANS), coumarin and coumarin derivatives, Lucifer yellow, Texas red, tetramethylrhodamine, 5-carboxyrhodamine, cyanine dyes (such as Cy5) and the like.
  • FAM 6-carboxyfluoroscein
  • TET 5 '-tetrachlorofluorescein phosphoroamidite
  • VIC and JOE tetrachloro-6- carboxyfluoroscein
  • EDANS 5-(2'-aminoethyl) aminonaphthalene-1-sulphonic acid
  • Pairs of fluorophores suitable for use as FRET pairs include, but are not limited to, fluorescein/rh ⁇ damine, fluorescein/Cy5, fluorescein/Cy5.5, fluorescein/LC Red 640, fluorescein/LC Red 750, and phycoerythrin/Cy7.
  • Quenchers include, but are not limited to, 4'-(4-dimethylaminophenylazo)benzoic acid (DABCYL), 4- dimethylaminophenylazophenyl-4 -maleimide (D ABMI), tetramethylrhodamine, carboxytetramethylrhodamine (TAMRA), BHQTM dyes and the like.
  • the polynucleotides can be prepared using conventional solid-phase synthesis using commercially available equipment, such as that available from Applied Biosystems USA Inc. (Foster City, California), DuPont, (Wilmington, Del.), or Milligen (Bedford, Mass.). Methods of coupling fluorophores and quenchers to nucleic acids are also in the art.
  • the probe polynucleotide is a molecular beacon.
  • molecular beacons are at least 17 nucleotides in length.
  • the molecular beacon probe is typically between about 17 and about 40 nucleotides in length.
  • the loop sequence that corresponds to or is complementary to the target sequence typically is about 7 to about 32 nucleotides in length, while the stem (or arm) sequences are each between about 4 and about 9 nucleotides in length.
  • part of the stem sequences of a molecular beacon may also be complementary to the target sequence.
  • the loop sequence of the molecular beacon is between about 10 and about 32 nucleotides in length. In other embodiments, the loop sequence of the molecular beacon is between about 15 and about 30 nucleotides in length.
  • the loop region of the molecular beacon probe comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 12 or 36, or the complement thereof. In another embodiment of the present invention, the loop region of the molecular beacon probe comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ JD NO: 13 or 37, or the complement thereof. In a specific embodiment, the loop region of the molecular beacon probe comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ JD NO:17, 19, 22, 24, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 or 63, or the complement thereof. Amplification and Detection
  • E. coli detection involves subjecting a test sample to an amplification reaction in order to obtain an amplification product, or amplicon comprising the target sequence.
  • an "amplification reaction” refers to a process that increases the number of copies of a particular nucleic acid sequence by enzymatic means.
  • Amplification procedures are well-known in the art and include, but are not limited to, polymerase chain reaction (PCR), TMA, rolling circle amplification, nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA) and Q-beta replicase amplification.
  • PCR polymerase chain reaction
  • TMA rolling circle amplification
  • NASBA nucleic acid sequence based amplification
  • SDA strand displacement amplification
  • Q-beta replicase amplification Q-beta replicase amplification.
  • SDA primers comprise additional nucleotides near the 5' end that constitute a recognition site for a restriction endonuclease.
  • NASBA primers comprise additional nucleotides near the 5' end that are not complementary, to the target sequence but which constitute an RNA polymerase promoter.
  • the target sequence is amplified by PCR.
  • PCR is a method known in the art for amplifying a nucleotide sequence using a heat stable polymerase and a pair of primers, one primer (the forward primer) complementary to the (+)-strand at one end of the sequence to be amplified and the other primer (the reverse primer) complementary to the (-)- strand at the other end of the sequence to be amplified.
  • Newly synthesized DNA strands can subsequently serve as templates for the same primer sequences and successive rounds of strand denaturation, primer annealing, and strand elongation, produce rapid and highly specific amplification of the target sequence.
  • PCR can thus be used to detect the existence of a defined sequence in a DNA sample.
  • PCR refers to the various forms of PCR known in the art including, but not limited to, quantitative PCR, reverse-transcriptase PCR, real-time PCR, hot start PCR, long PCR, LAPCR, multiplex PCR, touchdown PCR, and the like.
  • Real-time PCR refers to a PCR reaction in which the amplification of a target sequence is monitored in real time by, for example, the detection of fluorescence emitted by the binding of a labelled probe to the amplified target sequence.
  • the present invention thus provides for amplification of a portion of an E. coliprpR gene of less than about 500 nucleotides in length and comprising at least 65 consecutive nucleotides of the sequence set forth in S ⁇ Q JD NO.T2 using a pair of polynucleotide primers, each member of the primer pair comprising at least 7 nucleotides of the sequence as set forth in any one of S ⁇ Q ID NOs:2-l 1, or the complement thereof.
  • the present invention further provides for amplification of a portion of an E.
  • coli ykgM 3 region of less than about 500 nucleotides in length and comprising at least 65 consecutive nucleotides of the sequence set forth in S ⁇ Q ID NO:36 using a pair of polynucleotide primers, each member of the primer pair comprising at least 7 nucleotides of the sequence as set forth in any one of S ⁇ Q JD NOs:26-35, or the complement thereof.
