Hybridization probes derived from the spacer region between the 16S and 23S rRNA genes for the detection of non-viral microorganisms

Claim 2

Probe according to claim 1, for use in a hybridization assay, liable to be obtained in the process which comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a sequence of the spacer region between rRNA genes, particularly the spacer region between the 16S rRNA gene and the 23S rRNA gene, selected to be unique to non-viral organisms, particularly prokaryotic organisms, more particularly bacteria, sought to be detected, said sequence of the spacer region between rRNA genes being selected - either by * comparing the nucleotide sequence of the spacer region between the rRNA genes of the sought organism with the nucleotide sequence of the spacer region between the rRNA genes of the closest neighbours, * selecting a sequence of at least 15 oligonucleotides, and preferably from about 15 to about the maximum number of oligonucleotides of the spacer region, and more preferably from about 15 to about 100 oligonucleotides of the spacer region between rRNA genes of the sought organism which presents at least one mismatch with the spacer region between the rRNA genes of at least one of the closest neighbours, - or by * deleting, in the spacer region between the rRNA genes of the organism to be sought, the tRNA genes and possibly the signal sequences, to obtain a shortened spacer region and * determining by trial and error a specific nucleotide sequence of at least about 15 oligonucleotides, and preferably from about 15 to about the maximum number of oligonucleotides of the spacer region, and more preferably from about 15 to about 100 oligonucleotides, from the shortened spacer region, said sequence being able to hybridize specifically with the nucleic acids (DNA and/or RNAs) of the sought organism.

Claim 3

Probe according to anyone of claims 1 or 2, containing - either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes itself of from 15 to the maximum number of nucleotides of the selected nucleic acid : - or a variant sequence which distinguishes of any of the preceding sequences : * either by addition to or removal from any of their respective extremities of one or several nucleotides; * or changing within any of said sequences of one or more nucleotides; * or both; yet provided that in any of the above circumstances said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.

Claim 4

Probe for detecting one or more Neisseria gonorrhoeae strains, containing : - either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes itself of from 15 to the maximum number of nucleotides of the selected nucleic acid : - or a variant sequence which distinguishes of any of the preceding sequences : * either by addition to or removal from any of their respective extremities of one or several nucleotides; * or changing within any of said sequences of one or more nucleotides; * or both; yet provided that in any of the above circumstances said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.

Claim 5

Process for detecting Neisseria gonorrhoeae strains in a biological sample, wherein said process comprises contacting said biological sample in which the nucleic acids (DNAs and/or RNAs) of the strains have been made accessible to hybridization, if need be, under suitable denaturation conditions, said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, particularly in which at least one of the probes of claim 4 is used as amplification primer, with a probe according to anyone of claim 4 under conditions enabling hybridization between the probe and complementary nucleic acids of the Neisseria gonorrhoeae strains, which may be present in the sample, and detecting the hybrids possibly formed, particularly with a probe hybridizing both DNA and RNA of Neisseria gonorrhoeae strain which may be present in the biological sample.

Claim 6

Process for detecting Neisseria gonorrhoeae , in a biological sample, according to claim 5, wherein: the hybridization medium contains about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) about 25 mM of phosphate buffer pH 7.1, 20% deionized formamide, 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and about 0.1 mg/ml sheared, denatured salmon sperm DNA, and/or wash medium contains about 3 x SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 4, the hybridization temperature being suitable adjusted to the range of about 50°C and/or the wash temperature to the range of about 50°C, and particularly wherein said target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT) respectively are as follows: GCGAAGTAGA ATAACGACGC ATCG HT and/or WT : 50 °C.

Claim 7

Process for detecting Neisseria gonorrhoeae strains from other organisms, wherein said process comprises contacting said biological sample, in which the nucleic acids (DNAs and/or RNAs) have been made accessible to hybridization, if need be, under suitable denaturation conditions, with two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Neisseria gonorrhoeae and which is selected from any one of the probes of claim 4, whenever required under hybridization and washing conditions adjusted such as to ensure specific hybridization with complementary nucleic acids of the Neisseria gonorrhoeae strains, which may be present in the sample, yet not with complementary DNA or RNA of other organisms and detecting the hybrids possibly formed.

