Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

• ⁇ -lactam antibiotics e.g., penicillins and cephalosporins
• ⁇ -lactamases are enzymes that open the ⁇ -lactam ring of penicillins, cephalosporins, and related compounds, to inactivate the antibiotic.
• the production of ⁇ -lactamases is an important mechanism of resistance to ⁇ -lactam antibiotics among Gram-negative bacteria.
• Expanded-spectrum cephalosporins have been specifically designed to resist degradation by the older broad-spectrum ⁇ -lactamases such as TEM-1, 2, and SHV-1.
• ESBLs extended-spectrum ⁇ -lactamases
• CTX-M ⁇ -lactamases are spreading throughout North America and have been found in a wide variety of isolates within the family Enterobacteriaceae.
• Organisms producing CTX-M ⁇ -lactamases are typically resistant to cefotaxime, have lower minimum inhibitory concentration (MIC) values to ceftazidime and elevated MIC values to cefepime.
• MIC minimum inhibitory concentration
• these enzymes are capable of hydrolyzing the newer cephalosporins and aztreonam.
• NCLS National Committee for clinical Laboratory Standards
• ceftazidime an initial screening with either cefpodoxime, cefotaxime, ceftazidime, ceftriaxone, or_aztreonam followed by a confirmation test using both cefotaxime and ceftazidime in combination with clavulanate (NCCLS performance standards for antimicrobial and susceptibility testing; 12 th informational supplement, M100-S12 (2002)), and that a practice among some clinical laboratories is to use ceftazidime as the initial screening drug and ceftazidime with clavulanate as the confirmation test (Brenwald et al., /. Antimicrob. Chemother., 5i:195-196 (2003); Dandekar et al., Diagn. Microbiol, Infect.
• the present invention is directed to the use of oligonucleotide primers specific to nucleic acids characteristic of (typically, genes encoding) certain ⁇ - lactamases.
• the present invention uses primers of the invention to identify family-specific ⁇ -lactamase nucleic acids (typically, genes) in samples, particularly, in clinical isolates of Gram-negative bacteria. Even more specifically, the present invention provides primers to specifically identify groups within the CTX-M ⁇ -lactamase family.
• the present invention is directed to a primer selected from the group of: 5' - GAC GAT GTC ACT GGC TGA GC - 3' (SEQ ID NO:l); 5' - AGC CGC CGA CGC TAA TAC A - 3' (SEQ ID NO:2); 5' - GCG ACC TGG TTA ACT ACA ATC C - 3' (SEQ ID NO:3); 5' - CGG TAG TAT TGC CCT TAA GCC - 3' (SEQ ID NO:4); 5' - GCT GGA GAA AAG CAG CGG AG - 3' (SEQ ID NO:5); 5' - GTA AGC TGA CGC AAC GTC TG - 3' (SEQ ID NO:
• the present invention is directed to a primer pair including one primer selected from the group of: 5' - GAC GAT GTC ACT
• GGC TGA GC - 3' (SEQ ID NO: 1); 5' - AGC CGC CGA CGC TAA TAC A - 3' (SEQ ID NO:2); 5' - GCG ACC TGG TTA ACT ACA ATC C - 3' (SEQ ID NO:3); 5' - CGG TAG TAT TGC CCT TAA GCC - 3' (SEQ ID NO:4); 5' - GCT GGA GAA AAG CAG CGG AG - 3' (SEQ ID NO:5); 5' - GTA AGC TGA CGC AAC GTC TG - 3' (SEQ ID NO:6); 5' - CGC TTT GCC ATG TGC AGC
• the present invention is directed to a primer selected from the group of 5' - GAC GAT GTC ACT GGC TGA GC - 3' (SEQ ID NO: 1); 5' - AGC CGC CGA CGC TAA TAC A - 3' (SEQ ID NO:2); and full-length complements thereof; a primer selected from the group of 5' - GCG ACC TGG TTA ACT ACA ATC C - 3' (SEQ ID NO:3); 5' - CGG TAG TAT TGC CCT TAA GCC - 3' (SEQ ID NO:4); and full-length complements thereof; a primer selected from the group of 5' - GCT GGA GAA AAG CAG CGG AG - 3' (SEQ ID NO:5); 5' -
• a nucleic acid characteristic of a ⁇ -lactamase enzyme includes a gene or a portion thereof.