  • the product of the amplification reaction can be detected by a number of means known to individuals skilled in the art. Examples of such detection means include, for example, gel electrophoresis and/or the use of polynucleotide probes. In one embodiment of the invention, the amplification products are detected through the use of polynucleotide probes. Such polynucleotide probes are described in detail above.
  • a further embodiment of the invention therefore, provides for amplification and detection of a portion of an E. coliprpR gene of less than about 500 nucleotides in length and comprising at least 65 consecutive nucleotides of the sequence set forth in SEQ JD NO: 12 using a combination of polynucleotides, the combination comprising one or more polynucleotide primers comprising at least 7 nucleotides of the sequence as set forth in any one of SEQ JD NOs:2-l 1, or the complement thereof, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 12, or the complement thereof.
  • the present invention provides for amplification and detection of a portion of an E. coli ykgM 3 ' region of less than about 500 nucleotides in length and comprising at least 65 consecutive nucleotides of the sequence set forth in SEQ JD NO: 36 using a combination of polynucleotides, the combination comprising one or more polynucleotide primers comprising at least 7 nucleotides of the sequence as set forth in any one of SEQ JD NOs:26-35, or the complement thereof, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ JD NO:36, or the complement thereof.
  • the present invention provides for concurrent amplification and detection of a portion of an E. coliprpR gene and a portion of an E. coli ykgM 3 ' region using primers and probes as described above.
  • the present invention thus provides for methods to specifically amplify and detect E. coli nucleic acid sequences in a test sample in a single tube format using the polynucleotide primers, and optionally one or more probes, described herein.
  • Such methods may employ dyes, such as SYBR ® Green or SYBR ® Gold that bind to the amplified target sequence, or an antibody that specifically detects the amplified target sequence.
  • the dye or antibody is included in the reaction vessel and detects the amplified sequences as it is formed.
  • a labelled polynucleotide probe such as a molecular beacon or TaqMan® probe
  • a labelled polynucleotide probe distinct from the primer sequences, which is complementary to a region of the amplified sequence, may be included in the reaction, or one of the primers may act as a combined primer/probe, such as a Scorpion probe.
  • test sample as used herein is a biological sample suspected of containing, or known to contain, one or more E. coli target nucleotide sequences.
  • a method using the polynucleotide primers and probes or primer/probes is provided to specifically amplify and detect an E.coli target nucleotide sequence in a test sample, the method generally comprising the steps of:
  • the method employs one or more probe capable of specifically hybridising to a portion of an E.coli prpR target nucleotide sequence and one or more polynucleotide primer capable of amplifying said portion of the prpR target nucleotide sequence, in combination with one or more probe capable of specifically hybridising to a portion of an E. coli ykgM target nucleotide sequence and one or more polynucleotide primer capable of amplifying said portion of the ykgM target nucleotide sequence.
  • the method employs one or more labelled probes in step (a).
  • amplification reagents includes conventional reagents employed in amplification reactions and includes, but is not limited to, one or more enzymes having nucleic acid polymerase activity, enzyme cofactors (such as magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, nucleotides such as deoxynucleotide triphosphates (dNTPs; for example, deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and deoxythymidine triphosphate) and other reagents that modulate the activity of the polymerase enzyme or the specificity of the primers.
  • enzyme cofactors such as magnesium or nicotinamide adenine dinucleotide (NAD)
  • NAD nicotinamide adenine dinucleotide
  • salts such as magnesium or nicotinamide adenine dinucleotide (NAD)
  • step (b) of the above method can be repeated several times prior to step (c) by thermal cycling the reaction mixture by techniques known in the art and that steps (b), (c) and (d) may take place concurrently such that the detection of the amplified sequence takes place in real time.
  • the polynucleotide probe may be a combined primer/probe, or it may be a separate polynucleotide probe, in which case two different polynucleotide primers are used.
  • test sample may undergo enrichment, extraction and/or purification steps to isolate nucleic acids therefrom prior to the amplification reaction, and/or the amplified product may be submitted to purification/isolation steps or further amplification prior to detection, and/or the results from the detection step (d) may be analysed in order to quantify the amount of target present in the sample or to compare the results with those from other samples.
  • the method is a real-time PCR assay utilising two polynucleotide primers and a molecular beacon probe.
  • the real-time PCR assay employs a combination of two polynucleotide primers and a molecular beacon probe for amplification and detection of aprpR target sequence with two polynucleotide primers and a molecular beacon probe for amplification and detection of a ykgM target sequence.
