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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to the field of molecular diagnostics, in particular for the determination of the presence of beta-lactam-resistant bacteria in a biological sample.
• ESBLs Extended-spectrum beta-lactamases
• ESBLs are capable of conferring resistance to the most common beta-lactam antibiotics in use in institutional and outpatient care, such as peni- cillins, cephalosporins, and monobactams.
• ESBLs may also confer multi-drug resistance to non-beta-lactam antibiotics such as quinolones, aminoglycosides and trimethoprim, narrowing treatment options.
• ESBL-producing bacteria were first discovered in the early 1980s among hospitalized patients, particularly in the most vulnerable patients in in- tensive care units, but have now spread also among community-based patients.
• ESBL-associated infectious syndromes include mainly urinary tract infections and secondly intra-abdominal infections. Upon escape to the bloodstream, ESBL-producing bacteria may cause sepsis or other infections serious enough to warrant hospitalization.
• CTX-M-encoding blacrx-M gene originates from chro- mosomally encoded enzymes of the Kluyvera spp but exists today as a plas- mid-conjugated gene with the ability to move between different bacterial popu- lations.
• Primary carriers of blacrx-M include Escherichia coli, Klebsiella pneu- moniae, Salmonella typhimurium, and Proteus mirabilis, but further blacrx-M- carrying bacterial strains emerge increasingly.
• CTX-M types To date over 124 different CTX- M types have been reported. There is no consensus on the precise classification of CTX-M types but according to D'Andrea (Int. J. Med., 2013.
• the main CTX-M groups are CTX-M-1 , -2, -8, -9, -25, and KLUC.
• Amino acid variation within each subgroup is less than 5%, while the inter-group variation is at least 10%.
• Detection and identification of ESBL producers is crucial not only because delayed recognition and inappropriate treatment of infected patients has been associated with increased mortality but also to limit the spread of these multidrug-resistant organisms.
• Widely used sensitivity tests such as disk diffusion and dilution antimicrobial susceptibility tests, as well as confirmatory tests, mostly based on synergy between clavulanic acid and cephalosporin, are inexpensive and relatively easy to use. However, such tests are time- consuming and suffer from limited sensitivity.
• Assaying ESBLs at the genetic level represents an alternative approach for the identification and typing of blacrx-M genes.
• numerous PCR-based assays have been developed. Such assays are precise and sensitive but many of them require a battery of amplicon specific sequencing primers as well as labor-intensive and time-consuming analysis of the PCR product, e.g. by DNA sequencing.
• These drawbacks can be avoided, at least partly, by universal, degenerated CTX-M primers or by employing real-time PCR-based methods allowing simultaneous amplification and analysis of the PCR product as disclosed by Birkett et al. in J. Med. Microbiol., 2007, 56:52- 55.
• a further drawback associated with methods of identifying blacrx-M genotypes at the nucleotide level is concomitant detection of highly homologous non-blacTx-M gene sequences, particularly chromosomal K1 gene of Klebsiella oxytoca, causing false positive results and, thus, necessitating use of confirmatory tests.
• Patent Publication US 2009/0163382 discloses primers and probes for a microarray-based method of detecting antibiotic-resistant bacterial species on the basis of a number of different antibiotic resistance genes. However, only a part of the 124 different currently known CTX-M types are detected by this method. Also Fu et al. (J. Microbiol. Methods, 2012, 89:1 10-1 18) disclose a DNA microarray for drug-resistant gene detection. The array contains a total of 1 15 probes from 17 categories of drug-resistant genes. However, only a part of the 124 different currently known CTX-M types are detected by this method.
• the present invention relates to a method of determining the presence or absence of CTX-M producing bacteria in a biological sample.
• the method comprises the steps of:
• steps b) and c) may be performed either separately or simultaneously.
• the probe hybridizes to a region of blacrx-M gene, which region corresponds to that of nucleotides 503-539 of SEQ ID NO: 23 in CTX-M-15.
• a person skilled in the art can easily recognize corresponding blacrx-M regions in other CTX-M variants.
• the present embodiments are not limited to probes hybridizing to blacrx- M-is but encompass probes hybridizing to any blacrx-M variant.
• the probe may be bisected to a 5'-probe and a 3'-probe.
• said 5'-probe comprises a nucleic acid sequence set forth in SEQ ID NO: 34, 35, or 63
• said 3'-probe comprises a nucleic acid sequence set forth in SEQ ID NO: 44, 45, or 70.
• the probes may also have a sequence identity of at least 80% with the sequences disclosed herein as long as they retain their ca- pability to hybridize with blacrx-M genes under normal hybridization conditions.
• the amplification step is performed with a forward primer comprising a nucleic acid sequence set forth in SEQ ID NO: 1 , 2, or 61 and a reverse primer comprising a nucleic acid sequence set forth in SEQ ID NO: 1 1 or 12.
• the primers may also have a sequence identity of at least 80% with the sequences disclosed herein as long as they retain their capability to amplify blacrx-M genes under normal PCR primer hybridization conditions.
• the present invention provides oligonucleotide primers and probes capable of amplifying and hybridizing to blacrx-M., respec- tively.
• the probes may comprise a nucleic acid sequence having at least 80% sequence identity to the sequence set forth in any of SEQ ID NO:34, 35, 44, 45, 63, or 70, or to at least 10 consecutive nucleotides of SEQ ID NO:24 or SEQ ID NO: 72, as long as they retain their capability to hybridize with blacrx-M genes under normal hybridization conditions.
• an oligonucleotide probe mixture comprising at least three different oligonucleotide molecules each comprising, independently form each other, at least 10 consecutive nucleotides of a nucleic acid sequence comprised in SEQ ID NO: 24 or SEQ ID NO: 72.
• probes derived from SEQ ID NO:24 or SEQ ID NO: 72 may exist as bisected dual probes.
• the primers may comprise a nucleic acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:1 , 2, 1 1 , 12, or 61 , as long as they retain their capability to amplify blacrx-M genes under normal PCR primer hybridization conditions.
• Figure 1 is a schematic drawing illustrating the principle of chelate- complementation-based detection methods (Karhunen et al., Anal. Chem., 2010, 82:751 -754).
• Figure 1A no target oligonucleotide is present and, thus, chelate complementation does not occur.
• Figure 1 B a target oligonucleotide is present and chelate complementation is enabled upon binding of the lantha- nide-ion-carrier-chelate-conjugated probe and the antenna-conjugated probe in a close proximity to the target oligonucleotide.
• Figure 2 illustrates the performance of the present primers in a SYBR® Green l-based PCR.
