MXPA00000197A - Methods for rapidly detecting methicillin resistant staphylococci - Google Patents

Abstract

Compositions and methods are provided for determining the presence of an antibiotic resistant mecA gene in a biological sample, comprising the general steps of (a) treating cells contained within a biological sample to expose target single-stranded nucleic acid molecules;(b) reacting the target single-stranded nucleic acids with a scissile link-containing nucleic acid which is complementary to a portion of an antibiotic resistant mecA gene, and with an enzyme which cleaves double-stranded target-probe complexes, under conditions which allow the target and probe to hybridize to each other to form a double-stranded target-probe complex, the enzyme molecule being capable of cleaving the scissile link ofthe target-probe complex such that one or more fragments of the nucleic acid probe released from said complex;and (c) determining whether cleaved portions of the detecting probe fragments released from said nucleic acid probe are produced, and thereby detecting the presence of the antibiotic resistant mecA gene.

Description

METHODS FOR QUICKLY DETECTING METICILIN RESISTANT STAPHYLOCOCES TECHNICAL FIELD The present invention relates generally to medical diagnostics, and more specifically, to methods, probes and primers for detecting the mecA gene of methicillin-resistant staphylococci.

BACKGROUND OF THE INVENTION Methicillin-resistant Staphylococcus aureus (MRSA) is an extremely virulent antibiotic-resistant form of Staphylococcus aureus, which has spread rapidly around the world. MRSA has been found to be common in many hospitals, with a range of frequency from less than 1% to more than 80% (Pittet and Waldvogel, QJ Med. 239-241, 1997). Unfortunately, MRSA is no longer considered only a pathogen associated with the hospital, as the community with MRSA acquired is also on the rise. Resistance to methicillin by Staphylococcus aureus is caused by the expression of a low affinity penicillin binding protein (PBP) called PBP2a or PBP2 ', in addition to the usual PBPs (Hartman and Tomasz, J. Bacteriol., 158: 513- 516; 1984; Murakami and Tomasz, J. Bacteriol., 171: 874-879, 1989). The responsible mecA gene encoding PBP2 ', mecA, is present in resistant strains but not in susceptible ones (Hartman and Tomasz, J. Bacteriol 158.513-516, 1984, Matsuhashi et al., J. Bacteriol. 167: 975- 980, 1986), and has only a slight variation of sequence from strain to strain (Ryffel et al., Gene 94: 137-138, 1990). MRSA strains that carry the mecA gene also tend to show resistance to other antibiotics, such as, aminoglycosides, macrolides, quinolones, beta-lactams, monobactams, imipenem, meropenem (Maple et al., Lancet 1: 537-540, 1989 Kayser, Chemotherapy 42 (suppl 2): 2-12, Chambers et al., Cited in Bowler, QJ Med 90: 243-246, 1997). Some strains of S. aureus may exhibit heterogeneity of expression of the mecA gene (Hartman et al., Antimicrob Agents Chemother, 29: 85-92, 1986, Hiramatsu et al., Microbiol. Immunol. 36: 445-453, 1992, Ryffel et al., Antimicrob, Agents Chemother, 38: 724-728, 1994). Within these populations, although all cells carry mecA, most cells are susceptible to low concentrations of the antibiotic and only a minority of the cells (1 in 105-106) express resistance (Hackbarth et al., Antimicrob. Agents Chemother 33: 991-994, 1989; Ryffel et al., Antimicrob Agents Chemother, 38: 724-728, 1994). Consequently, these strains exhibit low MIC and may be incorrectly characterized as sensitive to methicillin. Conversely, some strains of S. aureus lacking the mecA gene may exhibit a low or high level of resistance to methicillin due to alternative mechanisms of resistance (Chambers et al., Antimicrob Agents Chemother, 33: 424-428, 1989; Hiramatsu et al., Supra). The low-level resistance to methicillin due to the production of β-lactamase, production of methicillinase or the synthesis of modified PBP (Knapp, et al., J. Clin Microbiol 34: 1603-1605, 1996; McDougal, et al. al., J. Clin. Microbiol., 23832-839, 1986; Tomasz. iritilMiMiaMiferita MUtoHMtfMMIÜbltl et al., Antimicrob. Agents Chemother. 33: 1869-1874, 1989), can be misdiagnosed as MRSA by conventional susceptibility testing. Rapid detection, both for the prevention of transmission and treatment of methicillin-resistant Staphylococcus species (MRS) has become a priority around the world. There are a number of techniques available in the diagnostic field to detect MRS, including immunological tests and conventional biochemical tests. For example, an accepted method for the detection of MRS is the classification of isolates on Mueller-Hinton agar containing 4% NaCl and 6 μg / ml oxacillin. Many laboratories now use automated classification systems, such as Microscan APA (Dade) and Vitek GPS-SA card (bioMerieux, Hazlewood, MO), which provide susceptibility results within 24 hours (Knapp et al., Supra). The BBL® CrystalMR MRSA ID test (Beckton Dickinson, Cockeysville, MD) is a more rapid susceptibility test that allows diagnosis after 4 to 6 hours (Wallet et al., J. Antimicrobiol. Chemother, 37: 901-909).; Martínez et al, Rev. Esp. Quimioterap. 9: 130-133, 1996). However, none of the susceptibility tests can reliably differentiate between heterogeneous MRSA and limit oxacillin-resistant S. aureus (BORSA, Knapp et al., Supra, Lencastre et al., Eur. J. Clin. Microbiol. Infecí. Dis. 12: S13-S18). Many of these techniques also have time-related disadvantages, lack specificity and detection sensitivity. To direct such emissions, the detection of the mecA gene using the polymerase chain reaction ("PCR") has also been attempted. Briefly, PCR has been used in several studies to detect the mecA gene, but the results of the test using this method are expensive and can take up to 6 hours (Wallet, supra). In addition, there are a number of problems associated with such methods, including contamination of substrates, remanent contamination, thermocyclization, specific room requirements, embarrassing from the point of view of the number of initiators and probes required, the number of steps and time involved in the process. processing of the samples and the special training required. Despite the wide use of PCR in the area of molecular biology due to its high sensitivity and multiple applications, there are only a few PCRs approved by FDA or other diagnostic products based on target amplification. The main factor limiting the use of these technologies in the establishment of clinical diagnosis is the inherent problem of amplicon contamination. There are several chemical or physical methods that were developed to reduce or eliminate the pollution problem. In general, these methods add extra steps and are expensive. Although the above methods can be used to detect MRSA, there is an urgent need for a reliable and user-friendly method to detect the mecA gene in nosocomial and non-nosocomial settings. The present invention provides probes, primers and methods for detecting the mecA gene that meet these needs. Additionally, the present invention provides other related advantages.

BRIEF DESCRIPTION OF THE INVENTION As briefly mentioned, the present invention provides com positions and methods for detecting the mecA gene of methicillin-resistant staphylococci. Within one aspect of the present invention, methods are provided for determining the presence of an antibiotic-resistant mecA gene in a biological sample, comprising the steps of (a) treating cells contained within a biological sample to expose the single-filament nucleic acid molecule targets, (b) reacting the single-filament nucleic acid targets with a nucleic acid probe containing heel-binding, which is complementary to a portion of an antibiotic-resistant mecA gene, and with an enzyme that cuts dual-filament probe-target complexes, under conditions that allow the target and the probe to hybridize with each other to form a double-filament probe-target complex, the enzyme molecule being able to cut the cleavable link of the probe-target complex so that one or more fragments of the n-nucleic acid probe are released from said complex, and (c) determine if produce the cut portions of the nucleic acid probe, and thereby detect the presence of an antibiotic-resistant mecA gene. Within several modalities, the determination of whether the cut probe is produced can be achieved by directly detecting the cut portions of the nucleic acid probe and / or detecting a decrease in the amount of non-detected probe. cut. ^^^^ ¡^ ^^^^^ ¿^^ & "Within one embodiment, the Probe comprises at least a portion of the nucleotide sequence GACGATAATA GCAATACAAT CGCACATACA TTAATAGAGA AAAAGAAAAA AGATGGCAAA GATATTCAAC TAACTATTGA TGCTAAAGTT CAAAAGAGTA TTTATAAC 5 (SEQ ID NO: 13). Within a further embodiment, the probe consists essentially of at least a portion of the nucleotide sequence GAACTTTAGC ATCAATAGTT AGTTGAATAT CTTTGCCATC TTTTTTTCTTT TTCTCTATTA ATGTATGTGC GATTGTATTG CTATTATCG (SEQ ID NO: 4). Other representative probes include: AATAGAGAAA AAGAAAAAAG ATGGCAAAG (SEQ ID NO: 1); and AATAGAGaaaaAGAAAAAAGATGGCAAAG-3 '(SEQ ID NO: 5), wherein the uppercase letters represent deoxyribonucleotides and the lower case letters represent ribonubleotides. As used herein, a probe should be at least 8 nucleotides in length, and may be 10, 12, 14, 15, , 18, 20, 30 or even 100 or more nucleotides in length. Within several modalities, the mecA gene is from a staphylococcus species, either positive coagulase (for example, S. aureus), or negative coagulase (for example, S. epidermidis, S. sciuri). Within other related aspects of the present invention, it is provide probes for detecting the presence of an antibiotic-resistant mecA gene in a biological sample, wherein said probe comprises at least a portion of the sequence GACGATAATA GCAATACAAT CGCACATACA TTAATAGAGA AAAAGAAAAA AGATGGCAAA GATATTCAAC TAACTATTGA TGCTAAAGTT 25 CAAAAGAGTA TTTATAAC (SEQ ID NO: 13). They are also provided sets that comprise such probes, together with an enzyme (for example, RNase H), which cleaves cleavable links. Within further aspects, sets are provided for detecting the presence of a antibiotic-resistant mecA gene in a biological sample, comprising (a) one or more nucleic acid probes containing cleavable links, and (b) an enzyme capable of cleaving the link split when the probe is attached to a target. Within several modalities, the enzyme is RNase H. Within additional modalities, the mecA gene is of a species of staphylococcus. These and other aspects of the present invention will become apparent upon reference to the following detailed description and accompanying drawings. In addition, several references are set forth herein, which describe in more detail certain procedures or com positions (eg, plasmids, etc.) and are consequently incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DIAMETERS FIG. 1 is a schematic illustration of a modality of a cyclic probe reaction. Figure 2 is a histogram q ue sho ws LTS resu the frequency distribution of the classification of 285 isolates of Staphylococcus, including S. aureus and S. epidermidis mecA gene for the used raw reaction using Cycling Technology sampled. The chimeric probe labeled with 32P was mecA945-29 (SEQ ID NO: 6) and the reaction mixture contained the acid combination ?? II IBIÍÍIÍII an i I i ii iiiifí iiliiiliiii - • MftÜi ^^ ii ethylenebis (oxietilonitrilo) -tetracético 1 0 mM (EGTA) and spermine 2.0 m M. The isolates can be divided into positive and negative mecA mecA, based on the Percent I tried Cut (Percentage of probe cut). Figure 3 is a schematic illustration of the non-isotopic CPT assay. The simple filament lens (I) serves as a catalyst for C PT. In the presence of the probe (F-DNA-RNA-DNA-B) (I I) and RNase H (11), the RNA portion of the probe-target complex (IV) is cut by RNase H. The shorter cut probe fragments dissociate from the target, thereby regenerating the target DNA for additional cyclization (V). the anti-fluorescein antibody coupled to horseradish peroxidase (anti-F-HRP) is added (VI) and the reaction is transferred to streptavidin-coated plates. The uncut probe attached to anti-F-H RP is captured using the plates (Vi l). The excess antibody is washed (VI I I) and the HRP substrate is added (IX) to measure the amount of uncut probe. The absorbance, or development of color (X), is inversely proportional to the amount of target DNA. Figure 4 is a histogram showing the frequency distribution of January 20 clinical isolates of S. aureus based on the detection of mecA gene by assay CPT not isotypic using fluoresceinated probe and biotinylated q uimérica FmecA945-29B (SEQ ID NO: 5). The assay was performed on the raw ones and the isolates can be differentiated into M RSA (positive mecA) and MSSA (negative mecA) as two overlapping populations based on the absorbance value at 650 nm (650).