• a "gene” as used herein, is a segment or fragment of nucleic acid (e.g., a DNA molecule) involved in producing a peptide (e.g., a polypeptide and/or protein).
• a gene can include regions preceding
• coding region i.e., regulatory elements
• intervening sequences introns, e.g., non-coding regions
• coding region is used broadly herein to mean a region capable of being transcribed to form an RNA, the transcribed RNA can be, but need not necessarily be, further processed to yield an mRNA.
• a method for identifying a ⁇ -lactamase in a clinical sample is provided.
• the clinical sample provided is characterized as Gram- negative bacteria with resistance to ⁇ -lactam antibiotics.
• the method of the present invention for identifying a ⁇ -lactamase in a clinical sample includes, providing a pair of oligonucleotide primers specific for one or more groups within the CTX-M ⁇ -lactamase family, wherein one primer of the pair is complementary to at least a portion of the ⁇ -lactamase nucleic acid in the sense strand and the other primer of each pair is complementary to at least a portion of the ⁇ -lactamase nucleic acid in the antisense strand; annealing the primers to the ⁇ -lactamase nucleic acid; simultaneously extending the annealed primers from a 3' terminus of each primer to synthesize an extension product that is complementary to the nucleic acid strands annealed to each primer wherein each extension product after separation from the ⁇ -lactamase nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair; separating the amplified products; and analyzing the separated
• the CTX-M ⁇ -lactamase family includes several groups of ⁇ - lactamases within the family.
• the present invention provides a method for identifying one or more groups within the CTX-M ⁇ -lactamase family.
• the method described above, employs oligonucleotide primer pairs that are specific for one or more groups within the CTX-M family of ⁇ -lactamases, particularly primer pairs specific for one or more groups of CTX-M ⁇ - lactamases such as the ⁇ -lactamases of Group 1: CTX-M-1, 3, 10-12, 15 (also known as UOE-1), 22, 23, 28, 29, and 30; Group 2: CTX-M-2, 4-7, 20, and TOHO-1, Group 3: CTX-M-8; and Group 4: CTX-M-9, 13, 14, 16-19, 21, 27, and TOHO-2.
• the primers may also be specific for nucleic acid of the ⁇ - lactamases of Group 5: CTX-M-25 and 26.
• the primer pairs may be specific for one group within the CTX-M ⁇ -lactamase family or more than one group within the CTX-M ⁇ -lactamase family (e.g., a primer pair specific for two groups within the CTX-M ⁇ -lactamase family); however, none of the primer pairs of the present invention are specific for all CTX-M ⁇ -lactamase family groups (in which case they would be only family-specific but not group-specific).
• the primer pairs of the present invention can distinguish a CTX-M ⁇ - lactamase from another ⁇ -lactamase family (e.g., a TEM, SHV, or OXA family), the primer pairs of the present invention can also distinguish between different groups within the CTX-M ⁇ -lactamase family.
• Real-time polymerase chain reaction PCR is recognized in the art as a useful tool that may provide advantages over traditional PCR, as described more thoroughly below.
• the present invention is also directed to a method for identifying a ⁇ -lactamase in a clinical sample, the method including: providing a primer pair comprising one primer selected from the group of: 5'- GAC GAT GTC ACT GGC TGA GC - 3'(SEQ ID NO:1); 5'- AGC CGC CGA CGC TAA TAC A - 3'(SEQ ID NO:2); 5'- GCG ACC TGG TTA ACT ACA ATC C - 3'(SEQ ID NO:3); 5'- CGG TAG TAT TGC CCT TAA GCC - 3' (SEQ ID NO:4); 5' - GCT GGA GAA AAG CAG CGG AG - 3' (SEQ ID NO:5); 5' - GTA AGC TGA CGC AAC GTC TG - 3' (SEQ ID NO:6); 5' - CGC
• kits useful in detecting a ⁇ - lactamase of interest in a clinical sample include packaging, containing, separately packaged: at least one primer pair capable of hybridizing to a ⁇ -lactamase nucleic acid selected from the group of members of
• Groups 1-5 of the CTX-M ⁇ -lactamase family a positive and negative control; and a protocol for identification of the ⁇ -lactamase nucleic acid of interest, wherein the at least one primer pair is specific for one or more groups within the CTX-M ⁇ -lactamase family.