Claim 8

Kit for the detection in vitro of a large number, preferably all Neisseria gonorrhoeae strains in a biological sample, said kit containing : either - at least one probe selected among any of those according to claim 4; - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a large number, preferably all strains of Neisseria gonorrhoeae to be carried out; - when appropriate means for detecting the hybrids resulting from the preceding hybridization, or - at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Neisseria gonorrhoeae and which is selected from any one of the probes of claim 4, - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Neisseria gonorrhoeae to be carried out, - when appropriate means for detecting the hybrids resulting from the preceding hybridization.

Claim 9

Probe for detecting one or more Neisseria meningitidis strains, containing : - either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes itself of from 15 to the maximum number of nucleotides of the selected nucleic acid : - or a variant sequence which distinguishes of any of the preceding sequences : * either by addition to or removal from any of their respective extremities of one or several nucleotides; * or changing within any of said sequences of one or more nucleotides; * or both; yet provided that in any of the above circumstances said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.

Claim 10

Process for detecting Neisseria meningitidis strains in a biological sample, wherein said process comprises contacting said biological sample in which the nucleic acids (DNAs and/or RNAs) of the strains have been made accessible to hybridization, if need be, under suitable denaturation conditions, said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, particularly in which at least one of the probes of claim 9 is used as amplification primer, with a probe according to any one of claim 9 under conditions enabling hybridization between the probe and complementary nucleic acids of the Neisseria meningitidis strains, which may be present in the sample, and detecting the hybrids possibly formed particularly with a probe hybridizing both DNA and RNA of Neisseria meningitidis strain which may be present in the biological sample.

Claim 11

Process for detecting Neisseria meningitidis , in a biological sample, according to claim 10, wherein: the hybridization medium contains about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) about 25 mM of phosphate buffer pH 7.1, 20% deionised formamide, 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and about 0.1 mg/ml sheared, denatured salmon sperm DNA, and/or wash medium contains about 3 x SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 10, the hybridization temperature being suitable adjusted to the range of about 40 to 58°C and/or the wash temperature to the range of about 40 to 58°C, and particularly, wherein said target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT) respectively are as follows:

Claim 12

Process for detecting Neisseria meningitidis strains from other organisms, wherein said process comprises contacting said biological sample, in which the nucleic acids (DNAs and/or RNAs) have been made accessible to hybridization, if need be, under suitable denaturation conditions, with two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Neisseria meningitidis and which is selected from any one of the probes of claim 9, whenever required under hybridization and washing conditions adjusted such as to ensure specific hybridization with complementary nucleic acids of the Neisseria meningitidis strains, which may be present in the sample, yet not with complementary DNA or RNA of other organisms and detecting the hybrids possibly formed.

Claim 13

Kit for the detection in vitro of a large number, preferably all Neisseria meningitidis strains in a biological sample, said kit containing : either - at least one probe selected among any of those according to claim 9; - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a large number, preferably all strains of Neisseria meningitidis to be carried out; - when appropriate means for detecting the hybrids resulting from the preceding hybridization, or - at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Neisseria meningitidis and which is selected from any one of the probes of claim 9, - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Neisseria meningitidis to be carried out, - when appropriate means for detecting the hybrids resulting from the preceding hybridization.

Claim 14

Probe for detecting one or more Haemophilus ducreyi strains, containing : - either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes itself of from 15 to the maximum number of nucleotides of the selected nucleic acid : - or a variant sequence which distinguishes of any of the preceding sequences : * either by addition to or removal from any of their respective extremities of one or several nucleotides; * or changing within any of said sequences of one or more nucleotides; * or both; yet provided that in any of the above circumstances said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.

Claim 15

Process for detecting Haemophilus ducreyi strains in a biological sample, wherein said process comprises contacting said biological sample in which the nucleic acids (DNAs and/or RNAs) of the strains have been made accessible to hybridization, if need be, under suitable denaturation conditions, said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, particularly in which at least one of the probes of claim 14 is used as amplification primer, with a probe according to any one of claim 14 under conditions enabling hybridization between the probe and complementary nucleic acids of the Haemophilus ducreyi strains, which may be present in the sample, and detecting the hybrids possibly formed, particularly with a probe hybridizing both DNA and RNA of Haemophilus ducreyi strain which may be present in the biological sample.