Figure 5 is a histogram sho ws the frequency distribution of 58 cl ínicos isolated S. aureus based on the detection of mecA gene by assaying direct fluorescence using the chimeric CPT fluoresceinated probe and biotinylated using plate format . The chimeric probe was FmecA945-29 (XL) 4B3 (SEQ I D NO: 5) and the CPT assay was performed on raw materials. Isolates may differ in MRSA (positive mecA) and MSSA (negative mecA) as two non-overlapping populations based on relative fluorescence units (RFU). Figure 5 is a h Istog branch results intensities GET fluorescence comings by assaying direct fluorescence of CPT from a pants blind 1 20 isolates of Staphylococcus clinical aureus for the mecA gene, using the format tube sim ple. The chimeric probe was FmecA945-29 (XL) 4B3 (SEQ ID NO: 5), the CPT test was performed on raw used as described in exam ple 8. These results show that the isolated M RSA and MSSA can be easily differentiated as two populations without overlapping based on RFU. Figure 7 is a flow chart for performing a modality for detecting PCR amplicons by CPT. Figure 8 shows the results of the comparison of the CPT enzyme immunoassay (CPT-E IA, 8A) and the agar-stained gel with Et-Br (8B) for the detection of the PCR amplification standard.

Figure 9 shows the sensitivity comparison of the CPT-EIA PCR (9A) with the gels stained with ethyl bromide (9B) for the detection of the mecA gene from MRSA and MSSA. Figure 10 shows the results of the effect of numbers of PCR cycles in the detection by CPT-EIA (10A) and agarose gel stained with ethidium bromide (10B) for the detection of the mecA gene from MRSA and MSSA.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS "Nucleic acid molecule" refers to a polynucleotide or polymeric nucieotide, which can be either of natural or synthetic origin. Representative examples of nucleic acid molecules include DNA (ds- or ss-DNA), RNA, DNA-RNA hybrids, or nucleic acid molecules which are composed of, or contain a nucleic acid analogue (e.g. -enantiometics of nucleotides that occur naturally). Additionally, the nucleotides can be modified in their sugar portions, or in the pyrimidine or purine base portions. Examples of modification to sugar moieties include modification or replacement of, for example, one or more hydroxyl groups with another group. Modifications of base portions include alkyl or acylated purines and pyrimidines. In addition, the nucleic acid monomers can be linked by phosphodiester linkages, or analogs of such linkages (for example, phosphorothioate, phosphorodithioate, phosphoramidite, and the like). "Isolated nucleic acid molecule" refers to a nucleic acid molecule that is not integrated into the genomic DNA of an organism. Isolated nucleic acid molecules include, for example, probes and other nucleic acid molecules generated synthetically or recombinantly. "Shatter link" refers to a nucleic acid molecule, which is capable of being cut or broken without cutting or breaking any nucleic acid sequence of the molecule by itself or of the target nucleic acid sequence. The cleavable links include any chemical connection structure, which joins two nucleic acid sequences, and which is capable of being selectively cut without cutting the nucleic acid sequences to which it is linked. The cleavable link can be a simple union or a sequence of multiple units. An example of such a chemical structure is an RNA sequence.

Other chemical structures suitable as a cleavable link are a DNA sequence, an amino acid sequence, a sequence of abasic nucleotides or an abasic nucleotide, or any carbohydrate polymer, i.e., cellulose or starch. When the cleavable link is a nucleic acid sequence, it differs from the nucleic acid sequences of NAT and NA2 (described below). "Probe containing a cleavable link" refers to a synthetic nucleic acid molecule, which is constructed in view of a sequence known to be complementary or substantially complementary to * £ a target nucleic molecule. Within certain embodiments, the probe comprises the structure [Na! - S - NA2] n, where NAi and NA2 are different non-complementary nucleic acid molecules and S is a cleavable link, and n is an integer from 1 to 1 0. "Ribonuclease H" ("RNase H") refers to an enzyme capable of specifically cleaving the RNA strand in the hybrid RNA: DNA (see generally Crouch &Dirkensen in Nucleases, Linn &Roberts (Eds. ), pp. 21-1-241, Cold Spring Harbor Laboratory Press, Plainview, NY, 1 982). Thus, as noted above, methods are provided for determining the presence of an antibiotic-resistant mecA gene in a biological sample, comprising the steps of (a) treating cells contained within a biological sample to expose nucleic acid molecules of single objective filament, (b) reacting the target single filament nucleic acids with a nucleic acid probe containing a cleavable link, which is complementary to a portion of an antibiotic-resistant mecA gene, and with an enzyme that cuts complexes double-filament target probe, under conditions that allow the target and the probe to hybridize with each other to form a double-filament probe-target complex, the enzyme molecule being capable of cutting said cleavable link of a complex probe- objective, so that one or more fragments of the nucleic acid probe are released from said complex, and (c) determining whether the cut portions of the nucleic acid probe are produced, and thereby detecting the presence of a resistant mecA gene. antibiotic. Using such methods, someone of skill in the art can generate fast and accurate results, which can provide results on the same day from the test samples. This allows the proper use of antibiotics for patients, reducing with it the use of vancomycin. In addition, such methods can be used easily to monitor outbreaks of epidemics or for routine surveillance in nosocomial and non-nosocomial settlements. Such methods can be used to detect the presence of a desired target nucleic acid molecule within a biological sample. Representative examples of biological samples include cultured samples (eg, growth in a bacteriological medium) or clinical samples, including, for example, samples of nasal swabs, blood, urine, feces, abscess or spinal fluid. Methods for generating target nucleic acid molecules can be easily achieved by someone of ordinary skill in the art given the description provided herein (see, generally, Sam brook et al., Molecular Cloning: A Laboratory Manual : A laboratory manual) (2nd ed.), Cold Spring Harbor Laboratory Press, 1989). As noted above, within an aspect of the present invention, the target nucleic acid molecule is reacted with a probe of complementary single strand nucleic acids having a cleavable link. Briefly, a wide variety of nucleic acid probes having cleavable links can be used within the context of the present invention. Preferably, the probe is designed so that, on cutting by an enzyme, which is capable of cutting Specifically, the probe-target complex in the cleavable link, the probe portions are released, which are detectable (see US Patents Nos. 4,876, 1 87, 5,01 1, 769 and 5,403, 71 1). The preferred probe molecules of the present invention, generally have the structure [(ÑAU (S) z (-NA2) y] n, where NAT and NA2 are molecules composed of nucleic acids or nucleic acid analogues, -S- is a cleavable link and x, y and z are integers from 1 -100 and n is an integer of 1 -1 0. Within certain particularly preferred embodiments of the invention, NAT and NA2 may vary from 3 to 40 nucleotides, and when S is composed of Nucleic acids may vary in size from 2 to 20 nucleotides, Furthermore, it should be understood that as used within the context of the present invention, each of x, y and z may vary with each iteration of n. In the invention, a simple filament probe is used to react or hybridize to a simple filament target sequence, the methods described above should not be mimicked only to situations where the complementary probe and sequences target are paired to form a duplo. Simple filament nucleic acid molecules can be obtained and / or prepared directly from a target cell or organism using standard techniques (see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor, 1989), or are prepared using any of a wide variety of techniques, including, for example, PCR, NASBA, reverse transcription of RNA, SDA, branched-chain DNA and similar.