• the present invention is directed to a diagnostic kit for detecting a family of CTX-M ⁇ -lactamase which includes packaging, containing, separately packaged: at least one primer pair capable of hybridizing to a ⁇ - lactamase nucleic acid of interest; a positive and negative control; and a protocol for identification of the ⁇ -lactamase nucleic acid of interest; wherein the primers are selected from the group of: 5 ' -GAC GAT GTC ACT GGC TGA GC - 3 '
• the present invention is also directed to a diagnostic kit for detecting a family of CTX-M ⁇ -lactamase using real time polymerase chain reaction which includes packaging, containing, separately packaged: at least one primer pair capable of hybridizing to a ⁇ -lactamase nucleic acid of interest; a positive and negative control; and a protocol for identification of the ⁇ -lactamase nucleic acid of interest; wherein one primer of the pair of primers is selected from the group of: 5' -GAC GAT GTC ACT GGC TGA GC - 3' (SEQ ID NO:1); 5' -AGC CGC CGA CGC TAA TAC A - 3' (SEQ ID NO:2); 5' -GCG ACC TGG TTA ACT ACA ATC C - 3' (SEQ ID NO:
• Figures 1-2 are primers of the present invention.
• Figure 3 is a CTX-M PCR of four primer sets representing groups of b/ ⁇ CTXM genes.
• the present invention is directed to the detection of nucleic acid that is characteristic of (e.g., at least a segment of a gene that codes for) family-specific ⁇ -lactamase nucleic acid in samples (e.g., clinical isolates of Gram-negative bacteria). Specifically, the present invention is directed to the detection of ⁇ -lactamase nucleic acid (preferably, a gene or at least a segment of a gene) using unique primers and the polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• ⁇ -lactamases belonging to the CTX-M family e.g., ⁇ -lactamases of Groups 1-5 as indicated above
• the present invention provides primers to specifically identify groups within the CTX-M ⁇ -lactamase family. Accordingly, using the primers and methods of the present invention, not only can ⁇ -lactamases belonging to the CTX-M family be distinguished from other ⁇ - lactamases, but various groups within the CTX-M family (e.g., ⁇ -lactamases of Groups 1-5 as indicated herein) can be identified. This does not mean, however, that any one primer pair must be specific for only one group within the family, although this is preferred.
• Certain primers are specific for two (or more), but not all, groups, for example, which is still advantageous because it allows for narrowing the group identification.
• PCR amplification and sequencing of genes for use in characterizing organisms producing CTX-M- ⁇ -lactamases is known in the art, such as, for example, the molecular approach to screening ESBL-positive organisms for the presence of CTX-M genes as described in Edelstein et al. (Antimicrob. Agents Chemother., 47:3124-3132 (2003)).
• consensus primers i.e., "universal" primers
• which recognize all known variant genes of bla CTX _ M known in the art at the time are used to generate an amplified product of 544 base pairs.
• the specific groups of bla CTX _ M genes are identified by the use of restriction fragment length polymorphism (RFLP) analysis.
• RFLP restriction fragment length polymorphism
• the present invention provides an advantage over the above approach in that specific primer sets are used to detect various CTX-M- ⁇ -lactamase genes and/or groups of CTX-M- ⁇ -lactamase genes, negating the need to perform an additional identification step (at least for most embodiments). That is, it should be understood that for most embodiments of the present invention, an additional identification step is not necessarily needed, but one could be used if desired, particularly if the primers are specific for two or more groups and there is a desire to distinguish between these groups or to identify the specific CTX-M ⁇ -lactamase family member.
• the present invention provides amplification of a single DNA fragment for a CTX-M- ⁇ -lactamase gene, affording simple interpretation of results that can be adapted for use in reference laboratories for screening multiple isolates for the presence of CTX-M- ⁇ -lactamase genes.
• a selected primerjpair of the invention e.g., SEQ ID NO. 5 and SEQ ID NO.
• CTX-M- 14 ⁇ -lactamase is used to provide an amplified product, which, following a gel electrophoresis assay of the amplified products, provides identification of the ⁇ -lactamase of interest (i.e., the CTX-M- 14 ⁇ -lactamase) in a single step. That is, no additional identification steps, such as RFLP, WAVE analysis, sequence identification (Gold Standard), or single stranded conformational polymorphisms (SSCP), are required to identify the ⁇ -lactamases of interest, although such additional assays could be performed, if desired.
• the present invention is distinguished over the methods of Edelstein et al. as the Edelstein et al.