• Figure 3 illustrates the distinction of K1 amplification from blacrx-M amplification.
• Figure 3A shows a PCR amplification graph of a representative reaction with either 0 (circle), 1 000 (triangle), or 10000 (square) genome copies of CTX-M-1 -group-positive E. coli, or 10000 genome copies of K. oxytoca (cross).
• Figure 3B shows the melt curves of corresponding amplification prod- ucts. The curves for amplification products with initial 1 000 or 10000 genome copies of CTX-M positive E. coli are superimposed.
• RFU relative fluorescent unit.
• Figure 4 shows a chelate complementation-based PCR amplification graph as an average of three parallel reactions when two independent CTX-M-1 -group (circle), CTX-M-2-group (triangle), or CTX-M-9-group-positive (square) E. coli samples, three independent K. oxytoca samples (cross), and one CTX-M-negative E. coli sample (cross) were used as templates in an amount of 10000 genome copies per reaction.
• primer mixtures of SEQ ID NO:2 and SEQ ID NO:1 1 were used together with Eu-probe (SEQ ID NO:35) and antenna-probe (SEQ ID NO:45) mixtures (0.05 ⁇ of each individual probe). Only CTX-M-positive samples provided signals distinguishable from the background signal.
• Figure 5 demonstrates the analytical sensitivity of the present diagnostic chelate complementation-based PCR method as an average of three independent reactions.
• DNA from a CTX-M-1 -group-positive ( Figure 5A), CTX- M-2-group-positive ( Figure 5B), or CTX-M-9-group-positive ( Figure 5C) E. coli sample was used as a template at 10000 (circle), 1 000 (triangle), 100 (square), 10 (diamond), 1 (minus sign), 0.1 (plus sign), 0.01 (line with no symbol), or 0 (cross) genome copies per PCR reaction.
• the threshold cycle is the PCR cycle where the reaction signal exceeds the threshold level (signal-to- background 1 .5).
• Figure 6 presents the results of the analytical specificity of the chelate complementation-based PCR with extended CTX-M variant target variety with Eu-probe mixture of SEQ ID NO: 63 and antenna-probe mixture of SEQ ID NO:70 as an average of three independent reactions. Thirty CTX-M-positive samples (cross) were used as templates in an amount of 100 genome copies per reaction. Twenty five CTX-M-negative samples (triangles) were used as templates in an amount of 100 000 genome copies per reaction. Only CTX-M- positive samples produced signals distinguishable from the background signal.
• Figure 7 illustrates the analytical sensitivity of the chelate comple- mentation-based PCR with extended CTX-M variant target variety with Eu- probe mixture of SEQ ID NO: 63 and antenna-probe mixture of SEQ ID NO: 70 as an average of three independent reactions.
• DNA from a CTX-M-2-group- positive sample was used as a template at 100 000 (line with no symbol), 10000 (circle), 1 000 (triangle), 100 (square), 10 (diamond), 1 (minus sign), 0.1 (plus sign) or 0 (cross) genome copies per PCR reaction. Similar results were obtained with CTX-M-1 -group, CTX-M-8-group, CTX-M-9-group, or CTX-M-25- group-positive samples.
• Figure 8 demonstrates the performance of the chelate complementation-based PCR with extended CTX-M variant target variety with Eu-probe mixture of SEQ ID NO: 63 and antenna-probe mixture of SEQ ID NO: 70 with bacterial cells using CTX-M-2-positive cells as an example.
• the data points of the figure represent an average of three independent reactions. DNA was not isolated from the bacterial cells before analysis but the cells were added intact as a template to the PCR reaction at 300 000 (line with no symbol), 30000 (circle), 3 000 (triangle), 300 (square), 30 (diamond), 3 (minus sign), 0.3 (plus sign) or 0 (cross) colony forming units per PCR reaction.
• the present invention relates to methods and means for determining the presence of beta-lactamase, particularly extended spectrum beta- lactamase (ESBL), producing bacteria in a biological sample. More specifically, the ESBL-producing bacteria are CTX-M-producing bacteria.
• beta-lactamase particularly extended spectrum beta- lactamase (ESBL)
• ESBL extended spectrum beta- lactamase
• extended spectrum beta-lactamase or “ESBL” refers any enzyme capable of providing resistance to and deactivating the antibacterial properties of beta-lactam antibiotics such as penicillins, cephalosporins (e.g. cefotaxime, ceftriaxone, ceftazidime, cefepime, and oxy- imino-monobactam aztreonam), and monobactams by breaking down the ⁇ - lactam ring structure common to all beta-lactam antibiotics.
• beta-lactam antibiotics such as penicillins, cephalosporins (e.g. cefotaxime, ceftriaxone, ceftazidime, cefepime, and oxy- imino-monobactam aztreonam)
• monobactams by breaking down the ⁇ - lactam ring structure common to all beta-lactam antibiotics.
• CTX-M refers to any member of a subgroup of ESBLs, i.e. plasmid encoded enzymes having predominantly greater activity against cefotaxime than other oxyimino cephalosoprins.
• CTX-M encompasses not only the at least 124 different blacrx-M -encoded enzymes identified to date but also any CTX-M species to be discovered in the future.
• CTX-M species irrespective of the subgroup into which they have been classified on the basis of amino acid sequence alignments, are encompassed in the term "CTX-M".
• CTX-M-1 -group-positive sample refers to a sample containing bacteria carrying a gene for a CTX-M type belonging to the CTX-M-1 group.
• CTX-M-2, -8, -9, and -25 groups are main CTX-M groups, namely CTX-M-2, -8, -9, and -25 groups.
• CTX-M-2 ⁇ 92507 CTX-M-75* GQ 149244 Y10278, AB059404,
• CTX-M-3 AB098539, AF550415 CTX-M-76 AM982520
• CTX-M-7 AJ005045 CTX-M-80 EU202673
• CTX-M-8 AF189721 CTX-M-81 EU136031
• CTX-M-9 AF174129 CTX-M-82 EU545409, DQ256091
• CTX-M-1 1 AY0051 10, AJ310929 CTX-M-84 FJ214367
• CTX-M-14 AF31 1345 CTX-M-88 FJ873739
• CTX-M-17 AY033516 CTX-M-91 GQ870432
• CTX-M-27 AY156923 CTX-M-102 HQ398215
• CTX-M-29 AY267213 CTX-M-105 HQ833651
• CTX-M-34 AY515297 CTX-M-1 10 JF274242
• CTX-M-35 AB176533 CTX-M-1 1 1 JF274243
• CTX-M-36 AB177384 CTX-M-1 12 JF274246
• CTX-M-38 AY822595 CTX-M-1 14 GQ351346
• CTX-M-42 DQ061 159 CTX-M-122 JN790863
• CTX-M-48 AY847144 CTX-M-133 AB 185834
• the present oligonucleotides are not fully complementary to sequences marked with an asterisk.