Within one embodiment, NAi and NA2, as described above, are DNA molecules, which may or may not have the same sequence. Alternatively, NA- and NA2 can be constructed of RNA molecules, which may or may not have the same sequence, or a combination of RNA and DNA molecules. The DNA or RNA molecules used can be derived from naturally occurring sources, or they can be synthetically formed. Each of NA- and NA2 can be from about 5 bases to 10,000 bases in length. Within certain variants, the probe and target nucleic acid molecule do not need to be perfectly complementary, and in fact, they can be different, by the way, by one, two, three or more nucleic acids (see, for example, PCT publication). WO 95/141 06 and U.S. Patent No. 5,660,988). Within additional variants, the target nucleic acid molecule is present in a heterogeneous population of genomic nucleic acids. Within other embodiments, NAT or NA2 may be composed of nucleic acid analogues, such as, methyl phosphonates, carbamates, amidates, triesters or "Peptide nucleic acids" ("PNA"). For example, the PNA oligomers can hybridize to complementary target oligonucleotide sequences (DNA or RNA) with very high specificity. Such duplos are more stable than the corresponding dups of DNA-DNA or DNA-RNA (Egholm et al., Nature 365: 556-568, 1993). Additionally, PNA can be linked to double-strand DNA (ds) by filament displacement (Nielsen et al., Science 254: 1497-1 500, 1 991) and hence can obviate the requirement of ^^ MÉB ¡^ g ^ l ^ traditional double-filament denaturation in the preparation of the sample. Generally, the low salt concentration is preferred for the binding of PNA to dsDNA (< 50 mM / L Na +). The modaerated concentration of salt can inhibit binding through the displacement of the double strand of PNA to dsDNA. However, once the DNA / DNA dupls are joined, they are stable at high salt concentration. In addition, these dupes are also thermally stable compared to oligonucleotide / oligonucleotide dupls (PNA / DNA dupls are more stable at approximately 1 ° C per base pair compared with corresponding DNA / DNA). Based on the requirement of high sequence specificity to the target oligonucleotide, higher thermal stability and resistance to high double salt concentration once formed, PNAs are often ideal molecules for use in the methods described herein. Within certain embodiments, two short PNAs with cleavable link can be linked and used as a highly specific sequence probe. Probes of the present invention may also have one or more detectable labels attached to one or both ends (e.g., NA or NA2). The label can be virtually any molecule or reagent, which is capable of being detected, representative examples of which include radioisotopes or radiolabelled molecules, fluorescent molecules, fluorescent antibodies, enzymes, or incestuous chemolumin catalysts. Within certain embodiments of the invention, the probe may contain one or more labels, such as, a fluorescent or enzymatic label (e.g., extinct fluorescent pairs, or a fluorescent label and an enzyme label), or a label and a label. binding molecule, such as biotin (for example, the probe, either in its cut or uncut state, can be covalently or non-covalently bound to both a label and an ion molecule (see also, for example, U.S. Patent 5,731,146.) As noted above, the nucleic acid probe has a cleavable link, which is capable of being cut or broken without cutting or breaking any nucleic acid sequence of the molecule per se. m ism, or of the target nucleic acid sequence As used within the context of the present invention, a cleavable link is any chemical connection structure, which joins two nucleic acid sequences, and which is capable of being cleaved. or selectively without cutting the nucleic acid sequences to which it is attached. The cleavable link can be a simple union or a sequence of multiple units. An example of such a chemical structure is a Rna molecule. Other chemical structures, which may be suitable as a cleavable link are DNA molecules, an amino acid sequence, an abasic nucleotide molecule or any carbohydrate polymer (eg, cellulose or starch). When the cleavable link is a nucleic acid molecule, it should differ from the nucleic acid sequence of NAT and NA2. In the nucleic acid probes described above, when n is greater than one, the unit NA? -S-NA2 is repeated. As should be readily understood by one of ordinary skill in the art given the description described herein, the unit may be the same within. . ^^ of each repetition, or it can vary randomly in a defined pattern. In addition, the split link can also vary from unit to unit. For example, a cleavable link can be an amino acid sequence and another an RNA molecule. As noted above, the probes of the present invention can also be linked to a solid support either directly, or through a chemical linker. Representative examples of solid supports include lithium, cellulose, polymer-based, or plastic materials. Within a particularly preferred embodiment of the invention, the nucleic acid probes have the structure: [Na! - S - NA2] n where NAT and NA2 are nucleic acid sequences, S is a cleavable nucleic acid link, and n is an integer from 1 to 1 0. Within this embodiment, NAi and NA2 are different nucleic acid sequences , which are not complementary to each other, and -S- is a cleavable link, which is capable of being cut or broken by n cutting or breaking NAT O NA2, OR a target nucleic acid sequence capable of hybridizing to NA sequences ! or NA2, where if the cleavable link is a nucleic acid sequence it is RNA when both NAi and NA2 are DNA sequences or the cleavable link is DNA when both HA2 and NA2 are RNA sequences. Methods for constructing such nucleic acid probes can be easily accomplished by one of ordinary skill in the art, given the description provided herein. Particularly preferred methods are described, for example, by: Matteucci and Caruthers, J. Am. ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡Éü ^^ lí Chem. Soc. 103: 3185,1981, Beraucage and Caruthers, Tetrahedron Lett.22: 1859-1862, 1981, US Patents Nos. 4,876,187 and 5,011,769, Ogilvie et al. , Proc. Nati, Acad. Sci. USA 85: 8783-8798, 1987; Usman et al., J. Am. Chem. Soc. 109: 7845-7854, 1987; Wu et al., Tetrahedron Lett. 29: 4249 -4252, 1988; Chaix et al., Nuc.Aids Res., 17: 7381-7393, 1989; Wu et al., Nu.Aids Res. 17: 3501-3517, 1989; McBride and Caruthers, Tetrahedron Lett. : 245-248, 1983, Sinha et al., Tetrahedron Lett 24: 5843-5846, 1983, Sinha et al., Nuc.Aids Res. 12: 4539-4557, 1984, and Gasparutto et al., Nuc. Acids. Res. 20: 5159-5166, 1992. Particularly preferred probes (and synthetic targets) are based on the S. aureus mecA gene described by Song et al., FEBS Letters 221: 167-171, 1987 (EMBL Access No. Y00688) .MecA DNA sequences from different strains of MRSA show only minor variations (Ryffel et al., Gene 94: 137-138, 1990, S. aureus EMBL Access No. X52593 and S epidermidis EMBL Access no. X52592). The mecA gene is also distributed among the coagulase-negative staphylococci and associated with methicillin resistance (cited in Kobayashi et al., Epidemiol, Infect. 113: 259-266, 1994). Simple probes can be designed to detect the mecA gene, generally they can choose conserved regions or use of universal bases or abasic sites or other modifications, as described in the provisional US application (Attorney's case No. 480091.423P2).

"Briefly, the synthesis of oligonucleotides is achieved in cycles, where each cycle extends the oligonucleotide by a nucleotide, each cycle consisting of four steps: (1) unprotecting the endpoint. of the nucleoside or oligonucleotide in the solid support, (2) coupling the following nucleoside phosphoramidite to the immobilized nucleotide in the solid phase, (3) exceeding the small percentage of the 5'-OH groups of the nucleotides, which did not couple to the added phosphoramidite, and (4) oxidizing the oligonucleotide linkage to a phosphotriester linkage Representative methods for synthesizing oligonucleotides and biotinylation and fluoresceination of the oligonucleotides are shown in Example 1.

Detection Reactions As noted above, a wide variety of cyclized reactions can easily be performed for the detection of a desired target nucleic acid molecule according to the general steps outlined above (see also, US patents 5, 01 1, 769 and 5,403, 71 1). Within one aspect, such methods generally comprise the steps of (a) treating cells contained within a biological sample to expose the single-stranded, objective filament nucleic acid molecule, (b) reacting the target single-strand nucleic acids. with a nucleic acid probe containing a cleavable link, which is complementary to a portion of an antibiotic-resistant mecA gene, and with an enzyme, which cuts probe-target complexes ^ fc üH ^ i ^ double filament, under conditions that allow the target and probe to hybridize to each other to form a double-filament probe-target complex, the enzyme molecule being able to cut in the cleavable link of the probe-target complex, so that one or more fragments of the nucleic acid probe are released from said complex, and (c) determining whether the cut portions of the nucleic acid probe are produced, and thereby detecting the presence of an antibiotic mecA gene. Within other related aspects, the compositions and methods provided herein may be used in a wide variety of other methods / related methods (e.g., U.S. Patents Nos. 5,210,015, 5,487,972, 5,422,253, 5,691,142, 5,719,028, 5,130,238, 5,409,818, 5,554,517 5,589,332, 5,399,491, 5,480,784, 5,215,899, 5,159,766, 5,194,379, 5,474,916, 5,698,400, 5,656,430, and PCT publications nos. WO 88/10215, WO 92/08800, WO 96/02668, WO 97/19193, WO 97/09444; WO 96/21144; WO 92/22671). Within certain embodiments of the invention, the detection reactions provided herein may be performed using additives, such as polyamines (eg, spermine) or ribosomal proteins, which increase the sensitivity, specificity and / or reaction rate. These, as well as other related aspects, are described in the provisional US applications entitled "ADDITIVES FOR USE IN CYCLING PROBÉ REACTIONS", filed on May 18, 1998 (case of attorney No. 480094.419 P2); and "METHODS FOR ACCELERATING HYBRIDIZATION OF NUCLEIC ACID MOLECULES" • ArtÉfc "* •" "* - (Methods to accelerate the hybridization of nucleic acid molecules", presented May 1, 1998) (case of attorney No. 480094.422P2).

In another embodiment, CPT can be used to detect amplicons generated by any target amplification technology. Example 1 illustrates the use of the CPT enzyme immunoassay (CPT-E IA) for the detection of PCR amplicons. CPT allows rapid and accurate detection of PCR amplicons. In addition, CPT adds a second level of amplification but without further amplifying the target, and consequently, it is possible to use significantly fewer PCR cycles. This will reduce the chance of contamination and false positives. CPT adds a second level of specificity, which will prevent the detection of non-specific amplicons and primer dimer. The PCR-CPT method can also be used to detect unequal genes. Other variations of this test include "exponential" cyclization reactions, such as those described in U.S. Pat. 5,403, 71 1 (see also U.S. Patent No. 5, 747,255). The lateral flow device (strip or dipstick) as described in US Pat. Nos. 4855240 and 470301 7, for example, represents another embodiment for the detection format for the MRSA assay. Instead of detecting the mecA probe not cut in cavities coated with esptreptavidin (ie, EIA format), the uncut probe is captured by streptavidin impregnated in a membrane (i.e., strip format). There are several advantages to using this format. No additional detection reagents, fewer hands at a time, and a short detection time are required. Representative examples of formats . yes & yes: - * 3sF7 suitable additional tests including any of the above tests, which are performed on solid supports, such as immersion rods, magnetic beads and the like (see, generally, U.S. Patent Nos. 5,639,428; 5,635,362 5,578,270; 5,547,861; 5,514,785; 5,457,027 5,399,500; 5,369,036 5,260,025; 5,208,143; 5,204,601; 5,188,937 5,166,054; 5,139,934 5,135,847; 5,093,231; 5,073,340; 4,962,024 4,920,046; 4,904,583 4,878,710; 4,865,997; 4,861,728; 4,855,240; and 4, 847,194) In another embodiment, CPT can be performed using exponential formats with two sets of nucleic acid probe molecules, which are immobilized on solid support as described in U.S. Pat. 5,403,711. This would be advantageous since the assay can be performed in a simple container, the signal can be monitored over time and would result in a very fast and sensitive assay. Still in another embodiment, CPT-EIA can be used to detect MRSA by the use of reverse transcriptase to transcribe cDNA from mRNA expressed in the mecA gene followed by cyclization probe technology (RT-CPT) as described in US Pat. . 5,403,711. The uncut specific probe of the cDNA can then be detected by EIA. The following examples are offered by way of illustration and in no way as a limitation.