• the step of analyzing the separated amplified products includes a visual inspection of the gel produced by gel electrophoresis of the amplified products.
• the gel produced by the present methods typically provides a clear, unambiguous band; thus, the primers and methods of the present invention may be performed by personnel with lesser training and/or experience and still provide accurate results.
• the methods of Edelstein et al. typically do not produce as clear a product as do the methods of the present invention, as the results of RFLP analysis are often difficult to interpret and do not give the single band results provided by the primers and methods of the presentjnvention.
• the methods of Edelstein et al. generally provide more ambiguous results, which are typically more difficult for an untrained person to correctly interpret.
• the methods of the present invention provide primer pairs specific for one or more groups within the CTX-M ⁇ -lactamase family, which is defined herein to include primer pairs specific for one group (e.g., ⁇ -lactamases found in Group 1 of the CTX-M family) and also primer pairs specific for more than one group (e.g., ⁇ -lactamases found in Groups 3 and 5 of the CTX-M family); however, the primer pairs are not specific for all ⁇ -lactamase family groups.
• the present invention provides a method for identifying in a single step a ⁇ -lactamase included in the CTX-M ⁇ -lactamase family over a ⁇ - lactamase included in another family of ⁇ -lactamases (e.g., an OXA family), as well as provides a method of distinguishing one group of the CTX-M ⁇ - lactamase family over another group of the CTX-M ⁇ -lactamase family.
• This is distinguished over the methods of Edelstein et al., for example, which do not distinguish, and in particular do not distinguish in one step, between groups within the CTX-M ⁇ -lactamase family.
• Edelstein et al. for example, which do not distinguish, and in particular do not distinguish in one step, between groups within the CTX-M ⁇ -lactamase family.
• the present invention advantageously, provides the ability to identify in a clinical sample, in a single step, the presence of one group over another group of the CTX-M ⁇ -lactamase family.
• the methods of the present invention involve the use of the polymerase chain reaction sequence amplification method (PCR) using novel primers.
• PCR polymerase chain reaction sequence amplification method
• this amplification method relates to the treatment of a sample containing nucleic acid (typically, DNA) of interest from bacteria, particularly Gram-negative bacteria, with a molar excess of an oligonucleotide primer pair, heating the sample containing the nucleic acid of interest to yield two single-stranded complementary nucleic acid strands, adding the primer pair to the sample containing the nucleic acid strands, allowing each primer to anneal to a particular strand under appropriate temperature conditions that permit hybridization, providing a molar excess of nucleotide triphosphates and polymerase to extend each primer to form a complementary extension product that can be employed in amplification of a desired nucleic acid, detecting the amplified nucleic acid, and analyzing the amplified nucleic acid for a size specific amplicon (as indicated below) characteristic of the specific ⁇ -lactamase of interest.
• a size specific amplicon as indicated below
• the oligonucleotide primer pair which may include at least one primer selected from the group of SEQ ID NO. 1 to SEQ ID NO. 8, includes one primer that is substantially complementary to at least a portion of a sense strand of the nucleic acid and one primer that is substantially complementary to at least a portion of an antisense strand of the nucleic acid.
• the process of forming extension products preferably involves simultaneously extending the annealed primers from a 3' terminus of each primer to synthesize an extension product that is complementary to the nucleic acid strands annealed to each primer wherein each extension product after separation from the ⁇ -lactamase nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair.
• the amplified products are preferably detected by size fractionization using gel electrophoresis. Variations of the method are described in U.S. Patent No. 4,683,194 (Saiki et al.).
• oligonucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
• oligonucleotide refers particularly to the primary structure, and thus includes double and single-stranded DNA molecules and double and single- stranded RNA molecules.
• a “primer,” as used herein, is an oligonucleotide that is complementary to at least a portion of nucleic acid of interest and, after hybridization to the nucleic acid, may serve as a starting-point for the polymerase chain reaction.
• primer or "oligonucleotide primer,” as used herein, further refer to a primer, having a nucleotide sequence that possesses a high degree of nucleic acid sequence similarity to at least a portion of the nucleic acid of interest.
• “High degree” of sequence similarity refers to a primer that typically has at least about 80% nucleic acid sequence similarity, and preferably about 90% nucleic acid sequence similarity. Sequence similarity may be determined, for example, using sequence techniques such as GCG FastA (Genetics Computer Group, Madison, Wisconsin), Mac Vector 4.5 (Kodak/TBI software package) or other suitable sequencing programs or methods known in the art.
