Species specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories

A method for detecting the presence or amount of Escherichia coli in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Escherichia coli DNA and of any one of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Escherichia coli in said test sample.

A method for detecting the presence or amount of Moraxella catarrhalis in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Moraxella catarrhalis DNA and of SEQ ID NO. 29, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Moraxella catarrhalis in said test sample.

A method for detecting the presence or amount of Pseudomonas aeruginosa in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Pseudomonas aeruginosa DNA and of any one of SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Pseudomonas aeruginosa in said test sample.

A method for detecting the presence or amount of Staphylococcus epidermidis in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Staphylococcus epidermidis DNA and of SEQ ID NO. 36, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Staphylococcus epidermidis in said test sample.

A method for detecting the presence or amount of Staphylococcus aureus in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Staphylococcus aureus DNA and of SEQ ID NO. 37, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Staphylococcus aureus in said test sample.

A method for detecting the presence or amount of Streptococcus pneumoniae in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Streptococcus pneumoniae DNA and of any one of SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 35, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Streptococcus pneumoniae in said test sample.

A method for the detection, identification or quantification of a bacterial species bearing exotoxin A gene speA directly from a test sample or from bacterial colonies, which comprises the following steps:

• a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of the bacterial colonies isolated from the sample or inoculating said sample or said bacterial colonies on an inert support and lysing in situ said inoculated sample or bacterial colonies to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;
• b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotide sequence has at least twelve nucleotides in length and is capable of specifically and ubiquitously hybridizing with DNA from strains or representatives of a bacterial species bearing exotoxin A gene speA and capable of hybridizing with any one of SEQ ID NO. 33 and a sequence complementary thereof, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, and
• c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence or amount of a bacterial species bearing exotoxin A gene speA in said test sample.

A method for detecting the presence or amount of a bacterial species bearing exotoxin A gene speA in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of a bacterial species bearing exotoxin A gene speA DNA and of SEQ ID NO. 33, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of a bacterial species bearing exotoxin A gene speA in said test sample.

A method for detecting the presence or amount of Enterococcus faecalis in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Enterococcus faecalis DNA and of any one of SEQ ID NO. 1 and SEQ ID NO. 2, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Enterococcus faecalis in said test sample.

A method for detecting the presence or amount of any bacterial species in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing a pair of universal primers which sequence is defined in SEQ ID NO:
• 126 and SEQ ID NO:
• 127, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said any bacterial species DNA that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of said any bacterial species in said test sample.

A nucleic acid having the nucleotide sequence of any one of SEQ ID NO:

• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 29, SEQ ID NO:
• 36, SEQ ID NO:
• 37, a part thereof and variants thereof which, when in single stranded form, ubiquitously and specifically hybridize with a target bacterial DNA as a probe or as a primer.

An oligonucleotide having a nucleotide sequence of any one of SEQ ID NOs:

• 38 to 43, SEQ ID NOs:
• 83 to 86, SEQ ID NO:
• 118, SEQ ID NO:
• 119, SEQ ID NO:
• 127, SEQ ID NO:
• 131, SEQ ID NO:
• 132, SEQ ID NOs:
• 141 to 153, SEQ ID NO:
• 158, SEQ ID NO:
• 159 and SEQ ID NO:
• 160.

A method for detecting the presence or amount of Klebsiella pneumoniae in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Klebsiella pneumoniae DNA and of any one of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Klebsiella pneumoniae in said test sample.

A method for detecting the presence or amount of Proteus mirabilis in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Proteus mirabilis DNA and of any one of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Proteus mirabilis in said test sample.

A method for detecting the presence or amount of Staphylococcus saprophyticus in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Staphylococcus saprophyticus DNA and of any one of SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Staphylococcus saprophyticus in said test sample.

A method for detecting the presence or amount of Haemophilus influenzae in a test sample which comprises the following steps:

• a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Haemophilus influenzae DNA and of any one of SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
• b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level;
• and
• c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of Haemophilus influenzae in said test sample.

A method for the detection of the presence or amount of any bacterial species directly from a test sample or from bacterial colonies, which comprises the following steps:

• a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of the bacterial colonies isolated from this sample, or inoculating said sample or said bacterial colonies on an inert support, and lysing in situ said inoculated sample or to release the bacterial DNA, said bacterial DNA being in a substantially single stranded form;
• b) contacting said single stranded DNA with a universal probe which sequence is selected from the group consisting of SEQ ID NO:
• 122, SEQ ID NO:
• 123, SEQ ID NO:
• 124, SEQ ID NO:
• 125, SEQ ID NO:
• 128, SEQ ID NO:
• 129, SEQ ID NO:
• 130 and a sequence complementary thereof, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, and
• c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence or amount of said any bacterial species in said test sample.