#%. EXAMPLES EXAMPLE 1 CONSTRUCTION OF NUCLEIC ACIDS PROBES Nucleic acid molecules can be synthesized using standard chemistries in automated, solid-phase synthesizers, such as the PerSeptive Biosystems Expedite DNA synthesizer (Boston, MA), Model 391 DNA synthesizer from PE Applied Biosystems , Inc. or the DNA / RNA Synthesizer Model 394 from PE Applied Biosystems, Inc. (Foster City, CA). Preferably, the PerSeptive Biosystems Expedite DNA synthesizer is used and the manufacturer's modified protocol is made to make oligonucleotides. Reagents for oligonucleotide synthesis are commercially available from a variety of sources including synthesizer manufacturers, such as, PerSeptive Biosystems, PE Applied Biosystems Inc., Glen Research (Sterling, VA) and Biogenex. For DNA and RNA synthesis, the preferred fluorescein amidite, deoxy- and ribo-nucleoside phosphoramidites, 2'-O-methyl and reagents, such as activator, Cap A, Cap B, oxidant and trityl deblock reagent are available from PerSeptive Biosystems. Biotin-TEG-phosphoramidite and Biotin-TEG-CPG are available from Glen Research. The ammonium hydroxide (28%) used for the deprotection of the oligonucleotides is purchased from Aldrich. The 1 M tetrabutylammonium fluoride (TBAF) used to remove the 2'-O-tert-butyldimethylsilyl group is purchased from Aldrich and used after drying molecular sieves for 24 hours. All dampers are prepared from sterilized water in an autoclave and filtered through 0.2 μM filters. The following procedure is used to prepare biotinylated and / or fluorescein oligonucleotides. The biotin-TEG-CPG (1 μmol) is packed in a synthesis column. The nucleoside phosphoramidites are then linked to make the nucleic acid sequence defined using the Modified PerSeptive Biosystem protocol to make oligonucleotides. The fluorescein amidite is dissolved in acetonitrile at a final concentration of 0. 1 M. The fluorescein amide is loaded onto the synthesizer and added to the 5 'end of the oligonucleotide. Alternatively, the thio-linker containing phosphoramidite is added to the 5 'end of the chimeric probe using the modified protocol. After deprotection of the step described below, the probe is purified by reverse phase H PLC, using M illipore resin R-2, which retains the trityl-containing oligonucleotide. In order to generate free reactive thio group, the probe purified with H PL is treated with silver nitrate for 90 m inutes at room temperature followed by neutralization of silver nitrate with dithiothritol (DTT). The fluorescein supernatant is then added to the free thio group of the probe and then purified either by H PLC or by electrophoresis as described below. After synthesis of the oligonucleotide sequence, the oligonucleotide bound to the resin is initially treated with 25% ethanol-ammonium hydroxide (4 ml) at room temperature for 1 hour and subsequently at 55 ° C for 1 6 hours in a closed tube. The tube is cooled, the supernatant is removed and concentrated to dryness, in order to remove the ammonia. The residue is dissolved in 1 ml of water and filtered through a 0.2 μm filter. The OD260 is determined and an aliquot of about 2 OD2eo units is injected into the HPLC R-2 column of Biocad to obtain a baseline in the chromatogram for the tert-butyldimethylsilyl groups of the chimeric probe. The remaining probe solution is lyophilized using a centrifugal vacuum evaporator (Labconco) in a 1.5 ml microcentrifuge tube. The remaining oligonucleotide residue is deprotected with 1.0 M TBAF for 24 hours. To determine the degree of desilylation that has taken place, an aliquot of the TBAF reaction mixture is injected into the H PLC (column R-2) using an ineal gradient of 0 to 60% acetonitriyl in triethyl ammonium acetate. 50 μM (TEAA), pH 6.5 If only partial desilylation has occurred, the TBAF reaction mixture is allowed to proceed for an additional 1 to 16 hours for complete removal of the protecting groups. The reaction mixture of TBAF is quenched with 1 00 mM NaOAc, pH 5.5 and evaporated to dryness. The crude oligonucleotide product is desalted on a P-6 column (2 cm x 10 cm, Bio-Rad), the fractions are concentrated to approximately 1 ml and the concentration is measured at OD260. The crude oligonucleotide is purified by polyacrylamide gel electrophoresis (PAGE) using 20% polyacrylamide-7M urea. The run buffer in gel is 1 x TBE (Tris-Borate-ethylenediaminetetraacetic acid (EDTA), pH 8.3) and the electrophoresis is carried out at a current of 50 mA for 3.5 to 4 hours. The oligonucleotide band is visualized with UV light, cut, placed in a conical plastic tube of 15 ml and extracted to the tablet and soaked in 5 ml of 50 mM NaOAc (pH 5.5) for approximately 12 hours . The tubes are then centrifuged at 3000 RPM and the supernatant is carefully removed by a Pasteur pipette. The gel is rinsed with 2 ml of extraction buffer to remove any residual product. The combined extract is concentrated to a volume of approximately 1 ml and desalted in a P-6 column. The fractions containing the probe are deposited and concentrated to a final volume of approximately 2 μl. The analytical purity of oligonucleotides is verified by labeling the 5 'end of the oligonucleotide with [? 32P] -ATP and T4-polynucleotide kinase and then running the labeled oligonucleotide in PAGE E. OD260 is measured using the Hewlett Packard 845X UV spectrophotometer. The oligonucleotide solution is filtered through a 0.2 μM filter and stored at -20 ° C. Using the above procedure, the following oligomers can be synthesized. In these sequences, the letters in the upper box have been used to denote deoxyribonucleotides, the letters in the lower box to denote ribonucleotides, the letters underlined to denote 2'-O-methyl bonds, the letter F in the upper box to denote brand of fluorescein, letter B of the upper box to denote biotin, letters of upper box XL to denote the XL linker (available from Glen Research or Clontech), and the sub-box number to denote repeats of the indicated portion. mecA945-29D (SEQ ID NO: l) 5 * - AAT AGA GAA AAA GAA AAA AGA TGG CAA AG-3 ' mecA945T (SEQ ID NO: 2) 5'-CTT TGC CAT CTT TTT TCT TTT TCT CTA TT-3 ' mecA932-59R (SEQ ID NO: 3) 5 '-cgc here uac auu aau aga gaa aaa gaa aaa aga ugg caa aga uau uca acu aac uau ug-3' ccmecA915-89 (SEQ ID NO: 4) 5'-GAA CTT TAG CAT CAA TAG TTA GTT GAA TAT CTT TGC CAT CTT TTT TCT TTT TCT CTA TTA ATG TAT GTG CGA TTG TAT TGC TAT TAT CG-3 ' mecA945-29 (SEQ ID NO: 5) 5'-AAT AGA GAA AAA Gaa aaA AGA TGG CAA AG-3 ' mecA945-29AIgP (SEQ ID NO: 6) 5'-AAT AGA GAA AAA Gaa aaA AGA TGG CAA AGA, 8-3 * mecA945-29 (2 'OMe) (SEQ ID NO: 7) 5'-AAT AGA GAA AAA Gaa aaA AGA TGG CAA AG-3' mecA945-29R (SEQ ID NO: 8) 5'- aau aga gaa aaa gaa aaa aga ugg caa ag-3 ' mecA834-25 (SEQ ID NO: 9) 5 * -TGGTAAAAA GGGACT CGAAAAACT T-3 'mecAL1039-22 (SEQ ID NO: 10) 5'-GGT GGATAG CAGTAC CTGAGC C-3 * mecA869-29 (SEQ ID NO: l 1) 5'-AGC TCC AAC ATGAAGATG GCT ATC GTG TC-3 ' mecALl 042-30 (SEQ ID NO: 12) 5'-ACC TGT TTG AGG GTG GAT AGC AGT ACC TGA-3 ' FmecA945-29B (SEQ ID NO: 5) 5'-FAAT AGA GAA AAA Gaa aaA AGA TGG CAA AGB-3 ' F-mecA945-29 (XL) 4B3 (SEQ ID NO: 5) 5'-FAAT AGA GAA AAA Gaa aaA AGA TGG CAA AG (XL) 4B, -3 * mecA913-1020 (SEQ ID NO: 13) 5'-GACGATAA TAGCAATACA ATCGCACATA CATTAATAGA GAAAAAGAAA AAAGATGGCA - AAGATATTCA ACTAACTATT GATGCTAAAG TTCAAAAGAG TATTTATAAC-3 ' mecA938-36 (SEQ ID NO: 14) 5, -ATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAG-3, raecA938-36 (SEO ID NO: 15) , -ATACATTAATAGAGaaaaAGAAAAAAGATGGCAAAG-3, Table of other mecA probes Name of the probe Sequence ID No. 5 'to 3' sequence mecAL53-22 SEQ ID NO: 16 ACGGAGAAgaagTTGTAGCAGG mecA172-27 SEQ ID NO: 17 GGTGAAGTAgaaaTGACTGAAGTCCG mecA356-25 SEQ ID NO: 18 AAGATGGTATGTggaaGTTAGATTG mecA358-24 SEQ ID NO: 19 GATGGTATGTggaaGTTAGATTGG mecA360-26 SEQ ID NO: 20 TGGTATGTGgaagTTAGATTGGGATC mecA824-27 SEQ ID NO: 21 ATGCAGTTATTGGTaaaaAGGGACTCG ecALI 255-23 SEQ ID NO: 22 TGTTTGagggTGGATAGCAGTAC mecA1393-25 SEQ ID NO: 23 GAUAACAUUUucuuUGCUAGAGUAG 2'Omethyl, except for ribonucleotides mecA1735-28 SEQ ID NO: 24 CAAGTCGTAAATaaaaCACATAAAGAAG mecA1913-25 SEQ ID NO: 25 TACAAGATAAAggaaTGGCTAGCTA mecA1930-27 SEQ ID NO: 26 GCTAGCTACAATGCCaaaaTCTCAGGT EXAMPLE 2 REACTION OF ISOTOPIC CYCLING PROBE TECHNOLOGY The conditions and reaction of cyclization probe technology (CPT) are carried out using procedures modified from previously published methods (WO 95/14106; Bekkaoui et al., Bio Techniques 20 (2) '240-248, 1996). Briefly, a specified chimeric probe is 5 'labeled with radioactive 32 P using [32 P] -ATP (Du Pont, Sambrook et al., 1 990) and T4 polynucleotide kinase (RTG, Pharmacia Biotech, Piscataway, NJ). A simple tube of RTG is resuspended in 1 5 μl of water. One pmol of probe is combined with 5 μl of? -32P ATP and 3 μl of RTG. The final volume is adjusted to 10 μl with water and incubated at 37 ° C for 30 minutes. Unincorporated? -32P is separated from the kinase probe by using a Nick G50 column (Pharmacia). The recovered probe is adjusted to 0.1 x in SSC buffer (15 mM NaCl, 1.5 mM sodium citrate, pH 7.0) and stored at -20 ° C. Unless otherwise indicated, the CPT reaction is performed by adding in order, the following: TES cyclized buffer, chimeric labeled probe, RNase H to give a cyclized cocktail, which is then added to the Denatured sample to be tested. The cyclized reaction mixture contains the specified concentrations of nucleic acid, nucleic acid, either as purified genomic DNA or crude crude lysate, 3.3 μg of RNase H in TES cyclised buffer (TES-CB), which has the following final concentration: 0.05% Triton X-1 00®, 4 mM MgCl2, ethylenbis (oxyethonitrile) -tetraacetic acid 1 mM (EGTA), 20 mM TES buffer, pH 6.8. The sample preparations, addition of test additives and other components used in the cyclization reactions are described in the specific examples. Unless otherwise indicated, the CPT reactions are incubated for 30 minutes at 56 ° C and then stopped by addition of urea charge buffer (8 M urea, 1 00 mM ethylenediaminetetraacetic acid (E DTA) and 0.25% each of bromophenol blue and xylene cyanol, 80 mM phosphate buffer) at 56 ° C. The reaction mixtures are then resolved by 7M-20% acrylamide / bisacrylamide (9: 1) urea gel electrophoresis (SDS-PAGE) at 500 volts, with cooling. The gel is analyzed in a Phosphorl mager ™ R using a computer program l mageQuant ™ R (Molecular Dynamics, Sunnyvale, CA). The amount of cyclized probe is estimated by integration of the band areas corresponding to intact and cut probe. Unless indicated otherwise, in a CPT reaction, the percentage of cut of probe is the total amount of cut of probe in relation to the amount of input probe (Equation No. 1): Percentage of cut of probe = (probe cut / total input probe) x 100 (1) In a simple CPT system, the C 1 support refers to the percentage of probe cut in the reaction buffer without the presence of RNase H or white counterpart . C2 refers to the percentage of cut of probe in the presence of RNase H, but without homologous white (Equation No. 2): C2 = (probe cut / total input probe) X 100 (2) For the complex CPT system , C3 refers to percentage of probe cut in the sample (biological samples containing foreign components, such as, heterologous DNA or proteins) in the absence of RNase H. C4 refers to percentage of probe cut in the biological sample in the presence of RNase H, but in the absence of homologous target (Equation No. 3): C4 = (probe cut / total input probe) X 1 00 (3) The net percentage of probe cut is the percentage of probe cut due to the homologous target and is calculated by subtracting the support C2 (simple system), or C4 (complex system) from the cut percentage (Equations No.4 or 5, respectively). Net Probe Cutoff Percentage = Cut Percentage - C2 (4) Probe Cut Net Percentage = Cut Percentage - C4 (5) The signal to noise ratio (S: N) for CPT is defined as the percentage ratio of probe cut in the presence of the white homologous to C2 (simple system, Equation No. 6) or C4 (complex system, Equation No.7): S: N = percentage of cut / C2 (6) S: N = percentage of cut / C4 (7) EXAMPLE 3 PREPARATION OF THERMOSTABLE RNASE H The following example describes a representative method for preparing thermostable RNase H from Thermus thermophilus. The cloning of the thermostable gene and its expression is described in detail in WO 95/05480 and Bekkaoui et al., BioTechniques 20: 240-248, 1996 based on the modification of the method by Kanaya & Itaya, J. Biol. Chem. 267: 10184-10192, 1992. Briefly, the RNase H gene of T. thermophilus (Kanaya &Itaya, supra) is cloned by PCR into vector pT7-7 (plDB9) and is subcloned into the vector pET11a (Novagen) resulting in the plasmid plDB33. The plasmid plDB33 is subsequently transformed into the bacterial strain BL21 (DE3) (Novagen, Madison, Wl). Cells BL21 (DE3) containing plDB33 are grown at 37 ° C in LB medium (Sambrook et al, 1990) containing 0.1 mg / ml ampicillin. When the culture is at an OD600 of 0.6 - 0.8, I PTG is added to a final concentration of 0.5 mM and the cells are cultured for four more hours. RNase H is expressed in the inclusion bodies with the pl DB33 construct. The cells are harvested by centrifugation at 3000x for 1 5 minutes at 4 ° C. The cell pellets are resuspended at 1 g of fresh weight in 5 ml of TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA buffer). Cells are used in a dry ice / ethanol bath using a sonicator (Branson, model 450) and centrifuged at 1 5,000 x g for 30 m inutes at 4 ° C. The pellet is resuspended in 7 M urea in TE buffer, pH 8.0 and incubated with stirring for 2 hours at 4 ° C. the resuspended cells are sonicated for 2 minutes in ice, followed by centrifugation at 1 2,000xg for 10 minutes and the supernatant is collected and subjected to dialysis overnight against 1 l of sodium acetate buffer (8 M urea) , 20 mM sodium acetate, pH 5.5) with two changes. After a centrifugation for 20 minutes at 31,000x g, the clear protein supernatant solution (150 ml) is collected and mixed with approximately 25 ml of pre-swallowed phosphocellulose (2 x equilibrated in column buffer, P1). 1, Whatman I International Ltd., Kent, UK) for 3 hours. The resulting paste is washed twice with the sodium urea acetate buffer and emptied into a column. The column is connected to an FPLC system (Pharmacia) and washed twice in steps with 140 mM NaCl and 21 0 mM in the sodium urea urea buffer. The protein is then levigated using a linear gradient of NaCl 0.21 to 0.7 M in the sodium acetate urea buffer. At the end of the salt gradient, the column is maintained at 0.7 M NaCI until all the protein is levigated. The fractions are analyzed by SDS-PAGE and those containing RNase H are deposited and desalted using a Sephadex G-25 column with buffer containing 150 mM NaCl in 20 mM sodium acetate, pH 5.5. The levigated protein fractions are deposited, concentrated with a Centriprep 10 filter (Amicon, Beverly, MA), and stored at -20 ° C in glycerol storage buffer (40% glycerol, 150 mM NaCl and 20 mM sodium acetate). , pH 5.5).