• complement and “complementary” as used herein, refer to a nucleic acid that is capable of hybridizing to a specified nucleic acid molecule under stringent hybridization conditions.
• Stringent hybridization conditions include, for example, temperatures from about 50°C to about 65°C, and magnesium (Mg) concentrations from about 1.5 millimolar (mM) to about 2.0 mM.
• Mg magnesium
• mM millimolar
• a specified DNA molecule is typically “complementary” to a nucleic acid if hybridization occurs between the specified DNA molecule and the nucleic acid.
• “Complementary,” further refers to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding in double stranded nucleic acid molecules.
• the terms “amplified molecule,” “amplified fragment,” and “amplicon” refer to a nucleic acid molecule (typically, DNA) that is a copy of at least a portion of the nucleic acid and its complementary sequence. The copies correspond in nucleotide sequence to the original molecule and its complementary sequence.
• the amplicon can be detected and analyzed by a wide variety of methods. These include, for example, gel electrophoresis, single strand conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP), capillary zone electrophoresis (CZE), and the like.
• the amplicon can be detected, and hence, the type of ⁇ -lactamase identified, using gel electrophoresis and appropriately sized markers, according to techniques known to one of skill in the art.
• the primers are oligonucleotides, either synthetic or naturally occurring, capable of acting as a point of initiating synthesis of a product complementary to the region of the DNA molecule containing the ⁇ -lactamase of interest.
• the primer includes nucleotides capable of hybridizing under stringent conditions to at least a portion of at least one strand of a nucleic acid molecule of a ⁇ - lactamase selected from the group of CTX-M-1 , CTX-M-2, CTX-M-3, CTX-
• the primers of the present invention typically have at least about 15 nucleotides, preferably at least about 18 nucleotides, and more preferably at least about 20 nucleotides.
• the primers have no more than about 30 nucleotides, preferably no more than about 28 nucleotides, more preferably no more than about 26 nucleotides, even more preferably no more than about 24 nucleotides, and still more preferably no more than about 22 nucleotides.
• the primers are chosen such that they preferably produce a primed product of at least about 300 base pairs and preferably no greater than about 600 base pairs, more preferably no greater than about 500 base pairs.
• a primer used in accordance with the present invention includes a label constituent.
• the label constituent can be selected from the group of an isotopic label, a fluorescent label, a polypeptide label, and a dye release compound.
• the label constituent is typically incorporated in the primer by including a nucleotide having the label attached thereto.
• Isotopic labels preferably include those compounds that are beta, gamma, or alpha emitters, more preferably isotopic labels are selected from the group of 32 P, 35 S, and 125 I.
• Fluorescent labels are typically dye compounds that emit visible radiation in passing from a higher to a lower electronic state, typically in which the time interval between adsorption and emission of energy is relatively short, generally on the order of about 10 "8 to about 10 "3 second.
• Suitable fluorescent compounds that can be utilized include fluorescien and rhodamine, for example.
• Suitable polypeptide labels that can be utilized in accordance with the present invention include antigens (e.g., biotin, digoxigenin, and the like) and enzymes (e.g., horse radish peroxidase).
• a dye release compound typically includes chemiluminescent systems defined as the emission of absorbed energy (typically as light) due to a chemical reaction of the components of the system, including oxyluminescence in which light is produced by chemical reactions involving oxygen.
• PCR While PCR, through the use of repetitive multiplication of template molecules, is a sensitive and extremely useful analytical tool, such repetitive multiplication also provides a drawback in that small differences in the template can result in significant differences in the amount of product.
• One answer to this concern is the use of "real-time" PCR.
• primers, methods, and kits of the present invention may also be useful in carrying out real-time PCR.
• Real-time PCR monitors the fluorescence emitted during the PCR reaction as an indicator of amplicon production during the PCR cycle (i.e., in real-time) to provide data collected in the exponential phase of the reaction.
• Real-time PCR is based on the detection and quantitation of a fluorescent reporter or intercalation of a florescent dye, such as S YBR ® Green into the amplified PCR product.
• the signal increases in direct proportion to the amount of PCR product in a reaction and, by recording the amount of fluorescence emission at each cycle, the PCR reaction may be monitored during the exponential phase.
• real-time PCR substantially eliminates the need for post-PCR processing of PCR products, thus increasing throughput and decreasing the possibility of carryover contamination.