• This target region comprises short regions which show very high homology between different CTX-M-variants but differs clearly from a corresponding region in closely related non-CTX-M sequences used in the alignment.
• the alignment was used to design fully complementary probes and primers for more than 90% of the CTX-M variants.
• Designed probes and 3'- primers contained at least 2 to 6 nucleotide differences as compared to the closely-related non-CTX-M sequences used in the alignment. All probe sequences were designed to be complementary to and thus detect antisense strands because reverse primers amplifying antisense strands have more nucleotide mismatches against non-CTX-M sequences compared to forward pri- mers amplifying sense strands. In other words, the antisense strands are amplified more specifically than the corresponding sense strands.
• each oligonucleotide sequence was designed individually such that the total number of sequences required for identifying as many CTX-M variants as possible would be as low as possible.
• the present sequences differ from earlier CTX-M probes and primes not only by their length but also by significantly lower number of different primer and probe molecules required for broad-range CTX-M detection.
• the term "primer” refers to an oligonucleotide mole- cule comprising or consisting of at least 20 nucleotides designed to hybridize with a complementary portion of a target blacrx-M gene and to act as an initiation site for the amplification of the target nucleic acid molecule e.g. by PCR.
• the term "5'-primer”, i.e. "forward primer”, refers to a primer molecule which hybridizes to the antisense strand and amplifies the nucleotides of the sense strand of a blacrx-M gene
• forward primer refers to a primer molecule which hybridizes to the antisense strand and amplifies the nucleotides of the sense strand of a blacrx-M gene
• reverse primer refers to a primer molecule which hybridizes to the sense strand and amplifies the nucleotides of the antisense strand of a blacrx-M gene.
• the present invention provides 5'-primers comprising or consisting of the following nucleic acid sequence:
• Xi is either C or T, and X2 is either A or G;
• X 2 is either A or G
• X3 is G when Xi is C, and X2 is A or G; or
• X3 is G when Xi is T, and X2 is G;
• X 3 is T when Xi is T, and X2 is A;
• Xi is C when X 2 is A, X 3 is C, and X4 is G;
• Xi is T when X 2 is G, X3 is T, and X4 is G; or
• Xi is T when X 2 is A, X 3 is T, and X4 is T.
• primers may be presented in an alternative way, i.e. as mix- tures of forward primers comprising or consisting of nucleic acid sequences set forth in SEQ ID NO:s 3 to 6, SEQ ID NO:s 7 to 10, or SEQ ID NO:s 7 to 10 and 62, respectively (Table 2).
• any combination or any one of these primers or primer mixtures may be employed.
• Primers of SEQ ID NO:s 3 to 6 are encompassed in the forward primer mixture of SEQ ID NO:1
• primers of SEQ ID NO:s 7 to 10 are encompassed in the forward primer mixture of SEQ ID NO:2
• primers of SEQ ID NOs: 7 to 10 and 62 are encompassed in the forward primer mixture of SEQ ID NO: 61 .
• the present invention provides 3'-primers com- prising or consisting of a nucleic acid sequence:
• Xi is A when X 2 is either A or C, X3 is G, X4 is C, X 5 is A, and X 6 is C;
• Xi is G when X 2 is C, X3 is G, X4 is C, X 5 is either G or A, and
• Xi is G when X 2 is C
• X3 is A
• X4 is T
• X 5 is G
• ⁇ is G
• these primers may be set forth in an alternative way, i.e. as mixtures of reverse primers comprising or consisting of SEQ ID NO:s 13 to 17, or SEQ ID NO:s 18 to 22, respectively.
• any combination or any one of the primers set forth above may be employed.
• 5'- and 3'-phmers or primer mixtures may be used in any desired combination for the amplification of CTX-M target nucleic acids.
• 5'-primer mixture of SEQ ID NO:1 may be used together with 3'-primer mixture of SEQ ID NO:12; 5'-primer mixture of SEQ ID NO:2 and 3'-primer mixture of SEQ ID NO:1 1 ; and 5'-primer mixture of SEQ ID NO:61 and 3'-primer mixture of SEQ ID NO:1 1 .
• primer pairs include 5'-primer mixture of SEQ ID NO:1 and 3'-primer mixture of SEQ ID NO:1 1 ; 5'-primer mixture of SEQ ID NO:2 and 3'-primer mixture of SEQ ID NO:12; and 5'-primer mixture of SEQ ID NO:61 and 3'-primer mixture of SEQ ID NO:12. Replacing the primer mixture of SEQ ID NO:1 or 2 with the primer mixture of SEQ ID NO:61 extends the range of CTX-M-types to be amplified to cover also CTX-M- 1 14, if present in the sample.
• the primers do not have to be exactly complementary to the target strand but must be sufficiently complementary to hybridize therewith and retain the capability to amplify the 6/acT -M genes under normal primer hybridization conditions.
• hybridize or “bind” refers to the physical interaction between complementary regions of two single-stranded nucleic acid molecules creating a double-stranded structure.
• hybridize refers to interactions between present oligonucleotides and their target polynucleotides under hybridization conditions that allow complementary regions of the two molecules to interact by hydrogen bonding and remain engaged.
• hybridization conditions refers independently not only to the conditions of the hybridization step per se, but also to the conditions of one or more washing steps performed thereafter.
• Modifiable variables of the hybridi- zation conditions include, but are not limited to, duration (typically from some seconds to some hours), temperature (generally from 25°C to 70°C), salt composition and concentration (e.g., 2-4xSSC6xSSC, or SSPE), chaotropic agent composition (e.g., formamide, or dimethyl sulfoxide (DMSO)) and concentra- tion, and usage of substances that decrease non-specific binding (e.g., bovine serum albumin (BSA), or salmon sperm DNA (ssDNA)).
• duration typically from some seconds to some hours
• temperature generally from 25°C to 70°C
• salt composition and concentration e.g., 2-4xSSC6xSSC, or SSPE
• chaotropic agent composition e.g., formamide, or dimethyl sulfoxide (DMSO)
• concentra- tion e.g., concentra- tion
• usage of substances that decrease non-specific binding e
• hybridization stringency refers to the degree to which mismatches are tolerated in hybridization. The more stringent the conditions, the more likely mismatched DNA strands are to be forced apart, whereas less stringent hybridization conditions enhance the stability of mismatched strands. A person skilled in the art is able to select the hybridization conditions such that a desired level of stringency is achieved. Generally, the stringency may be increased by increasing temperatures, lowering the salt concentrations, and using organic solvents.