EXAMPLE 4 DETERMINATION OF METICILLIN RESISTANCE STATUS OF STAPHYLOCOCCUS AUREUS BY MEASURE GENE DETECTION USING CPT REACTION The following example demonstrates the utility of the chimeric probe mecA945-29 (SEQ ID NO: 5) and the effectiveness of spermine and EGTA in reaction of CPT for the detection of the mecA gene of the used raw of S. aureus isolates. This experiment was designed to examine the effect of spermine and EGTA on the CPT reaction for the detection of the mecA gene in MRSA isolates using raw materials. For this experiment, the MRSA isolates (ATCC 33592, American Type Culture Collection, Rockville, MD) and MSSA (ATCC 11632) were grown on Trypticase Soy Agar (TSA) plates with 5% sheep blood (PML Microbiologics, Richmond, BC) at 37 ° C overnight. . A sterile swab was used to remove the colonies from the TSA plate followed by resuspension of the cells in 2 ml of 0.05% Triton X-100® in 20 mM TES buffer (pH 6.8). The cell suspensions were then adjusted to microcentrifuge tubes. Lysis of the cells was performed with the addition of acromopeptidase (Wako Bioproducts, Richmond, VA) to a final concentration of 150 units / ml per sample. The suspensions were mixed and incubated at 37 ° C for 20 minutes. The mecA945-29 chimeric probe was synthesized and labeled as described in Examples 1 and 2. Thermostable RNase H was produced as described in Example 3. The CPT reactions and analysis were performed as in Example 2, except for Next: 1 .8 fmol of chimeric probe mecA945-29 was used and the concentrations of additives tested were 1 mM EGTA, 2 mM sperm or a combination of 1 mM EGTA and 2 mM spermine. Fifty μl of samples of crude was denatured with heat in a heating block at 95 ° C for 5 minutes, and then transferred directly to a water bath of 58 ° C (the reaction temperature was 56 ° C). The reaction cocktail (50 μl) was added immediately and the incubation continued for an additional 20 minutes. At the end of the incubation, an equal volume of charge dye containing 40 mM PB (1000 μl) was added to the samples in the water bath. The samples were then transferred to a heating block of 95 ° C for 5 minutes. The samples were wrung down briefly and 20 μl was loaded onto an acrylamide gel for electrophoresis.

Table 1 summarizes the results of the effect of sperm and EGTA on the CPT reactions for the detection of the mecA gene from used M RSA. Briefly, it was observed that in the absence of spermine or EGTA there was no differentiation between the isolates of M RSA and MSSA due to the high C4 support. The addition of EGTA alone reduced the percentage of probe cut in both M RSA and MSSA, but still did not allow differentiation between the two. The addition of spermine alone to the CPT reaction allowed the detection of M RSA as the C4 support decreased, resulting in a signal-to-noise ratio of approximately 5. Addition of both EGTA and sperm in the reaction CPT dramatically improved the detection of the target. As shown in Table 1, there was a greater reduction in the support of C4 and mecA MRSA could be detected with a remarkable signal-to-noise ratio of 20. These results clearly indicate the need to add both spermine and EGTA to the reaction of cyclized in order to obtain the clear differentiation between the isolates of M RSA and MSSA.

Table 1 . The effect of spermine and EGTA on CPT reactions for the detection of the mecA gene from raw MRSA.

The above example demonstrates the useful use of the isotopically-labeled chimeric mecA945-29 probe to differentiate MRSA from MSSA by detection of the mecA gene in the presence of the spermine and EGTA additives.

EXAMPLE 5 CLINICAL CLASSIFICATION FOR ISOLATES OF METICILIN RESISTANT STAPHYLOCOCES BY DETECTION OF GENE mecA USING CPT REACTION The following example demonstrates the successful use of isotopically labeled chimeric probe and the additives, spermine and EGTA, in CPT reactions for the detection of gene mecA of used raw of clinical isolates of staphylococci. This experiment examines the use of the 3 P chimeric probe labeled mecA945-29 (SEQ ID NO: 5) and the combination of spermine (2.0 mM) and EGTA (1.0 mM) in the CPT reaction for the detection of the mecA gene used of 285 isolates of staphylococci. These isolates were from the following sources: Wishart Memorial Hospital (Indianapolis, IN), Cleveland Clinic Foundation (Cleveland, Ohio), Vancouver General Hospital (Vancouver, BC) and 25 reference strains. In total there were 238 isolates of S. aureus and 47 of S. epidermidis. The crude lysate preparations, probe synthesis, CPT procedure and analysis were performed as described in Example 4, except that the cells were harvested from the TRSA blood plate with a 1 μl plastic loop (PM L Microbiological, Richmond, BC, Canada), were resuspended in 50 μl of 0.05% Triton X-1 00® in 20 mM TES buffer (pH 6.8) and used with the addition of acromopeptidase (Wako Bioproducts) as described in Example 4 The DNA was denatured with heat at 95 ° C for 5 minutes before use. The experiment was conducted as a blind operator study. Isolates were also tested with conventional oxacyl classification agar (PML Microbiological), Kirby-Bauere disk diffusion, minimum inhibitory concentration (MIC) using the E-Test with 4% NaCl Meu ller-Hi nton and S. aureus were tested with the BBL® CrystalM RM RSA ID test (Beckton Dickinson).

When the results of the CPT reaction were compared with the oxacillin agar classification, 4 discrepant samples were found.

It was observed that these isolates were positive on the agar screen, but negative on CPT. After the discrepant resolution by PCR (Example 7), it was confirmed that the mecA gene was absent in these isolates. The results of the previous experiment are shown in Figure 2 as a histogram of frequency distribution of the number of isolates versus percentage of cut of probe. Briefly, the frequency distribution of the probe cutoff percentage separated the isolates into two distinct populations based on the presence or absence of the mecA gene using the operator blinded study. Each of the susceptibility tests used in this study failed to correctly identify several staphylococcal isolates. The gold standard oxacillin agar screen identified 4 isolates of S. aureus as MRSA, although the mecA gene showed that it was not present. Each of these four isolates exhibited oxacillin bound resistance (M IC's 3-1 5 ug / ml) and were similarly misidentified by the M I C E test and oxacillin disk diffusion. One of these four isolates was also misidentified by the BBL Crystal I D M RSA System. About 31 additional S. aureus isolates lacking the mecA gene were designated as M RSA by the E test with oxacyl M Hc's 3-1 2 ug / ml and two of these isolates also failed by oxacillin disk diffusion. Each of these isolates of oxacillin-resistant S. aureus (BORSA) also showed to be susceptible to oxacillin in the presence of clavulanic acid by disk diffusion. Conventional susceptibility tests can not be reliably differentiated between isolates of S. aureus susceptible to oxacillin limit and isolates of heterogeneously resistant MRSA with low M IC.