  • the present invention provides for diagnostic assays using the polynucleotide primers and/or probes that can be used for highly specific and sensitive detection of one or more E. coli strains in a test sample.
  • the diagnostic assays comprise amplification and detection of E. coli nucleic acids as described above.
  • the diagnostic assays can be qualitative or quantitative and can involve real time monitoring of the amplification reaction or conventional end-point monitoring.
  • the invention provides for diagnostic assays that do not require post-amplification manipulations and minimise the amount of time required to conduct the assay.
  • a diagnostic assay utilising the primers and probes described herein, that can be completed using real time PCR technology in about 54 hours and generally less that 24 hours.
  • the present invention provides a rapid and sensitive diagnostic assay for the detection of E. coli contamination of a food sample.
  • Foods that can be analysed using the diagnostic assays include, but are not limited to, dairy products such as milk, including raw milk, cheese, yoghurt, ice cream and cream; raw, cooked and cured meats and meat products, such as beef, pork, lamb, mutton, poultry (including turkey, chicken), game (including rabbit, grouse, pheasant, duck), minced and ground meat (including ground beef, ground turkey, ground chicken, ground pork); eggs; fruits and vegetables; nuts and nut products, such as nut butters; seafood products including fish and shellfish; and fruit or vegetable juices.
  • the diagnostic assays may also be used to detect E. coli contamination of drinking water. While the primary focus of E. coli detection is food products, the present invention also contemplates the use of the primers and probes in diagnostic assays for the detection of E. coli contamination of other biological samples, such as patient specimens in a clinical setting, for example, faeces, blood, saliva, throat swabs, urine, mucous, and the like, as well as E. coli contamination of surfaces and instruments, such as surgical or dental instruments.
  • the diagnostic assays are also useful in the assessment of microbiologically pure cultures, and in environmental and pharmaceutical quality control processes.
  • the test sample can be used in the assay either directly (i.e. as obtained from the source) or following one or more pre-treatment steps to modify the character of the sample.
  • the test sample can be pre-treated prior to use, for example, by disrupting cells or tissue, extracting the microbial content from the sample (such as a swab or wipe test sample), enhancing/enriching the microbial content of the sample by culturing in a suitable medium, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, isolating nucleic acids, purifying nucleic acids, and the like.
  • test sample is subjected to one or more steps to isolate, or partially isolate, nucleic, acids therefrom.
  • test sample is subjected to an enrichment procedure to enhance the microbial content of the sample prior to use in the assay.
  • the polynucleotide primers and probes of the invention can be used in assays to quantitate the amount of an E. coli target nucleotide sequence in a test sample.
  • the present invention provides for a method to specifically amplify, detect and quantitate a target nucleotide sequence in a test sample, the method generally comprising the steps of:
  • the steps of this method may also be varied and may employ combinations of primers and probes for different target sequences as described above for the amplification/detection method.
  • the method employs one or more labelled polynucleotide probes in step (a) and steps (d) and (e) are as follows:
  • Step (e) analysing the amount of signal produced as an indication of the amount of target nucleotide sequence present in the test sample.
  • Step (e) can be conducted, for example, by comparing the amount of probe:target hybrid present to a standard or utilising one of a number of statistical methods known in the art that do not require a standard.
  • the standard can consist of a standard curve compiled by amplification and detection of known quantities of the E. coli target nucleotide sequence under the assay conditions.
  • relative quantitation can be performed without the need for a standard curve (see, for example, Pfaffl, MW. (2001) Nucleic Acids Research 29(9):2002-2007).
  • a reference gene is selected against which the expression of the target gene can be compared and an additional pair of primers and an appropriate probe are included in the reaction in order to amplify and detect a portion of the selected reference gene.
  • the reference gene is usually a gene that is expressed constitutively, for example, a house-keeping gene.
  • an internal standard in the reaction.
  • Such internal standards generally comprise a control target nucleotide sequence and a control polynucleotide probe.
  • the internal standard can further include an additional pair of primers that specifically amplify the control target nucleotide sequence and are unrelated to the polynucleotides of the present invention.
  • the control target sequence can contain primer target sequences that allow specific binding of the assay primers but a different probe target sequence. This allows both the E. coli target sequence(s) and the control sequence to be amplified with the same primers, but the amplicons are detected with separate probe polynucleotides.
  • the reference/control probe incorporates a detectable label that is distinct from the label incorporated into the E. coli target sequence specific probe(s).
  • the signals generated by these labels when they bind their respective target sequences can thus be distinguished.