• the present primers are designed to hybridize to their target sequences under normal PCR primer hybridization conditions. A person skilled in the art is able to determine and select such conditions easily.
• a further aspect of the present invention relates to oligonucleotide probes.
• the term "probe” refers to an oligonucleotide designed for detecting a target nucleic acid molecule in a sample to be analyzed.
• the present probes may be provided in different forms as well known in the art. For instance, each probe may be provided as a single probe molecule or as a dual probe consisting of two individual probe molecules, which hybridize next to each other to adjacent positions, preferably with zero to ten intervening nucleotides, in a complementary target sequence. In some embodiments, the dual probes have one intervening nucleotide in the complementary target sequence.
• the present probes may be provided as single oligonucleotide molecules, denoted hereinafter as mono-probes.
• the probes hybridize to the region of blacrx-M gene corresponding to nu- cleotides 397-617 (i.e. an amplicon obtainable by the present primers) or preferably nucleotides 503-539 of SEQ ID NO: 23 derived from CTX-M-15 variant (Gene Bank No. AY463958).
• the mono-probes comprise or consist of at least 10, preferably 10 to 30, nucleotides which hybridize to consecutive nucleotides in said region of nucleotides 397-617 or 503-539 of SEQ ID NO: 23.
• the present mono-probes comprise or consist of a nucleic acid sequence of SEQ ID NO: 24 SEQ ID NO: 72, or at least 10, preferably 10 to 30, consecutive nucleotides thereof.
• SEQ ID NO:24 and 72 are as follows:
• Xi is T when X 2 is T, X 3 is G, X4 is C, X 5 is A, X 6 is A, X 7 is C, Xs is T when X 2 is T, X 3 is G, X4 is G, X 5 is A, X 6 is A, X 7 is C, Xs is
• Xi is C when X 2 is T, X 3 is C, X4 is G or T, X 5 is G, ⁇ is A, X 7 is T,
• Xi is C when X 2 is T, X 3 is G, X4 is G, X 5 is G, ⁇ is A, , X 7 is T, X 8 is A, and X 9 is C;
• Xi is C when X 2 is T, X 3 is G, X4 is C, X 5 is A, X 6 is A, X 7 is C, Xs is when X 2 is C, X 3 is C, X 4 is G, X 5 is A, X 6 is A, X 7 is C, Xs is
• Xi is T when X 2 is A, X 3 is T, X4 is G, X 5 is A, X 6 is T, X 7 is G, Xs is G, X9 is A, X10 is A, X11 is G, Xi 2 is C; Xi 3 is A or G, and Xi 4 is T;
• Xi is T or C, when X 2 is A, X 3 is T, X4 is G, X 5 is A, X 6 is T, X 7 is G, Xs is C, X9 is A, X10 is A, X is A, Xi 2 is C; Xi 3 is A, and Xi 4 is C;
• Xi is T when X 2 is A, X 3 is T, X4 is G, X 5 is A, X 6 is T, X 7 is G, Xs is G, X9 is A, X10 is A, X is A, Xi 2 is C; Xi 3 is A, and Xi 4 is T;
• Xi is C when X 2 is A, X 3 is C, X4 is C, X 5 is A, X 6 is T, X 7 is G, Xs is G, X9 is A, X10, is A, X is A, Xi 2 is C; Xi 3 is A, and Xi 4 T;
• Xi is C when X 2 is A, X 3 is C, X4 is A, X 5 is A, X 6 is C, X 7 is G, Xs is
• X 9 is A, X10, is A, X is G, Xi 2 is C; Xi 3 is A, and Xi 4 T;
• Xi is C when X 2 is A, X 3 is T, X4 is C, X 5 is A, X 6 is T, X 7 is G, Xs is G or T, X 9 is G when Xi 0 , is A, X is A, Xi 2 is T; Xi 3 is A, and Xi 4 is C;
• Xi is C when X 2 is A, X 3 is T, X4 is G, X 5 is A, X 6 is T, X 7 is G, Xs is G, Xg is G, X10, is A, X is A, Xi 2 is T; Xi 3 is A, and Xi 4 is C;
• Xi is C, when X 2 is A, X 3 is T, X4 is G, X 5 is A, X 6 is T, X 7 is G, Xs is C, Xg is A, Xio is G, Xn is A, Xi 2 is C; X13 is A, and Xi 4 is C;
• Xi is C, when X 2 is A, X 3 is T, X4 is C, X 5 is A, X 6 is T, X 7 is A, X 8 is T, X 9 is A, X10 is A, X is A, X12 is C; X13 is A, and Xi 4 is C;
• Xi is C, when X 2 is A, X 3 is T, X4 is C, X 5 is A, X 6 is C, X 7 is G, Xs is T, Xg is A, X10 is A, X is A, Xi 2 is C; X13 is A, and Xi 4 is C;
• Xi is C, when X 2 is G, X3 is T, X4 is G, X 5 is A, X 6 is T, X 7 is G, Xs is C, Xg is A, X10 is A, X is A, Xi 2 is C; X13 is A, and Xi 4 is C;
• Xi is C, when X 2 is A, X 3 is T, X4 is G, X 5 is T, X 6 is T, X 7 is G, Xs is C, Xg is A, X10 is A, X is A, Xi 2 is C; X13 is A, and Xi 4 is C; and
• Xi is C, when X 2 is A, X 3 is T, X4 is G, X 5 is A, X 6 is T, X 7 is A, X 8 is
• Xg is A, X10 is A, X is A, Xi 2 is C; X13 is A, and Xi 4 is C.
• probes may be set forth in an alternative different way, i.e. as a mixture of oligonucleotide probes comprising or consisting of SEQ ID NO:s 25 to 33, or SEQ ID NO:s 25-30 and 73-79, respectively (Table 4).
• any combination or any one of the probes set forth herein may be employed.
• Probes of SEQ ID NO:s 25 to 33 are encom- passed in the mono-probe mixture of SEQ ID NO:24, while probes of SEQ ID NO:s 25-33 and 73-79 are encompassed in the mono-probe mixture of SEQ ID NO:72.
• the present mono-probes may be bisected to form any desired dual-probes.
• Such dual- probes do not have to be contiguous, i.e any appropriate number of nucleotides around the bisection site may be omitted from the probes.
• a person skilled in the art can easily choose a suitable bisection site taking into account specific requirements of the detection method to be employed.