Table 2. Isolates incorrectly identified by susceptibility tests The CPT assay accurately detected the mecA gene in isolates of S. aureus and S. epidermidis and allowed the correct identification of methicillin-resistant staphylococci. This trial was also able to differentiate the mecA positive MRSA from BORSA, which does not contain the mecA gene. The previous example demonstrates the sensitivity and specificity of the mecA945-29 probe labeled isotopically for the mecA gene of the crude used in both clinical isolates of positive and negative coagulase staphylococci.

EXAMPLE 6 PCR DETECTION OF GENE mecA PCR was performed for discrepant analysis by the following method. A pair of oligonucleotide primers mecA834-25 and mecAL1030-22 (SEQ ID Nos. 9 and 10), specific for the mecA sequence of MRSA, were synthesized, as described in Example 1. The crude ones of ATCC isolates from MRSA and MSSA were used as controls and PCR was performed after warm start with Taq polymerase. Hot-start PCR was performed in a volume of 50 μl by adding Taq polymerase at 80 ° C after denaturation for 5 minutes at 95 ° C. The final PCR reaction mixture contained the following: 200 μM of each of the dNTP mixture (dATP, dGTP, dCTP, dTTP, Pharmacia), 1.5 mM MgCl2, 50 mM KCl, 20 mM Tris HCl, pH 8.4, (1x PCR buffer) and 2 ng of the sample of crude lysate of Staphylococcus DNA in a final reaction volume of 50 μl. The samples were cyclized in the thermal lifting cycler (PTC 1 00, MJ Research I nc) using a cycle of 94 ° C for 40 seconds, 53 ° C for 40 seconds and 72 ° C for 90 seconds. The amplification was performed for 50 cycles. After amplification, the samples were analyzed electrophoretically using 1.8% agarose gel containing 0.5 μg / ml ethidium bromide. A molecular weight marker was also included. The sample was considered positive if the 227 bp amplicon was detected. This am plicon was detected in the control of M RSA of ATCC, but not in the control of MSSA of ATCC or any of the isolates of S. aureus discrepante.

EXAMPLE 7 NON-ISOTOPIC MRSA ASSAY The following example demonstrated a rapid non-isotopic CPT assay for the detection of the mecA gene in clinical isolates of S. aureus. The non-isotope MRSA assay, which combines CPT with an enzyme immone assay (CPT-EIA) is illustrated schematically in Figure 3. The Rapid MRSA Test uses a fluorescent, biotinylated chimeric probe, which provides a sensitive cleavable link to RNase H, when bound to the complementary base sequence of the mecA gene. The uncut probe (negative mecA) is detected by attaching the probe to a solid surface and binding an antibody conjugated with horseradish peroxidase, which converts a substrate to a colored final product. The cutting of the probe (positive mecA) prevents the binding of the probe-antibody complex to the solid surface, thus preventing the formation of the final colored product. A result is generated in 90 minutes. This experiment examines the use of the fluorescein-labeled chimeric probe FmecA945-29B (S EQ ID NO: 5) and the combination of spermine and EGTA in the CPT reaction for the detection of the mecA gene of crude 1 02 isolates of staphylococci . The source of isolates of S. aureus were from the following: Wishard Memorial Hospital, Cleveland Clinic Foundation, Vancouver General Hospital and ATCC. In total, there were 51 MRSA isolates and 51 MSSA isolates. Effective lysis of both MRSA and MSSA was developed and optimized. The composition of the Lysis Reagent is as follows: 200 an ions / ml of acyclopeptidase (Wako Bioproducts, Wako), 0.02 mg / ml of lysostaphin (Sig ma, St. Louis, MO), 0.05% (v / v) Triton X 1 00® and 20 mM TES buffer, pH 8.5. The chemical probe FmecA945-29B was synthesized, fluoresceinated and biotinylated as described in Example 1. The purified thermostable RNase H was prepared as in Example 3. The isolates were grown as described in Example 4 and 1 μl of growing culture loop was placed in 1.5 ml of microcentrifuge tube containing 50 μl of the Reagent. of Lysis. The samples were incubated at 54 ° C or room temperature for 10 minutes. It was denatured at 95 ° C for 2 minutes prior to the CPT reaction. The optimized CPT reaction reagents for the subtractive assay were as follows: 20 fmol / reaction of FmecA945-29B, 50 μl of crude lysate, 0.05% Triton X 100®, MgCl22 mM, EDTA 25 μM, spermine 625 μM, 1.65 μg / reaction of RNase H, 20 mM TES, pH 6.8, in a final reaction volume of 100 μl. The controls used were S. aureus mecA positive (ATCC 33592) and S. aureus mecA negative (ATCC 11632). The CPT reaction was performed at 54 ° C for 25 minutes. After cyclization, 100 μl of Peroxidase Stabilizing Buffer Reagent, DAKO, Mississauga, ON), polyclonal sheep horseradish peroxidase-conjugated anti-fluorescein antibody (1/750 dilution, NEN, Boston) was added to the tubes. , MA). Detection was performed using stripe cavities coated with streptavidin (Boehringer Mannheim GmbH, Germany, Boeringer). The CPT reaction was transferred to streptavidin-coated strip cavity, mixed for 10 seconds and incubated for 10 minutes at room temperature. The liquid was discarded and washed twice with 300 μl of Wash Buffer (137 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, Na2HPO410.1 mM, 0.5% Tween 20, pH 7.3). This was followed by the addition of 200 μl of substrate (Tetramethylbenzidine / H2O2, Sigma) and allowed to develop for 3 minutes at room temperature. Development was stopped using 100 μl of Detection Stop Reagent (750 mM Tris, 1.5% (w / v) sodium dodecyl sulfate, pH 7.7). The plate is read using a Vmax plate reader (Molecular Devices) set at 650 nm OD (OD650). Table 3 and Figure 4 summarize the screen results for the MRSA mecA gene when using the subtractive CPT assay.

Table 3. The average and standard deviations (SD) of absorbance at 650 nm (A650) of the experiment by examining the detection of used clinical isolates of MRSA (n = 51) and MSSA (n = 51) using the subtractive CPT assays.

The results indicate that all the data points of the 51 MRSA or MSSA fall within 3 standard deviations (SD) of their corresponding averages. Consequently, there is no overlap between the MRSA average plus 3x SD and the MSSA average minus 3x SD. This allowed the unambiguous test result, which separates the MRSA population from the MSSA population. The above example demonstrates that the fluorescent and biotinylated chimeric mecA945-29 probe can be successfully used in a non-isotopic CPT-EIA to differentiate MRSA from MSSA upon detection of the mecA gene.

EXAMPLE 8 MRSA TEST: FORMAT, REAGENT AND COMPOSITION OF SET The following is a representative example of a set for detecting the mecA gene of MR§A. This set allows the rapid detection of MRSA by detecting the mecA gene using the non-isotopic CPT assay (CPT-EIA) and has been optimized to detect MRSA isolates (discrepants) that are difficult to lyse or those that produce nucleases. The Rapid MRSA Test Set (48 tests) is comprised of the following items: * MRSA Lysis Reagent (2) * Streptavidin coated microcavities (48) * MRSA Cycle Reagent (48) * Washdown Absorber (1) X 50 ml) * MRSA Lysis Reconstitution Damper (1 X 3 ml) * Detection Substrate Reagent (1 X 12 ml) * Reconstitution Cycle Absorber (1 X 6 ml) * Detection Stop Reagent (1 X 5.5 ml) * MRSA Cycle Stop Reagent (1 X 6.8 ml) * Transfer pipette (50) * 50 μl Dropstira (75) * 50 μl Dropstira (75) * 200 μl Dropstir (50) * 200 μl Dropstir (fifty) The following describes the composition, reagents and materials that are part of the set: * MRSA Lysis Reconstitution Damper: Water and 20 ppm ProClin 300 R (Sigma). * MRSA lysis reagent (lyophilized): TES, Triton X-100®, Trehalose (Sigma), Acromopeptidase (Wako) and EGTA * Reconstitution Cycle Buffer: MgCI24 mM and 20 ppm ProClin 300MR. * MRSA cycle reagent (lyophilized): Trehalose, Polyvinylpyrrolidone, TES, Triton X-100®, spermine, mecA probe, RNase H and bovine serum albumin. * MRSA Cycle Stop Reagent: Buffered Salt Solution (DAKO) containing the anti-fluorescein antibody conjugated with horseradish peroxidase (1/1000 final dilution). * Microcavity Coated with Streptavidin (Boehringer). * Washing buffer: 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, Na2HPO410.1 mM, 0.5% Tween 20 and 20 ppm ProClin 300. * Detection Substrate Reagent: Tetramethylbenzidine (Sigma) and H2O2.

* Detection Stop Reagent: 0.75 mM Tris and 1.5% sodium dodecyl sulfate.

The procedure for performing the assay for detecting the mecA gene of the crude S. aureus Uses is as follows: A. Reconstitution of MRSA Lysis Reagent: 1. To a vial of MRSA Lysis Reagent pipette 1.5 ml of MRSA Lysis Reconstitution Buffer. 2. Swirl to dissolve. 3. Allow to settle at room temperature for 2 to 3 minutes before use. 4. Once reconstituted, a vial of Lysis Reagent from M RSA can be used for 2 weeks when stored at 2 ° C-8 ° C.

B. Reconstitution of MRSA Cycle Reagent 1. Reconstitution should be done during the incubation steps of the Specimen Preparation. 2. To a vial of RSA M Cycle Reagent add 2 drops of the Reconstitution Cycle Buffer. 3. Remolinear to dissolve. 4. This is a simple reagent. This reagent should be used within 30 minutes of reconstitution.

C. Sample preparation 1. Using a 50 μl Dropstir, add one drop of reconstituted M LA RSA Reagent to each 1.5 ml microcentrifuge tube (one tube per sample) 2. Add a loop of 1 μl of growth from a culture of 1 8 to 24 hours on a tryptic soy agar plate containing 5% sheep blood. Mix well to completely suspend cell growth. 3. Place at 55 ° C for 20 minutes. 4. Place at 95 ° C for 5 minutes.

D. Cyclization probe technology 1. Transfer tubes with lysate at 55 ° C. 2. Using a 50 μl Dropstir, add one drop of reconstituted RSA M Cycle Reagent to each tube. 3. I discovered at 55 ° C for 25 minutes. 4. Add 3 drops of MRSA Cycle Stop Reagent with tubes at 55 ° C.

Detection 1 Place the required number of Microcavities Coated with Streptavidin (one Microcavity per sample) in the frame of Microcavities. 2. Transfer the full-cycle reaction to the Mycoplasma Coated with Streptavidin using a transfer pipette. 3. Icubcubar at room temperature for 10 minutes. 4. I nvert the M icrocavity Coated with Streptavidin to discard the product. 5. Fill each Microcavity Coated with Streptavidin completely with Wash Absorber. 6. Ivert the Microcavity Coated with Streptavidin to discard the fluid. 7. Tap each Microcavity Coated with Streptavidin 5 times on a dry paper towel. 8. Repeat steps 5 - 7. -? ' 9. Using a 200 μl Dropstir, add one drop of Detection Substrate Reagent to each of the Microcavities Coated with Streptavidin. 1 0. Place at room temperature for 5 minutes. eleven . Add 4 drops of Detection Stop Reagent to each Microcavity Coated with Streptavidin. 12. Mix for 10 seconds. 1 3. Icubcubar at room temperature for 3 minutes. 14. Within 30 minutes, read visually and record the color area or measure / record the OD650.