  • a control target nucleotide sequence is a nucleic acid sequence that (i) can be amplified either by the E.coli target sequence specific primers or by control primers, (ii) specifically hybridizes to the control probe under the assay conditions and (iii) does not exhibit significant hybridization to the E.coli target sequence specific probe(s) under the same conditions.
  • the actual nucleic acid sequences of the control target nucleotide and the control probe are not important provided that they both meet the criteria outlined above.
  • the diagnostic assays can be readily adapted for high-throughput.
  • High-throughput assays provide the advantage of processing many samples simultaneously and significantly decrease the time required to screen a large number of samples.
  • the present invention contemplates the use of the polynucleotides of the present invention in high-througliput screening or assays to detect and/or quantitate E. coli target nucleotide sequences in a plurality of test samples.
  • reaction components are usually housed in a multi- container carrier or platform, such as a multi-well microtitre plate, which allows a plurality of assays each containing a different test sample to be monitored simultaneously. Control samples can also be included in the plates to provide internal controls for each plate.
  • a multi- container carrier or platform such as a multi-well microtitre plate
  • Control samples can also be included in the plates to provide internal controls for each plate.
  • Many automated systems are now available commercially for high-throughput assays, as are automation capabilities for procedures such as sample and reagent pipetting, liquid dispensing, timed incubations, formatting samples into microarrays, microplate thermocycling and microplate readings in an appropriate detector, resulting in much faster throughput times.
  • kits for detecting E. coli in a variety of samples comprise one or more pairs of primers and one or more probe capable of amplifying and detecting E. coli target sequence(s) as described above.
  • one of the primers and the probe may be provided in the form of a single polynucleotide, such as a Scorpion probe, as described above.
  • the probe provided in the kit can be unlabelled, or can incorporate a detectable label, such as a fluorophore or a fluorophore and a quencher, or the kit may include reagents for labelling the probe.
  • the primers/probes can be provided in separate containers or in an array format, for example, pre-dispensed into microtitre plates.
  • kits comprising a combination of primer and probe sets, each of the sets being capable of amplifying and detecting an E. coli target sequence associated with a different gene.
  • kits can optionally include amplification reagents, such as buffers, salts, enzymes, enzyme co-factors, nucleotides and the like.
  • amplification reagents such as buffers, salts, enzymes, enzyme co-factors, nucleotides and the like.
  • Other components such as buffers and solutions for the enrichment, isolation and/or lysis of bacteria in a test sample, extraction of nucleic acids, purification of nucleic acids and the like may also be included in the kit.
  • One or more of the components of the kit may be lyophilised and the kit may further comprise reagents suitable for the reconstitution of the lyophilised components.
  • kits are provided in suitable containers. As indicated above, one or more of the containers may be a microtitre plate. Where appropriate, the kit may also optionally contain reaction vessels, mixing vessels and other components that facilitate the preparation of reagents or nucleic acids from the test sample.
  • the kit may additionally include one or more controls.
  • control polynucleotides primers, probes, target sequences or a combination thereof
  • the kit can additionally contain instructions for use, which may be provided in paper form or in computer-readable form, such as a disc, CD, DVD or the like.
  • kits described above may be provided as part of a package that includes computer software to analyse data generated from the use of the kit.
  • the prpR gene coding regions from 10 different E. coli isolates were sequenced and aligned using the multiple alignment program Clustal WTM. The resulting alignment was used to identify short DNA regions that were conserved within the E. coli group, yet which are excluded from other bacteria.
  • Figure 1 depicts a sample of such an alignment in which a portion of the prpR gene of 10 different E. coli strains have been aligned.
  • a 120 nucleotide conserved sequence was identified as described above (SEQ JD NO: 12).
  • primer target sequences Within the conserved 120 nucleotide sequence identified as described in Example 1, regions that could serve as primer target sequences were identified. These primer target sequences were used to design primers to allow efficient PCR amplification. The primer sequences are shown below:
  • Reverse primer #1 5'- GATGAGATTGGCGAAATG -3' [SEQ JD NO: 15]
  • Forward primer #2 5'-CTACCGGAACAGGCTGATG-3' [SEQ JD NO:20]
  • forward primer #1 starts at position 469 and ends at position 490 of the alignment.
  • Reverse primer #1 represents the reverse complement of the region starting at position 571 and ending at position 588.
  • Example 3 Generation of Molecular Beacon Probes Specific for the E. coli rpR Consensus Sequence
  • a region within the consensus sequence described above was identified which not only was highly conserved in all E. coli isolates but was also exclusive to E. coli isolates. This sequence consisted of a 23 nucleotide region that would be suitable for use as a molecular beacon target sequence. The sequence is provided below:
  • a molecular beacon probe having the sequence shown below was synthesized by Integrated DNA Technologies Inc.
  • prpR molecular beacon probe #1
  • S ⁇ Q ID NO: 18, shown below can also be used as a molecular beacon probe for detecting E.coli.
  • the starting material for the synthesis of the molecular beacons was an oligonucleotide that contains a sulfhydryl group at its 5' end and a primary amino group at its 3' end.
  • DABCYL was coupled to the primary amino group utilizing an amine-reactive derivative of DABCYL.
  • the oligonucleotides that were coupled to DABCYL were then purified.
  • the protective trityl moiety was then removed from the 5'-sulfhydryl group and a fluorophore was introduced in its place using an iodoacetamide derivative.
  • Table 3 provides an overview of the thermodynamics of the folding of molecular beacon probe #1. Calculations were made using MFOLDTM software, or the Oligo Analyzer software package available on Integrated DNA Technologies Inc. web site.
  • Figure 2 shows the arrangement of PCR primers and the molecular beacon probe in the prpR consensus sequence. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature on the PCR product generated with the forward primer #1 and reverse primer #1.
  • a further prpR specific molecular beacon suitable for the detection of E. coli was also prepared as described above.
  • the sequence is shown below (nucleotides in lower case represent the nucleotides that make up the stem of the beacon):
  • prpR molecular beacon probe #2 5'-cgtgcGTGACCTCTTTTTCTTCCAGCACCgcacg-3' [S ⁇ Q JD NO:21]
  • Time of manipulation 3 hours. Proceed to prepare PCR reaction for real-time detection.
  • Example 5 Amplification of the prpR Target Sequence and Hybridization of Molecular Beacon Probe #1 in Real-Time PCR amplification was undertaken using the conditions described in Tables 4 and 5 below.
  • the intensity of fluorescence emitted by the fluorophore component of the molecular beacon was detected at the annealing stage of each amplification cycle.
  • Table 4 note that the PCR buffer contains 1.5 mM magnesium chloride (final concentration). Inclusion of additional magnesium chloride brings the final concentration to 4 mM in the reaction mixture.
  • Table 5 presents an overview of the cycles used for each step of the PCR amplification.
  • prpR forward primer #1, reverse primer #1 and molecular beacon probe #1 for amplifying and detecting E. coli isolates was demonstrated as described generally below.
  • prpR molecular beacon probe #1 Seven strains out of a total of 262 yielded an amplification product under these conditions (two strains of Citrobacter braakii and five strains of E. fergusonii) and gave a positive signal under these conditions, however, when the upper limit of detection was set at 30 Ct, no hybridization of molecular beacon #1 was observed (i.e. specificity of 100%). This upper limit also helped to eliminate false positives resulting from the amplification of trace amounts of E. coli DNA present in the recombinant Taq polymerase used in the PCR reaction.
  • Salmonella Salmonella saintpaul B04 -
  • Salmonella Salmonella saintpaul B05 -
  • Regions of the E. coli genome encompassing part of the ykgM gene from 10 different E. coli isolates were sequenced and aligned as described fox prpR in Example 1.
  • Figure 4 depicts a sample of such an alignment in which a portion of the genome encompassing the 3 ' end of the ykgM gene of 10 different E. coli strains have been aligned.
  • a 144 nucleotide conserved sequence (SEQ JD NO: 36) was identified from the multiple sequence alignment.
  • primer target sequences Within the conserved 144 nucleotide sequence identified as described in Example 8, regions that could serve as primer target sequences were identified. These primer target sequences were used to design primers to allow efficient PCR amplification. The primer sequences are shown below:
  • Reverse primer #2 5'-GAGAACAGTGGCATCAGAAG-3' [SEQ JD NO:39]
  • forward primer #3 starts at position 212 and ends at position 232 of the alignment.
  • Reverse primer #2 represents the reverse complement of the region starting at position 336 and ending at position 355.
  • a molecular beacon probe having the sequence shown below was synthesized by Integrated DNA Technologies hie.
  • SEQ ID NO:42 The complement of this sequence (SEQ ID NO:42, shown below) can also be used as a molecular beacon probe for the detecting E. coli.
  • the molecular beacons were designed as described in Example 3.
  • Table 8 provides a general overview qf the characteristics of molecular beacon probe #3.
  • the beacon sequence shown in Table 8 indicates the stem region in lower case and the loop region in upper case.
  • *R indicates A or G.
  • Table 9 provides an overview of the thermodynamics of the folding of molecular beacon probe #3. Calculations were made using MFOLDTM software, or the Oligo Analyzer software package available on Integrated DNA Technologies Inc. web site.
  • Figure 5 shows the arrangement of PCR primers and the molecular beacon probe in the ykgM consensus sequence. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature on the PCR product generated with the forward and reverse primers.
  • ykgM 7 «olecular beacon probe #4: 5'-cctgcCATCACGCCCCCTTTTTCGgcagg-3' [S ⁇ Q JD NO:44]
  • Genomic DNA from the species and strains presented in Table 11 below was isolated as described in Example 4. Amplification was conducted as described in Example 5 with the exception that ykg forward primer #3 and reverse primer #2 and the following PCR mix were used.
  • thermophilus B01 Bifidobacterium thermophilus B01 -