• Members of the present dual-probes may be denoted as 5'-probes and 3'-probes reflecting their order in a corresponding blacrx-M sense sequence. In other words, the sequence of a 5'-probe lies upstream from the sequence of a 3'- probe.
• 5'-probes according to the present invention comprise or consist of a nucleic acid sequence:
• Xi is C when X 2 is C or T, and X3 is C;
• Xi is T when X 2 is A, X 3 is T, X4 is G, X 5 is A, X 6 is T, and X 7 is G;
• Xi is C when X 2 is A, X 3 is C, X4 is C, X 5 is A, X 6 is T, and X 7 is G;
• Xi is C when X 2 is A, X 3 is T, X4 is C or G, X 5 is A, X 6 is T, and X 7 is
• Xi is C when X 2 is A, X 3 is T, X4 is C, X 5 is A, X 6 is C, and X 7 is G;
• Xi is C, X 2 is G, X 3 is T, X 4 is G, X 5 is A, X 6 is T or X 7 is G;
• Xi is C when X 2 is A, X 3 is C, X4 is A, X 5 is A, X 6 is C, and X 7 is G; Xi is C when X 2 is A, X 3 is T, X4 is G, X 5 is T, X 6 is T, and X 7 is G.
• probes may be set forth in an alternative different way, i.e. as a mixture of oligonucleotide probes comprising or consisting of SEQ ID NO:s 36 to 39, SEQ ID NO:s 40 to 43, or SEQ ID NO:s 40-43 and 64-69, respectively (Table 5).
• any combination or any one of the probes set forth herein may be employed.
• the probe mixture of SEQ ID NO: 63 extends the target recognition to samples positive for CTX-M-8, CTX-M-40, CTX-M-63, CTX-M-67, CTX-M-78, CTX-M-86, or CTX-M-121 .
• the former probes provide detection of about 90% of the CTX-M variants
• the latter probe extends the detection rate to over 90% of the existing CTX-M variants.
• Probes of SEQ ID NO:s 36 to 39 are encompassed in the 5'-probe mixture of SEQ ID NO:34, while probes of SEQ ID NO:s 40 to 43 are encompassed in the 5'-probe mixture of SEQ ID NO:35, and while probes of SEQ ID NO:s 40 ⁇ 13 and 64-69 are encompasses in the 5'-probe mixture of SEQ ID NO: 63.
• 3'-probes according to the present invention comprise or consist of a nucleic acid sequence:
• Xi is A when X 2 is A, X 3 is G, X4 is C; X 5 is A or G, and Xe is T;
• Xi is A when X 2 , is A, X 3 is A, X4 is C; X 5 is A, and Xe is C or T;
• Xi is G when X 2 , is A, X 3 is A, X4 is T; X 5 is A, and Xe is C; and
• Xi is A when X 2 is G, X 3 is A, X4 is C; X 5 is A, and Xe is C.
• probes may be set forth in an alternative different way, i.e. as a mixture of oligonucleotide probes comprising or consisting of SEQ ID NO:s 46 to 50, SEQ ID NO:s 51 to 55, or SEQ ID NO:s 51-55 and 71 , respectively (Table 6).
• any combination or any one of the probes set forth herein may be employed.
• the probe mixture of SEQ ID NO: 70 extends the target recognition to samples positive for CTX-M-1 10. Table 6. Designed 3'-probes.
• Probes of SEQ ID NO:s 46 to 50 are encompassed in the 5'-probe mixture of SEQ ID NO:44, while probes of SEQ ID NO:s 51 to 55 are encompassed in the 5'-probe mixture of SEQ ID NO:45; or wherein probes of SEQ ID NO:s 51-55 and 71 are encompassed in the 5'-probe mixture of SEQ ID NO: 70.
• oligonucleotide analogues such as peptide nucleic acids (PNA), or oligonucleotides comprising modified bases may be comprised in the present primers or probes.
• PNA peptide nucleic acids
• various chemical compounds or groups (e.g., amino groups) or other molecules, such as labels necessary for the detection can be attached to the primers or probes, or they can be entirely unmodified.
• antiparallel sequences of these oligonucleotide sequences are equally suitable, as is obvious to a person skilled in the art.
• the present primers and probes encompass also those, which have at least 80 % identity, preferably at least 85 %, more preferably at least 90% identity to the present primers and probes. More preferably, the sequences have at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, most preferably 100% identity to the oligonucleotide sequences disclosed herein. In particular there may be differences in the 5'-ends of the primers.
• the percent identity between two amino acid or two nucleic acid sequences is equivalent to the percent homology between the two sequences.
• the comparison of sequences and de- termination of percent identity between two sequences can be accomplished using standard methods known in the art.
• the present primer and probe sequences may lack one or more nucleotides, have one or more additional nucleotide, or have one or more change in the nucleotide sequence compared to the primer and probe sequences disclosed herein as long as they retain their functional characteristics.
• the present primers and probes may be produced using any method known in the art suitable for that purpose.
• biological sample refers to any sample of biological, preferably human, origin including, but not limited to, swab and brush samples of mucosae from different body parts, pus samples, and samples of different bodily fluids enabling a local infection to be detected.
• bodily fluids include synovial fluid, peritoneal fluid, cerebrospinal fluid (CSF), urine and blood.
• Said biological sample may also be a surface sample, such as a wipe sample taken e.g. in a hospital environment. Food samples and soil samples are also contemplated.
• DNA has to be extracted from the biological sample to be studied prior to any amplification reaction.
• DNA may be extracted from the biological sample by using well-known DNA extraction methods or commercially available kits, such as NucleoSpin® Tissue kit (Macherey-Nagel) or chloroform-phenol extraction.
• the DNA can also be obtained directly from the sample to be analysed without separate isolation.
• any appropriate technique comprising an amplification phase and a detection phase may be employed in the present diagnostic method.
• the amplification and detection phases may be performed either simultaneously or sequentially.
• detection of CTX-M producing bacteria is performed utilizing chelate complementation technology disclosed in International Patent Publication WO2010/109065.
• the present probes are provided as dual probes, which hybridize next to each other to adjacent positions, preferably with zero to ten, intervening nucleotides, in a complementary target sequence.
• One member of the dual probe is labelled with a lanthanide ion carrier chelate, such as a cyclic or non-cyclic aminopolycarboxylic acid chelate of Eu(lll), Sm(lll), Tb(lll) and Dy(lll), while the other member is labelled with a light- harvesting antenna ligand, such as monodentate, bidentate, tridentate or tetradentate.
• the lanthanide ion carrier chelate and the antenna ligand are brought to a close proximity enabling chelate complementation, i.e. formation of a highly fluores- cent mixed lanthanide complex, which consequently increases the intensity of the lanthanide luminescence.