• real-time PCR provides a wider dynamic range of an assay (i.e., determination of how much the target concentration can vary and still be quantified), and, thus, improved accuracy in quantitation of the PCR product.
• Real-time PCR may also be used for applications that would be less effective with traditional PCR (Dorak, Real-Time PCR, available on the World Wide Web at http//dorakmt.tripod.com/genetics/realtime.html, 2004).
• Real-time PCR methods typically use primers that are shorter than those used in traditional PCR methods; however, smaller amplicons are generally provided, typically at least about 150 base pairs, more preferably at least about 100 base pairs, and preferably no greater than about 200 base pairs.
• Real-time PCR may be used with primers and methods of the present invention wherein the primer pair used includes one primer selected from the primers of SEQ ID No. 1 to SEQ ID NO. 8, and another primer, resulting in an amplicon having, typically, no fewer than about 100 base pairs and no more than about 250 base pairs.
• the present invention provides a kit for use with real-time PCR, the kit including a primer pair wherein one primer of the primer pair is selected from the group of SEQ ID NO. 1 to SEQ ID NO. 8.
• primers of the present invention specific for certain ⁇ -lactamases are as follows, wherein "F” in the designations of the primers refers to a 5 'upstream primer and “R” refers to a 3' downstream primer.
• F in the designations of the primers refers to a 5 'upstream primer
• R refers to a 3' downstream primer.
• hybridization conditions utilizing primers of the invention include, for example, a hybridization temperature of at least about 50°
• primers are specific for nucleic acid characteristic of the following primers.
• CTX-M-1 3, 10-12, 15 (UOE-1), 22, 23, 28, 29 and 30 ⁇ -lactamase enzymes.
• CTX-M-2 4, 5, 6, 7, and 20 ⁇ -lactamase enzymes, as well as TOHO-1 ⁇ - lactamase.
• a size-specific amplicon of 351 base pairs will typically be obtained.
• the following primers are specific for nucleic acid characteristic of the CTX-M-9, 13, 14, 16, 17, 18, 19, 21, and 27 ⁇ -lactamase enzymes, as well as TOHO-2 ⁇ -lactamase.
• Primer Sequence 5' -GTA AGC TGA CGC AAC GTC TG - 3' (SEQ ID NO:6) Employing a primer pair containing the primer sequences of SEQ ID NO:6
• CTX-M-8, 25, and 26 ⁇ -lactamase enzymes are examples of CTX-M-8, 25, and 26 ⁇ -lactamase enzymes.
• Primer Sequence 5' -GCT CAG TAC GAT CGA GCC - 3' (SEQ ID NO:8) Employing a primer pair containing the primer sequences of SEQ ID NO:8
• a size-specific amplicon of 307 base pairs will typically be obtained.
• Various other primers, or variations of the primers described above, can also be prepared and used according to methods of the present invention.
• alternative primers can be designed based on targeted ⁇ -lactamase genes known or suspected to contain regions possessing high G/C content (i.e., the percentage of guanine and cytosine residues).
• a "high G/C content" in a target nucleic acid typically includes regions having a percentage of guanine and cytosine residues of about 60% to about 90%.
• changes in a prepared primer will alter, for example, the hybridization or annealing temperatures of the primer, the size of the primer employed, and the sequence of the specific resistance gene or nucleic acid to be identified. Therefore, manipulation of the G/C content, e.g., increasing or decreasing, of a primer or primer pair may be beneficial in increasing detection sensitivity in the method.
• Oligonucleotides of the invention can readily be synthesized by techniques known in the art (see, for example, Crea et al., Proc. Natl. Acad. Sci.
• a nucleic acid is isolated from a bacterial control strain known to express or contain the resistance gene.
• This control strain refers to a "positive control" nucleic acid (typically, DNA).
• a "negative control" nucleic acid typically, DNA
• the designed primers are employed in a detection method, as described above, and used in the positive and negative control samples and in at least one test sample suspected of containing the resistance gene of interest.
• the positive and negative controls provide an effective and qualitative (or grossly quantitative) means by which to establish the presence or the absence of the gene of interest of test clinical samples. It should be recognized that with a small percentage of primer pairs, possible cross-reactivity with other ⁇ -lactamase genes might be observed. However, the size and/or intensity of any cross-reactive amplified product will be considerably different and can therefore be readily evaluated and dismissed as a negative result.