Abstract

L'invention concerne des amorces et des sondes polynucléotidiques pour l'amplification et la détection spécifiques d'E. coli dans des échantillons. Ces amorces et ces sondes polynucléotidiques sont ciblées sur les gènes d'E. coli prpP et/ou ykgM et permettent de détecter une multitude de souches d'E. coli. Lesdites amorces et sondes peuvent être utilisées dans des méthodes diagnostiques en temps réel en vue d'une détection rapide d'E. coli dans diverses situations, et permettent de distinguer E. coli de Shigella spp. L'invention concerne également des trousses comprenant ces amorces et ces sondes.
EP05742611A 2004-05-07 2005-05-09 Polynucleotides pour la detection d'escherichia coli Withdrawn EP1759000A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US56919404P 2004-05-07 2004-05-07
PCT/CA2005/000694 WO2005108578A1 (fr) 2004-05-07 2005-05-09 Polynucleotides pour la detection d'escherichia coli

Publications (2)

Publication Number Publication Date
EP1759000A1 true EP1759000A1 (fr) 2007-03-07
EP1759000A4 EP1759000A4 (fr) 2008-05-07

Family

ID=35320234

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05742611A Withdrawn EP1759000A4 (fr) 2004-05-07 2005-05-09 Polynucleotides pour la detection d'escherichia coli

Country Status (4)

Country Link
EP (1) EP1759000A4 (fr)
AU (1) AU2005240681A1 (fr)
CA (1) CA2569085A1 (fr)
WO (1) WO2005108578A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114277167A (zh) * 2021-12-31 2022-04-05 杭州千基生物科技有限公司 一种用于细菌性肠道病原体的量子点核酸检测的试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1260592A1 (fr) * 2001-05-17 2002-11-27 MWG -Biotech AG Biopuce
JP2002355074A (ja) * 2001-01-24 2002-12-10 Univ Tsukuba 腸管出血性病原性大腸菌ｏ１５７：ｈ７に特異的な核酸分子およびポリペプチド並びにこれらの使用方法
EP1321530A2 (fr) * 2001-12-19 2003-06-25 Dr. Chip Biotechnology Incorporation Méthode pour détecter Escherichia coli
WO2003062464A2 (fr) * 2002-01-23 2003-07-31 Her Majesty, The Queen In Right Of Canada, As Represented By The Minister Of Health Detection du facteur majeur de virulence et du sous-type de la verocytotoxine de type 2 a partir d'isolats cliniques du e. coli au moyen d'une pcr multiplex en une seule passe