• fluorescence may be excited at one wavelength and the emission measured at another wavelength at the same time or, in time-resolved fluorometry, after a short delay after excitation.
• Chelate complementation technique may be employed both in iso- thermal and thermocycled nucleic acid amplification reactions, such as realtime quantitative PCR or homogeneous end-point PCR.
• a person skilled in the art can easily select ion carrier chelates having high enough thermodynamic and kinetic stability in order to be suitable for use in PCR.
• a preferred detection method for determining the presence or ab- sence of a CTX-M producing bacteria in a biological sample is a chelate complementation based real time PCR, the principle of which is illustrated in Figure 1 .
• oligonucleotide probes conjugated with an Europium (Eu) ion carrier chelate, preferably 7d-DOTA-Eu'" comprise or consist of a nucleic acid sequence set forth in SEQ ID NO: 34, 35, or 63
• the probes conjugated with a light harvesting antenna ligand, preferably 4-((4- isothiocyanatophenyl)ethynyl)-pyridine-2,6-dicarboxylic acid comprise or consist of a nucleic acid sequence set forth in SEQ ID NO:s 44, 45, or 70, or vice versa.
• CTX-M producing bacteria include homogeneous fluorescence-based nucleic acid hybridization assays typically based on either a quenched probe, i.e. TaqMan® probe, or two energy-transfer probes.
• a quenched probe i.e. TaqMan® probe
• two energy-transfer probes i.e. the quenched probes, i.e.
• probes containing both a fluo- rescent moiety and a quencher moiety may be utilized in real time quantitative PCR, wherein the fluorescent moiety is cleaved by the nuclease action of nucleic acid polymerase upon hybridisation during nucleic acid amplification resulting in a detectable fluorescence signal.
• Preferred probes for use as quenched probes are the mono-probes designed herein, such as those com- prising or consisting of a nucleic acid sequence set forth in SEQ ID NO:24 or 72, or at least 10, preferably 10 to 30, consecutive nucleotides thereof.
• the probes comprise or consist of a nucleic acid sequence set forth in SEQ ID NO:s 34, 35, 44, 45, 63, or 70.
• the other probe molecule is labelled with an energy donor, while the other probe molecule is labelled with an energy acceptor.
• the emission spectrum of the donor should overlap with the excitation spectrum of the acceptor.
• a person skilled in the art can easily select appropriate donor-acceptor-pairs suitable for use in real time quantitative PCR or any other applicable DNA amplification method.
• the donor- and acceptor- labelled probes hybridize next to each other to adjacent positions creating a detectable fluorescence signal resulting from fluorescent energy transfer (FRET) from the donor to the acceptor. Dual probes according to the present invention are preferred when this detection technology is to be utilized.
• Probes comprising or consisting of SEQ ID NO:34, 35, or 63 may be labelled with an acceptor, such as TAMRATM or Cy5TM, while the probes comprising or consisting of SEQ ID NO: 44, 45, or 70 may be labelled with a donor, such as FAMTM, TETTM, or Cy3TM, or vice versa.
• an acceptor such as TAMRATM or Cy5TM
• a donor such as FAMTM, TETTM, or Cy3TM
• molecular beacons which are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure.
• the loop contains a nucleic acid probe sequence according to any of the present embodiments, while the stem is formed by annealing of complementary arm sequences that are located on either side of the probe sequence.
• One of the arms is labeled with a fluorophore, while the other arm is labeled with a quencher.
• the stem keeps the probe in a closed conformation, causing the fluorescence to be quenched.
• the loop sequence will hybridize thereto thus linearizing the probe and causing the fluorophore and quencher to move away from each other leading to the restoration of a fluorescence signal.
• detection of CTX-M producing bacteria may be achieved by employing homogeneous detection with competitive hybridization.
• the present probes are provided as double-stranded probes, wherein the first strand comprises or consists of SEQ ID NO:24 or SEQ ID NO: 72, or at least 10 or 10 to 30 consecutive nucleotides thereof, or SEQ ID NO:34, 35, 44, 45, 63, or 70, and the second strand is complementary to the first strand.
• the probe may, additionally or alternatively, be a mixture of at least three different oligonucleotide molecules each having a first strand which comprises or consists of, independently form each other, a nucleic acid sequence comprised in SEQ ID NO: 24 or 72 or at least 10 or 10 to 30 consecutive nucleotides thereof.
• the first and second probe strands may be of equal or different length.
• the first strand is labelled with a fluor- ophore and the second strand with a quencher.
• the probe strands hybridize with each other and form a double-stranded probe molecule whose fluorescence is quenched.
• the first probe strand hybridizes to the target and, consequently, escapes from the quenching effect of the quencher probe and leads to a detectable signal.
• the level of the fluorescence signal is proportional to the amount of the target nucleic acid in the biological sample to be analysed.
• This technique may be combined both with real-time quantitative PCR or end-point PCR.
• a closed-tube platform comprising an integrated thermal cycler, a signal detection unit, such as a time-resolved fluorescence measurement unit, and software for the analysis of results, is employed.
• a signal detection unit such as a time-resolved fluorescence measurement unit
• LM-PCR ligation-mediated PCR
• two probe molecules hybridize next to each other to adjacent positions in a complementary target strand resulting in a double-stranded molecule with a single- stranded nick.
• the nick is ligated by a DNA ligase thereby connecting the probe molecules.
• no ligation reaction occurs and, con- sequently, the probe molecules will not be connected.
• DNA microarray technology may be employed.
• a DNA microarray or a DNA chip refers to a small substrate on which one or more of the present probes, preferably those comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:s 24 to 55, and 63 to 79 have been attached.
• the probes may be attached onto the surface of the microarray support, such a nitrocellulose membrane, nylon membrane, glass, or silicon, covalent or non-covalent binding with any commercially available arrayer that is suitable for this purpose, or they can be pi- petted manually onto the surface.
• the probes can be synthesized directly onto the surface by an appropriate in situ synthesis method, such as photolithography or ink-jet technology.
• any appropriate labeling method can be used in order to produce a labeled target strand or a labeled probe molecule.
• suitable labels include fluorescent labels (e.g., Cy5TM, Cy3TM, Cy2TM, TexasRedTM, FITC, Alexa Fluor® 488, TMR, FluorXTM, ROXTM, TETTM, or HEXTM), radioactive labels (e.g., 32P, 33P, or 33S), chemilumines- cent labels (e.g., HiLight Single-Color Kit), and colorimetric labels (e.g., enzyme labels).