• the invention also relates to kits for identifying a family-specific ⁇ - lactamase enzyme by PCR analysis.
• Kits of the invention typically include one or more primer pairs specific for a ⁇ -lactamase of interest, one or more positive controls, one or more negative controls, and protocol for identification of the ⁇ - lactamase of interest using polymerase chain reaction.
• Primer pairs useful in kits of the present invention include those selected from the group of SEQ ID NO. 1 to SEQ ID NO. 8.
• kits useful with real-time PCR methods include primer pairs wherein one primer of the pair is selected from the group of SEQ ID No. 1 to SEQ ID NO. 8.
• a negative control includes a nucleic acid (typically, DNA) molecule encoding a resistant ⁇ -lactamase gene other than the ⁇ -lactamase gene of interest.
• the negative control nucleic acid may be a naked nucleic acid (typically, DNA) molecule or inserted into a bacterial cell.
• the negative control nucleic acid is double-stranded; however, a single-stranded nucleic acid may be employed.
• a positive control includes a nucleic acid (typically, DNA) that encodes a ⁇ - lactamase gene from the family of ⁇ -lactamase genes of interest.
• the positive control nucleic acid may be a naked nucleic acid molecule or inserted into a bacterial cell, for example.
• the positive control nucleic acid is double-stranded, however, a single-stranded nucleic acid may be employed.
• the nucleic acid is obtained from a bacterial lysate.
• the present invention provides a kit for characterizing and identifying a family-specific ⁇ -lactamase gene that would have general applicability.
• the kit includes a polymerase (typically, DNA polymerase) enzyme, such as Taq polymerase, and the like.
• a kit of the invention also preferably includes at least one primer pair that is specific for a ⁇ -lactamase.
• a buffer system compatible with the polymerase enzyme is also included and are well known in the art.
• the at least one primer pair may contain a label constituent, a fluorescent label, a polypeptide label, and a dye release compound.
• the kit may further contain at least one internal sample control, in addition to one or more further means required for PCR analysis, such as a reaction vessel.
• a nucleic acid from the bacterial sample can be isolated and then subjected to PCR analysis using the provided primer set of the invention.
• family-specific ⁇ -lactamase genes in clinical samples particularly clinical samples containing Gram-negative bacteria, can be detected by the primers described herein in a "microchip" detection method.
• nucleic acid e.g., genes, of multiple ⁇ -lactamases in clinical samples can be detected with a minimal requirement for human intervention. Techniques borrowed from the microelectronics industry are particularly suitable to these ends.
• an object of the present invention is to provide a parallel screening method wherein multiple serial reactions are automatically performed individually within one reaction well for each of the plurality of nucleic acid strands to be detected in the plural parallel sample wells. These serial reactions are performed in a simultaneous run within each of the multiple parallel lanes of the device.
• Parallel as used herein means wells identical in function.
• the present invention provides multiple reaction wells, the reaction wells being reaction chambers, on a microchip, each reaction well containing an individualized array to be used for detecting a ⁇ -lactamase gene uniquely specified by the substrates provided, the reaction conditions and the sequence of reactions in that well.
• the chip can thus be used as a method for identifying ⁇ - lactamase genes in clinical samples.
• the quality control strains used for this study were E. coli ATCC 25922, E. coli 35218, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and K.
• K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as positive and negative controls, respectively.
• PCR Polymerase Chain Reaction
• Group IV primers amplified a 474 bp product from DNA prepared from strains 785D, EC984167, CFl, Rio-6, BM4493, ILT-2, and ILT-3. This primer set was also very specific resulting in no amplification of DNA when template was prepared from the other control strains. Using the group in primer set resulted in amplification of DNA prepared from only one strain, Rio-3, which produced CTX-M-8. An isolate producing CTX-M-25 was requested but not obtained. Therefore, we were unable to examine the ability of the Group III primer set to amplify blacrx-M-25. A representative gel indicating the specificity of the primer pairs is shown in Fig. 2. These data indicate a high level of specificity for these group-specific primer pairs.
• Group I includes CTX-M-1, 3, 10-12, 15 (UOE-1), 22, 23, 28, 29, 30;
• Group II includes CTX-M-2, 4-7, 20, and TOHO-1;
• Group III includes CTX- M-8;
• Group IV includes CTX-M-9, 13, 14, 16-19, 21, 27, and TOHO-2; and
• Group V includes CTX-M-25 and 26.

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