EXAM PLO 9 DIRECT FLUORESCENT CPT TEST FOR M RSA mecA The following example demonstrates the use of a direct fluorescence assay for the detection of the mecA gene in MRSA isolates by CPT using raw materials. Two experiments were performed for the detection of the mecA gene from classical isolates using the direct fluorescence assay (DFA), combined with CPT (CPT-DFA). In the first experiment, 58 isolates were classified using a plate reader format, and in the second experiment, 1 20 isolates were classified using a simple tube reader format. Briefly, the assay uses a fluorescent and biotinylated chemistry probe complementary to the sequence within the mecA gene present in MRSA, but not in MSSA. The probe is labeled with fluorescein at the 5 'end and biotin at the 3' end. The CPT assay is performed with crude lysate preparations from staphylococcal cells in the presence of RNase H and the chimeric probe. After the cyclization reaction, the magnetic beads coated with streptavidin are added to join the uncut probe. The supernatant containing the fluorescein-labeled cut fragment is separated from the beads using a magnet and the supernatant is transferred to a glass tube or a microtiter plate for fluorescence measurement.

A. Source of clinical isolates and sample preparation The sources of the eighty-five clinical isolates were obtained from Cleveland Hospital, Wishard Memorial and Vancouver General Hospital. All the cells of MRSA and MSSA were prepared and used essentially as described in Example 4, except that the used ones were prepared either as standard McFarland No. 2 density or No. 5 lacing of 1 μl of cells in 50 μl in volume . A loop of 1 μl of cells is approximately equivalent to 5x standard McFarland No. 5.

B. Union of biotinylated probes using streptavidin-coated magnetic beads Streptavidin-coated magnetic beads (Dynal M-280, Oslo, Norway) were prepared by washing twice in an equal volume of cyclized TES buffer (4 mM MgCl 2, 0.05% Triton X-1 00®, 20 M TES, pH 6.8). The pearls were then suspended again in 1 0 times «SMfe the original volume with cyclized buffer containing NaCl. The diluted beads were added to the reactions containing the biotinylated probe at a final concentration of 50 μg beads / reaction and a final NaCl concentration of 1.5 M. The ionization was carried out in a thermomixer (Eppendorf) at 37 ° C with mixing at a speed of 1 3 x 1 00 minutes "1, for 10 minutes The supernatant was then separated from the beads using an MPC magnet (Dynal) .

C. Detection of Fluorescence of the Probe after One-Step The probe was detected after binding using the Fluoroskan fluorescence plate reader (Labsystems, Needham, MA) by placing the supernatant containing the cut probe into a well cavity. Round black 'U' MicroFluor background (Dynatech, Chantilly, VA). To each cavity containing a sample, 1 50 μl of 0.22 M DEA pH 1 0 was added. Any bubble in the cavities was removed using the edge of a Kimwipe (Roswell, GA). The plate was scanned in the plate reader with an excitation wavelength of 485 nm, an emission wavelength of 538 nm and a tracking time / cavity of 20 x 0.1 s, at room temperature. Similar detection was performed using the Beacon-2000 tube reader (Panvera, Madison, Wisconsin), by placing the supernatant containing the probe in a borosilicate glass tube (Kimble, Toledo, OH), containing 1 1 μl of DEA 2.0 M, pH 1 0. Then the samples are vortexed and the fluorescence intensity was measured using the static mode of the Beacon-2000 instrument. The excitation and emulsion wavelengths were 485 nm and 51 5 nm respectively and an average of 10 read cycles at 25 ° C was used.

D. CPT procedure with raw MRSA or MSSA uses The assay used as a chimeric probe FmecA945-29 (XL) B3 (SEQ ID NO: 5) labeled with fluorescein at the 5 'end and three biotins at the 3' end. The cyclised reactions with crude lysate were carried out in a 50 μl reaction containing 25 μl of crude. The used ones were denatured at 95 ° C for 2 minutes. The CPT reaction was performed and the final conditions were as follows: 1 pmol of chimeric probe, 25 μl of crude, 50 μM EDTA, 0.5 μM spermine, 1.5 μg of RNase H in a final reaction volume of 50 μl. All the reactions were cyclized in a water bath of 60 ° C, in microcentrifuge tubes (uncovered) for 50 m inutes. The reactions were stopped with the addition of an equal volume of diluted beads (50 μg beads / reaction in Cyclized Buffer and NaCl), while still in the water bath. The one ion and detection were performed as described above. The net relative fluorescence units are calculated as the difference between the fluorescence intensities of MRSA and MSSA. The Fluoroskan plate reader was used to measure fluorescence on the initial clinical screen of 58 isolates. These results are summarized in Figure 5 as a frequency distribution histogram. Briefly, these results show that the MRSA and MSSA isolates could easily be differentiated as two non-overlapping populations and it was concluded that the assay had 1 00% sensitivity and 1 00% specificity for the detection of the relative gene. to the cut and resolution of PCR (see Example 5). In a subsequent trial, isolates of S. aureus (either MSSA or MRSA) were tested by DFA using 1 pmol of probe, with the Beacon-2000 tube fluorometer. The classification results of 1 20 isolates using the Beacon-2000 are shown in Figure 5. The range of MSSA samples is 58.1 - 1 01 .7 RFU and the range of M RSA samples are 144.2 - 1 91. 9, with a net opening between the highest MSSA sample and the lowest MRSA sample of 42.5 RFU. On this screen, a susceptible isolate was misidentified as a resistant isolate, giving 100% sensitivity and 98% specificity. This misidentification was due to an incomplete lysis of an MSSA sample. This strain was correctly identified when it was re-tested using a longer lysis time (data not shown). The above example demonstrates that the mecA945-29 probe sequence is very specific and sensitive for the detection of the mecA gene of Staphylococcus species in crude oils using the direct CPT fluorescence assay.

EXAM PLO 1 0 The following example demonstrates the utility of CPT for the rapid detection of PCR amplicons after a low number of PCR cycles.

The mecA gene of M RSA was used as a model system. Initially PCR was performed and then it was followed by CPT and detection was performed by an enzyme immunoassay (CPT-EIA). The CPT-EIA assay has been previously described in Example 7 and is illustrated in Figure 3. The procedure for the detection of amplicons by CPT-E IA is summarized in FIG. 7. Crude strains of MSSA (ATCC 1 1632) and MRSA (ATCC 33592) strains were grown overnight at 37 ° C on tryptic soy agar with 5% sheep blood (PM L Microbiologicals, Tualati n, OR). A cell suspension was made in TES buffer (Tes 20 mM (pH 6.8), 0.05% Triton X-1 00) to obtain a cell density equivalent to a standard 5x McFarland # 5 turbidity (7.5 x 1 09 cells / μl). The cells were used with 1 50 U / μl of acromopeptidase (Wako) for 25 min at 37 ° C. After lysis, the cells were stored at -20 ° C. Adequate dilutions were made in sterile deionized water. The PCR reaction was performed in a 25 μl reaction containing 20 mM ris (pH 8.3), 1.5 mM MgCl2, 0.2 mM dNTPs (Pharmacia), 6.25 pmol of each primer (Table 1) and 0 5 U AmpliTaq Gold (Perkin Elmer). The programmable thermal cycler PTC-1 00 (MJ Research, I nc.) Was used with the following PCR program: 95 ° C for 1 0 min, 20x (94 ° C for 40 s, 53 ° C for 40 s and 872 ° C for 1.5 minutes). The reaction was subjected to a final extension step at 72 ° C for 5 min and then held at 4 ° C before analysis. The PCR reactions were analyzed by CPT as described below or using a 2% agarose gel and stained with ethidium bromide (Et-Br). -. &- The 227 bp mecA PCR product was used as a standard to determine the sensitivity of CPT-EIA and gels stained with Et-Br. The amplicon was prepared as follows: several 1000 μl reactions were subjected to 30 cycle PCR and the amplicon was purified using a Qiagen PCR purification conjugate, as recommended by the manufacturer. The 227 bp product was quantified by spectrophotometer and used as a standard. The quantity of amplicon products was estimated using the following formula: N = N0 (1 + E) n, where N is the number of amplicon molecules, N0 is the initial quantity of the target molecules, n is the number of cycles and E is the efficiency of PCR.