• fluorescent labels e.g., Cy5TM, Cy3TM, Cy2TM, TexasRedTM, FITC, Alexa Fluor® 488, TMR, FluorXTM, ROXTM, TETTM, or HEXTM
• radioactive labels e.g., 32P, 33P, or 33S
• chemilumines- cent labels e.g., HiLight Single-Color Kit
• the microarray can be analyzed by any equipment or reader appli- cable to this purpose. If the target strand is fluorescently labelled, the analysis can also be performed for example by a fluorescence microscope. If a radioactive label has been used, the array or membrane can be analyzed by autoradiography.
• hybridization-based detection methods may be classified into two categories, i.e. to those performed in a solution and to those performed on a solid support.
• suitable exemplary detection methods only microarray-based techniques belong to the latter category in which the detection probes are attached onto a solid support which binds DNA.
• suitable solid surfaces include nitrocellulose or nylon membranes and glass or silicon based surfaces.
• the present probes do not essentially self-hybridize or form other unwanted secondary structures which would prevent or compromise their suitability for use in in-solution-hybridization techniques for detection purposes.
• the present invention provides a kit for use in any of the present methods of detecting the presence of CTX-M producing bacteria in a biological sample.
• a kit may comprise one or more of the present probes comprising or consisting of a nucleic acid sequence set forth in SEQ ID NO:24 or SEQ ID NO: 72, at least 10 or 10 to 30 consecutive nucleotides thereof, SEQ ID NO; 34, 35, 44, 45, 63, or 70.
• the probe may be a mixture of at least three different oligonucleotide molecules each comprising or consisting of, independently form each other, a nucleic acid sequence comprised in SEQ ID NO: 24 or SEQ ID NO: 72 or at least 10 or 10 to 30 consecutive nucleotides thereof.
• the present primers, or any combina- tion thereof, may or may not be included in the kit.
• the kit may also be suitable for use in any known platform utilizing integrated amplification and detection. It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways.
• the invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims. Examples
• DNA of inactivated bacterial cells was extracted using NucleoSpin® Tissue kit according to the manufacturer's instructions except for a pre-lysing step and wash and elution volumes.
• the pre-lysing was performed by adding 155 ⁇ Buffer T1 and 25 ⁇ Proteinase K to 45 ⁇ bacterial suspension, and incubating three hours at 56 °C. Buffers B5 and BE were used in volumes of 550 ⁇ and 80 ⁇ , respectively.
• DNA concentrations were determined with Quant- iTTM PicoGreen® dsDNA assay kit, and by exciting the samples at 480 nm and collecting the fluorescence emission at 520 nm for 1 .0 second.
• DNA yield varied between 5 ⁇ g and 28 ⁇ g as determined with standard curves generated with known DNA amounts.
• Primer mixtures (SEQ ID NO:1 , 2, 1 1 , and 12) were ordered from
• Alternative templates used for testing the combination of primer mixtures of SEQ ID NO: 2 and SEQ ID NO: 12 included DNA isolated from a K. oxytoca sample (10000 genome copies per reaction) and a CTX-M-1 -positive E. coli sample (either 0, 1 000, or 10000 genome copies per reaction).
• each reaction mixture contained 0.4 mM dNTP (Bio-Rad), 0.4 ⁇ Phire® Hotstart II DNA Polymerase (Thermo Fisher Scientific), 50 mM KCI, 1 .5 mM MgCI 2 , 2 mg/ml BSA, and SYBR® Green I (Life Technologies) in GenomEra PCR buffer (Abacus Diag- nostica) in a total volume of 20 ⁇ .
• Each amplification reaction was carried out in triplicate using C1000 TouchTM thermal cycler combined with CFX96 TouchTM Real-Time PCR Detec- tion System (Bio-Rad).
• the cycling consisted of initial denaturation at 98°C for 2.5 min, followed first by 9 cycles of 62°C for 15 s, 73°C for 10 s, and 98°C for 10 s, and then 18 cycles of 25°C for 30 s, 73°C for 10 s, 98°C for 10 s, 62°C for 15 s, 73°C for 10 s, and 98°C for 10 s.
• the fluorescence was measured at the end of the extension step in every second cycle starting at cycle 10.
• Figure 2 shows the results obtained using different primer mixture combinations at a total primer concentration of 0.5 ⁇ for both forward and reverse primer mixtures (the concentration of individual primer oligos varied between 0.10 ⁇ and 0.13 ⁇ ).
• template DNA was used in an amount of 10000 genome copies per reaction. Results obtained with the other template DNA amounts tested were in perfect concord with the results shown in Figure 2, and differences in the performance of parallel reactions were close to non-existing.
• the Eu-probes were labeled with 7d-DOTA-Eu'" (2,2',2"-(10-(3- isothiocyanatobenzyl)-1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)tri(acetate)- europium(lll)) at the 3'-end via the aminoC6 linker essentially as described earlier e.g. by Karhunen et al. in Acta Chimica Acta 772 (2013) 87-92.
• each labeling reaction contained 2.2 pg/ ⁇ of oligonucleotide probe and 20-fold molar excess of the DOTA-Eu'" in 50 mM carbonate buffer, pH 9.8.
• the reactions were incubated overnight at a temperature of +37 °C in slow shaking.
• the ion carrier chelate labeled probe was prepurified with a NAPTM-5 gel filtration column (GE Healthcare) according to the manufacturer's instructions using an elution buffer containing 10 mM Tris (pH 7.5), 50 mM NaCI and 10 ⁇ EDTA.
• the eluates were purified with reverse-phase HPLC with a 150 mm ⁇ 4.6 mm AerisTM PEPTIDE column (Phenomenex) using a gradient from 95% of solution A (50 mM triethylammonium acetate; TEAA; Sigma- Aldrich) and 5% solution of B (95% acetonitrile in 50 mM TEAA) to 86% of A and 14% of B in 1 min, and to 70% of A and 30% of B in 7 min, and finally to 100% of B in one min with a flow rate of 2.00 ml/min. After washing with 100% of B for 1 min, the percentage of B was lowered to 5% in 1 min, and the col- umn was equilibrated with 95% of A for 8 min. The collected fractions were dried in miVac Duo (GeneVac Ltd.).
• each labeling reac- tion contained 1 .4 ⁇ / ⁇ of oligonucleotide probe and 100-fold molar excess of the antenna ligand dissolved in ⁇ , ⁇ -dimethylformamide using a Hielscer ultrasonic homogenizer.
• the labeling reactions were carried out in 50 mM carbonate buffer, pH 9.8. The reactions were incubated overnight at a tempera- ture of +50 °C in slow shaking and prepurified similarly to the 7d-DOTA-Eu'" labeling reactions.