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6696254B2 (en) * 2001-11-21 2004-02-24 Kimberly-Clark Worldwide, Inc. Detection and identification of enteric bacteria

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002355074A (ja) * 2001-01-24 2002-12-10 Univ Tsukuba 腸管出血性病原性大腸菌ｏ１５７：ｈ７に特異的な核酸分子およびポリペプチド並びにこれらの使用方法
EP1260592A1 (fr) * 2001-05-17 2002-11-27 MWG -Biotech AG Biopuce
EP1321530A2 (fr) * 2001-12-19 2003-06-25 Dr. Chip Biotechnology Incorporation Méthode pour détecter Escherichia coli
WO2003062464A2 (fr) * 2002-01-23 2003-07-31 Her Majesty, The Queen In Right Of Canada, As Represented By The Minister Of Health Detection du facteur majeur de virulence et du sous-type de la verocytotoxine de type 2 a partir d'isolats cliniques du e. coli au moyen d'une pcr multiplex en une seule passe

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAYASHI T ET AL: "COMPLETE GENOME SEQUENCE OF ENTEROHEMORRHAGIC ESCHERICHIA COLI O157:H7 AND GENOMIC COMPARISON WITH A LABORATORY STRAIN K-12" DNA RESEARCH, UNIVERSAL ACADEMY PRESS, JP, vol. 8, no. 1, 28 February 2001 (2001-02-28), pages 11-22, XP009036572 ISSN: 1340-2838 *
See also references of WO2005108578A1 *

Also Published As

Publication number Publication date
EP1759000A4 (fr) 2008-05-07
WO2005108578A1 (fr) 2005-11-17
CA2569085A1 (fr) 2005-11-17
AU2005240681A1 (en) 2005-11-17

Similar Documents

Publication Publication Date Title
Perelle et al. Detection by 5′-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157: H7, associated with the world's most frequent clinical cases
US20080254449A1 (en) Polynucleotides For the Detection of Escherichia Coli 0157
Watterworth et al. Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli
Woubit et al. Novel genomic tools for specific and real-time detection of biothreat and frequently encountered foodborne pathogens
US8088572B2 (en) Polynucleotides for the detection of Escherichia coli O157:H7 and Escherichia coli O157:NM verotoxin producers
EP1759000A1 (fr) Polynucleotides pour la detection d'escherichia coli
US20080108060A1 (en) Polynucleotides for the Detection of Staphylococcus Aureus
CA2613159A1 (fr) Polynucleotides pour la detection de l'espece listeria
JP2775663B2 (ja) 細菌検出のためのオリゴヌクレオチドおよびそれを用いた検出法
EP1344833B1 (fr) Méthode pour la détection de Staphylococcus aureus
US20070141572A1 (en) Polynucleotides for the detection of listeria moncytogenes
US20070134659A1 (en) Polynucleotides for the detection of salmonella species
WO2006029522A1 (fr) Polynucleotides pour la detection d'especes de campylobacter
Kouguchi et al. Sequence-specific detection using a universal probe system based on the formation of a four-way junction structure
JP3141976B2 (ja) 赤痢菌検出のためのオリゴヌクレオチドおよびそれを用いた検出法
JP2993314B2 (ja) 黄色ブドウ球菌検出のためのオリゴヌクレオチドおよびそれを用いた検出法
Moore et al. Molecular characterisation of the rec A locus in clinical isolates of verocytotoxigenic E. coli O157: H7
Real Time Identification and characterization of Shiga Toxin (Verocytotoxin)-producing Escherichia coli (STEC/VTEC) by Real Time PCR amplification of the main virulence genes and the genes associated with the serogroups mainly associated with severe human infections
Winters Polymerase chain reaction for detection of Listeria monocytogenes
JPH119281A (ja) 細菌検出のためのオリゴヌクレオチドおよびそれを用いた検出法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20061204

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20080407

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20080707