Table 4. PCR initiators and CPT probe sequences. The letters indicate: upper square is DNA, lower square is RNA, F is fluorescein and B is biotin. Name SEQ ID NO. 5 'to 3' sequence mecA834-25 9 TGGTAAAAAGGGACTCGAAAAACTT mecAL 1 039-22 1 0 GGTGGATAGCAGTACCTGAGCC FmecA945-29B 5 F-AATAGAGAAAAAGaaaaAAGATGGCAAAG-B CPT-EIA. The 25 μl PCR reaction was denatured with heat to 95 ° C for 2 m in and then it was placed at 54 ° C. The components of CPT in 25 μl were added to the PCR reaction to give a CPT reaction volume of 50 μl. In addition to the PCR components, the reaction contained: 20 mM TES, pH 6.8, 4 mM MgCl 2, 0.05% Triton X-1 00, .3 £ £ isS. - 1 mM EGTA, 1 mM Spermine, 10 ftmJ of probe FmecA945-29B (Table 4) and 1.42 μg of RNase H. The CPT reaction was incubated at 54 ° C for 30 min and stopped with 100 μl of binding reagent containing a 1/600 dilution of anti-fluorescein-HRP conjugate (NEN, Boston, MA) in Peroxidase Stabilizing Buffer (DAKO, Mississauga, ON). After removal of 54 ° C, the samples were allowed to cool for 3 minutes at room temperature. The samples were then transferred to a strip cavity coated with streptavidin (Boehringer Mannheim) and incubated at room temperature for 10 min with shaking. The cavities were washed 2x with wash buffer (137 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, 10.1 mM Na2HPO4, 0.5% Tween 20, 20 ppm ProClin 300, pH 7.3)) and then 200 μL of EIA substrate was added. TMB Single Component Peroxidase (Biorad, Hercules, CA). After 3 minutes, color development was discontinued using the detection stop reagent (750 mM Tris, 1.5% SDS, 20 ppm ProClin 300, pH 7.7) and plates were read at OD 650 nm using a Vmax spectrophotometer (Molecular Devices, Sunnyvale, CA). The net OD650 (= ODcontro? -ODsample) was used to characterize the presence of the specific target. The number of times the target is cyclized (T) was estimated as follows: T = number of molecules cut / number of target molecules. It was found that the detection sensitivity of CPT-EIA using the 227 bp amplicon of standard mecA was 50 amol or 7.6 pg (Figure 8A?). Using the same amplicon, the limit of detection using Et-Br stained agarose gel was 50,000 amol or 7.6 ng (Fig. 8B?). The sensitivity of the CPT EIA detection was, consequently, 3 logs better than that of the Et-Br detection. In addition, it took approximately 1 hour to analyze the standard am plicons by CPT-EIA, compared to 1.5 hours for gel analysis. The analysis by CTP-EIA is semi-quantitative, while the analysis by gel is qualitative. CPT-EIA was performed using the PCR amplicon prepared from a serial dilution of crude MSSA and MRSA. The results in Fig. 9 showed that CPT-EIA could detect the amplicon from a 20-cycle PCR reaction starting with 100 or more MRSA cells. The signal observed with MSSA was equivalent to support. Similar reactions analyzed by gel required PCR reactions starting with 1000 cells to detect a band with Et-Br staining. (Figure 1 0). It was estimated that 80% of the probe was cut with the 1000 equivalent samples of MRSA cells. Assuming that the PCR efficiency is 1 00%, the number of molecules generated in the reaction would be 1 75 ammol. Thus, the number of times the target is cyclized in CPT is at least 50. With this amplification, it is possible to use significantly fewer cycles (20) than a regular PCR experiment (30 or more cycles). Using fewer cycles has two advantages. The first is that the remaining am plicon opportunity is reduced, as mentioned and second, that the time for the PCR reaction is shortened by 1 hour. Although very fast thermocyclers have recently been developed, most thermocyclers require approximately 3 hours during a 30-cycle PCR compared to 2-hours for 20 cycles. The total time required for PCR-CPT is below 3 hours. The number of PCR cycles was varied between 20 and 40 using 100 equivalents of MRSA or MSSA cells. CPT-EIA detected MRSA samples after 20 cycles of POlf? F? F? G. 11 A), whereas agarose gel analysis detected samples only after 30 cycles (Fig. 11B). The number of amplicons generated in 20 versus 30 PCR cycles is 175 amol compared to 175 fmol, respectively, which corresponds to a 1000-fold difference in sensitivity, which is in agreement with the experiments described above. or absence of the specific target without the requirement of an instrument (data not shown) The presence of the specific target is indicated by a very light blue color or its absence due to the cut of the probe while the absence of the target results in a blue color In addition to its speed and reduced risk of contamination, PCR-CPT adds a second level of specificity to PCR, in fact, CPT is based on hybridization and under the conditions used, it will not be detected at mpllicons not reduced. This method is a step forward and can eliminate the use of Southern or nested PCR in certain applications, and the use of highly specialized detection means. CPT allows rapid and accurate detection of PCR amplicons. In addition, CPT adds a second level of amplification, but without further amplifying the target, and consequently, it is possible to use significantly fewer number of PCR cycles. This will reduce the chance of contamination and false positive. CTP adds a second level of specificity, which will avoid the detection of non-specific amplicons and starter dimer. The PCR-CPT method can also be used for the detection of mismatched genes.

From the foregoing, it will be appreciated that while specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

LIST OF DECISIONS (1) GENERAL INFORMATION: (i) APPLICANT: Bekkaoui, Faouzi Cloney, Lynn P. (i) TITLE OF THE INVENTION: METHODS TO DETECT QUICKLY METICILLIN RESISTANT STAPHYLOCOCCLES (iii) NUMBER OF SEQUENCES: 26 (iv) ADDRESS FOR CORRESPONDENCE: (A) DESTINY: SEED and BERRY LLP (B) STREET: 6300 Columbia Center, 701 Fifth Avenue (C) CITY: Seattle (D) STATE: Washington (E) COUNTRY: US (F) POSTAL CODE: 98104 (v) LEGIBLE COMPUTER FORM: (A) TYPE OF MEDIA: FLEXIBLE DISC (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PACKAGE: Patent in release # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) SUBMISSION DATE: June 22, 1998 (C) CLASSIFICATION : (vii i) ATTORNEY / AGENT INFORMATION: (A) NAME: McMasters, David D. (B) REGISTRATION NUMBER: 33,963 (C) REFERENCE NUMBER / CASE: 480094.424P3 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: (206) 622-4900 (B) TELEFAX: (206) 682-6031 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO.1 AATAGAGAAA AAGAAAAAAG ATGGCAAAG 29 (2) INFORMATION FOR SEQ ID jNO: 2: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2 CTTTGCCATC TTTTTTCTTT TTCTCTATT 29 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 CGCACAUACA UUAAUAGAGA AAAAGAAAAA AGAUGGCAAA GAUAUUCAAC UAACUAUUG 59 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 89 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 GAACTTTAGC ATCAATAGTT AGTTGAATAT CTTTGCCATC TTTTTTCTTT TTCTCATATT 60 ATGTATGTGC GATTGTATTG CTATTATCG 89 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 par.es of bases (B) TYPE: nucleic acid '(C) FILAMENTO: simple (D) TOPOLOGY: linear (ix) CHARACTERISTICS. (A) NAME / KEY: - (B) LOCATION: 14..17 (D) OTHER INFORMATION: / note = "where N is an adenine ribonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 AATAGAGAAA AAGNNNNAAG ATGGCAAAG 29 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 AATAGAGAAA AAGAAAAAAG ATGGCAAAGA AAAAAAAAAA AAAAAAA 47 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (x) CHARACTERISTICS: (A) NAME / KEY: - (B) LOCATION: 14..17 (D) OTHER INFORMATION: / note = "where N is an adenine ribonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 AATAGAGAAA AAGNNNNAAG ATGGCAAAG 29 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 AAUAGAGAAA AAGAAAAAAG AUGGCAAAG 29 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 TGGTAAAAAG GGACTCGAAA AACTT 25 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 GGTGGATAGC AGTACCTGAG CC 22 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 AGCTCCAACA TGAAGATGGC TATCGTGTC 29 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 ACCTGTTTGA GGGTGGATAG CAGTACCTGA 30 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 108 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 GACGATAATA GCAATACAAT CGCACATACA TTAATAGAGA AAAAGAAAAA AGATGGCAAA 60 GATATTCAAC TAACTATTGA TGCTAAAGTT CAAAAGAGTA TTTATAAC 108 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 ATACATTAAT AGAGAAAAAG AAAAAAGATG GCAAAG 36 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ix) CHARACTERISTICS: (A) NAME / KEY: - (B) LOCATION: 15..18 (D) OTHER INFORMATION: / note = "where N is an adenine ribonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15 ATCCTTAAT AGAGNNNNNAG AAAAAAGATG GCAAAG 36 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 ACGGAGAAGA AGTTGTAGCA GG 22 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 GGTGAAGTAG AAATGACTGA ACGTCCG 27 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 AAGATGGTAT GTGGAAGTTA GATTG 25 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEO ID NO: 19 GATGGTATGT GGAAGTTAGA TTGG 24 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20 TGGTATGTGG AAGTTAGATT GGGATC 26 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 ATGCAGTTAT TGGTAAAAAG GGACTCG 27 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22 TGTTTAGGGT GGATAGCAGT AC 22 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23 GAUAACAUUU UCUUUGCUAG AGUAG 25 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 CAAGTCGTAA ATAAAACACA TAAAGAAG 28 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION 'SEQ ID NO: 25 TACAAGATAA AGGAATGGCT AGCTA 25 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) ) FILAMENT: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26 GCTAGCTACA ATGCCAAAAT CTCAGGT 27

Claims (9)

1. REIVI NDICATIONS
2. 1 . A method for determining the presence of an antibiotic-resistant mecA gene in a biological sample, comprising: (a) treating cells contained within a biological sample to expose target single filament nucleic acid molecules; (b) reacting said target single filament nucleic acids with a nucleic acid probe containing the cleavable link, which is complementary to a portion of an antibiotic resistant mecA gene, and with an enzyme which cuts probe complexes. double filament target, under conditions that allow the target and probe to hybridize to each other to form a dual filament target probe complex, said enzyme molecule being capable of cutting said cleavable link of said probe-target complex, so that one or more fragments of the nucleic acid probe are released from complex d; and (c) determining whether the cut portions of a nucleic acid probe are produced, and thereby detecting the presence of an antibiotic-resistant mecA gene. The method according to claim 1, wherein the step of determining comprises detecting a decrease in the amount of uncut probe, and thereby determining whether the cut portions of said nucleic acid probe are produced.
3. 3. The method according to claim 1, wherein the step of determining comprises directly detecting cut portions of the nucleic acid probe. The method according to claim 1, wherein said probe consists essentially of at least a portion of the nucleotide sequence GACGATAATA GCAATACAAT CGCACATACA TTAATAGAGA AAAAGAAAAA AGATGGCAAA GATATTCAAC TAACTATTGA

   TGCTAAAGTT CAAAAGAGTA TTTATAAC (SEQ I D NO: 1 3). The method according to claim 1, wherein said probe consists essentially of at least a portion of the nucleotide sequence GAACTTTAGC ATCAATAGTT AGTTGAATAT CTTTGCCATC TTTTTTCTTT TTCTCTATTA ATGTATGTGC GATTGTATTG CTATTATCG (SEQ ID NO: 4). 6. The method according to claim 2, wherein said probe has the sequence of n ucleotides AATAGAGAAA AAGAAAAAAG ATGGCAAAG

   (SEQ I D NO: 1). The method according to claim 1, wherein said probe consists essentially of the nucleotide sequence AATAGAGaaaaAGAAAAA AGATGGCAAAG-3 '(SEQ ID NO: 5); where the capital letters represent deoxyribonucleotides and the lower case letters represent ribonucleotides. The method of claim 1, wherein the mecA gene is from a staphylococcus species. 9. The method of claim 8, wherein the mecA gene is from a positive coagulase staphylococcus species.

   0. The method of claim 9, wherein the mecA gene is from S. aureus. eleven . The method of claim 8, wherein the mecA gene is from the group consisting of coagulase-negative staphylococcus species. The method of claim 1, wherein the mecA gene is from S. epidermidis. The method of claim 1, wherein the biological sample is selected from the group consisting of a nasal swab, blood, urine, stool, abscess and spinal fluid. 14. The method of claim 1, wherein the biological sample was grown or isolated first in a bacteriological agent medium. 1 5. A probe to detect the presence of an antibiotic-resistant mecA gene in a biological sample, wherein said probe comprises the sequence GACGATAATA GCAATACAAT CGCACATACA TTAATAGAGA AAAAGAAAAA AGATGGCAAA GATATTCAAC TAACTATTGA

   TGCTAAAGTT CAAAAGAGTA TTTATAAC (SEQ I D NO: 1 3). 16. A set for detecting the presence of an antibiotic-resistant mecA gene in a biological sample, comprising (a) one or more nucleic acid probes containing cleavable link, and (b) an enzyme capable of cleaving the cleavable link when said probe joins a target. 7. The assembly according to claim 16, wherein said enzyme is RNase H. 8. The assembly according to claim 16, wherein the second mecA gene is from a species of staphylococcus.