• the eluates were purified with reverse-phase HPLC with a 150 mm ⁇ 4.6 mm Hypersil® ODS C18 column (Thermo Fisher Scientific) using a gradient from 95% of solution A (50 mM triethylammonium acetate; TEAA; Sigma-Aldrich) and 5% solution of B (95% acetonitrile in 50 mM TEAA) to 86% of A and 14% of B in 1 min, and to 70% of A and 30% of B in 25 min, and finally to 100% of B in 2 min with a flow rate of 0.5 ml/min.
• solution A 50 mM triethylammonium acetate
• TEAA triethylammonium acetate
• Sigma-Aldrich 5% solution of B (95% acetonitrile in 50 mM TEAA)
• HPLC fractions were dissolved in 10 mM Tris (pH 7.5), 50 mM
• NaCI, 10 ⁇ EDTA and their oligonucleotide contents were determined by measuring absorbance at 260 and 330 nm with NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies) using UV-Vis Software.
• the hybridization reactions contained in a total volume of 60 ⁇ either 0 or 10 nM of a synthetic target oligonucleotide (Integrated DNA Technologies) and 25 nM of the corresponding Eu- and antenna-labeled probes in 50 mM Tris-HCI (pH 7.7), 600 mM NaCI, 0.1 % (vol/vol) Tween 20, 0.05% (w/v) NaN 3 , 30 ⁇ DTPA.
• the experiments were performed in yellow MaxiSorpTM plates (Nunc) by incubating the reactions for 10 min at RT, for another 20 min at +50 °C, and finally for 15 min at RT. The first and the third incubations were performed at slow agitation. Thereafter, Eu'" luminescence was measured with VictorTM 1420 plate reader (PerkinElmer Wallac).
• the present primers and probes were used at different concentrations and in different combinations.
• Eu-probe mixture of SEQ ID NO: 34 was used with antenna-probe mixture of SEQ ID NO: 44
• Eu-probe mixture of SEQ ID NO: 35 was used with antenna-probe mixture of SEQ ID NO: 45.
• Al- ternative templates included isolated DNA from two independent CTX-M-1 - group-positive, CTX-M-2-group-positive and CTX-M-9-group-positive E. coli samples, three independent K. oxytoca samples, and one CTX-M-negative E. coli sample. Template concentrations varied between 0 and 10000 genome copies per amplification reaction.
• Amplification reactions were built up as described above with some modifications.
• the reaction mixture contained 30 ⁇ DTPA but no SYBR® Green I. Further, owing to a greater reaction volume (40 ⁇ instead of 20 ⁇ ) 0.8 ⁇ Phire® Hotstart II DNA Polymerase was used.
• both forward and reverse primer mixtures (SEQ ID NO:2 and SEQ ID NO:1 1 , re- spectively) were used at a total primer concentration of 0.5 ⁇ .
• Each PCR was carried out in triplicate employing C1000 TouchTM thermal cycler.
• the cycling consisted of initial denaturation at 98°C for 2.5 min, followed first by 10 cycles of 62°C for 15 s, 73°C for 10 s, and 98°C for 10 s, and then 18 cycles of 25°C for 35 s, 73°C for 10 s, 98°C for 10 s, 62°C for 15 s, 73°C for 10 s, and 98°C for 10 s.
• the fluorescence was measured in every second cycle at 25°C starting at cycle 10.
• the measurements were performed with VictorTM X4 Multilabel Plate Reader (PerkinElmer) using excitation wavelength of 340 nm, measure- ment wavelength of 615 nm, delay time of 250 s, and measurement time of 750 MS.
• Results of analytical sensitivity tests performed with ten-fold dilution series of CTX-M-positive samples (either 0, 0.01 , 0.1 , 1 , 10, 100, 1 000, or 10000 genome copies per reaction) together with Eu-probe mixture of SEQ ID NO: 35 and antenna-probe mixture of SEQ ID NO: 45 (at individual probe con- centration of 0.05 ⁇ ) are shown in Figure 5.
• Each of the three parallel reactions provided signals, that exceeded the threshold signal level (signal-to- background 1 .5) when 10 or more genome copies per reaction were used as a template.
• CTX-M-9-group template even one genome copy per reaction was enough to provide a detectable signal in all three parallel samples, and 0.01 genome copies per reaction provided a signal in one of the parallel samples.
• Eu-probe mixture of SEQ ID NO: 63 was used with antenna-probe mixture of SEQ ID NO: 70.
• alternative templates included isolated DNA from twelve independent CTX-M-1 -group-positive, two independent CTX-M-2-group-positive, one CTX-M-8-group-positive, and twelve independent CTX-M-9-group-positive E. coli samples, two independent CTX- M-1 -group-positive E. cloacae samples, one CTX-M-25-group-positive K. pneumoniae sample, four independent K. oxytoca samples, and one CTX-M- negative sample of 21 different bacteria species (E. hormaechi, S. mutans, C. freundii, S. enteriditis, S.
• alternative templates included isolated DNA from one sample positive for either CTX-M-1 -group, CTX-M-2-group, CTX-M-8-group, CTX-M-9-group, or CTX-M- 25-group with template concentrations of either 0, 0.1 , 1 , 10, 100, 1 000, 10000, 100000 genome copies per reaction .
• Amplification reactions were performed as described above in Example 3. All reactions contained internal amplification control to verify successful amplification in negative reactions.
• Eu-probe mixture of SEQ ID NO: 63 was used with antenna-probe mixture of SEQ ID NO: 70.
• Alternative templates included bacterial cells from one CTX-M-1 -group-positive, one CTX-M-2-group-positive, one CTX-M-9- group-positive and one CTX-M-8-group-positive E. coli sample and one CTX- M-25-group-positive K. pneumoniae sample.
• Bacterial cells were cultured in 5 ml of SB medium in thermomixer at 37 °C until at visible turbidity but not overgrown. After centrifugation (2719xg, 2 min), the cell pellet was gently suspended in 2 ml of sterile PBS buffer. The growth of bacteria in 1 ml of the resulting suspension was halted by introducing 0.05% NaN 3 and the remaining bacterial suspension without NaN 3 was immediately used for the preparation of ten-fold dilution series in SB medium. One hundred ul of the SB dilutions were plated on LA plates and cultured at 37°C. Of the NaN 3 treated bacteria suspension, ten-fold dilution series were prepared in sterile water and stored refrigerated until analysis by PCR.
• CTX-M was detected in all samples with three or more colony forming units of bacteria.

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