Claim 16

Process for detecting Haemophilus ducreyi , in a biological sample, according to anyone of claim 15, wherein : the hybridization medium contains about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) about 25 mM of phosphate buffer pH 7.1, 20% deionized formamide, 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and about 0.1 mg/ml sheared, denatured salmon sperm DNA, and/or wash medium contains about 3 x SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 14, the hybridization temperature being suitable adjusted to the range of about 40°C and/or the wash temperature to the range of about 40°C, and particularly, wherein said target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT) respectively are as follows: CAATATGCCT CGCGCATAAT AA HT and/or WT : 40 °C.

Claim 17

Process for detecting Haemophilus ducreyi strains from other organisms, wherein said process comprises contacting said biological sample, in which the nucleic acids (DNAs and/or RNAs) have been made accessible to hybridization, if need be, under suitable denaturation conditions, with two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Haemophilus ducreyi and which is selected from any one of the probes of claim 22, whenever required under hybridization and washing conditions adjusted such as to ensure specific hybridization with complementary nucleic acids of the Haemophilus ducreyi strains, which may be present in the sample, yet not with complementary DNA or RNA of other organisms and detecting the hybrids possibly formed.

Claim 18

Kit for the detection in vitro of a large number, preferably all Haemophilus ducreyi strains in a biological sample, said kit containing : either - at least one probe selected among any of those according to claim 14; - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a large number, preferably all strains of Haemophilus ducreyi to be carried out; - when appropriate means for detecting the hybrids resulting from the preceding hybridization, or - at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Haemophilus ducreyi and which is selected from any one of the probes of claim 14, - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Haemophilus ducreyi to be carried out, - when appropriate means for detecting the hybrids resulting from the preceding hybridization.

Claim 19

Probe for detecting one or more Branhamella catarrhalis strains, containing : - either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes itself of from 15 to the maximum number of nucleotides of the selected nucleic acid : - or a variant sequence which distinguishes of any of the preceding sequences : * either by addition to or removal from any of their respective extremities of one or several nucleotides; * or changing within any of said sequences of one or more nucleotides; * or both; yet provided that in any of the above circumstances said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.

Claim 20

Process for detecting Branhamella catarrhalis strains in a biological sample, wherein said process comprises contacting said biological sample in which the nucleic acids (DNAs and/or RNAs) of the strains have been made accessible to hybridization, if need be, under suitable denaturation conditions, said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, particularly in which at least one of the probes of claim 19 is used as amplification primer, with a probe according to any one of claim 31 under conditions enabling hybridization between the probe and complementary nucleic acids of the Branhamella catarrhalis strains, which may be present in the sample, and detecting the hybrids possibly formed, particularly with a probe hybridizing both DNA and RNA of Branhamella catarrhalis strain which may be present in the biological sample.

Claim 21

Process for detecting Branhamella catarrhalis , in a biological sample, according to claim 20, wherein : the hybridization medium contains about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) about 25 mM of phosphate buffer pH 7.1, 20% deionized formamide, 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and about 0.1 mg/ml sheared, denatured salmon sperm DNA, and/or wash medium contains about 3 x SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 19, the hybridization temperature being suitable adjusted to the range of about 30°C and/or the wash temperature to the range of about 30°C, and particularly, wherein said target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT) respectively are as follows: CTTTGGTAAG ATGTTTAA HT and/or WT : 30°C.

Claim 22

Process for detecting Branhamella catarrhalis strains from other organisms, wherein said process comprises contacting said biological sample, in which the nucleic acids (DNAs and/or RNAs) have been made accessible to hybridization, if need be, under suitable denaturation conditions, with two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Branhamella catarrhalis and which is selected from any one of the probes of claim 19, whenever required under hybridization and washing conditions adjusted such as to ensure specific hybridization with complementary nucleic acids of the Branhamella catarrhalis strains, which may be present in the sample, yet not with complementary DNA or RNA of other organisms and detecting the hybrids possibly formed.

Claim 23

Kit for the detection in vitro of a large number, preferably all Branhamella catarrhalis strains in a biological sample, said kit containing : either - at least one probe selected among any of those according to claim 19; - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a large number, preferably all strains of Branhamella catarrhalis to be carried out; - when appropriate means for detecting the hybrids resulting from the preceding hybridization, or - at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Branhamella catarrhalis and which is selected from any one of the probes of claim 19, - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Branhamella catarrhalis to be carried out, - when appropriate means for detecting the hybrids resulting from the preceding hybridization.

Claim 24

Probe for detecting one or more Bordetella pertussis strains, containing : - either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes itself of from 15 to the maximum number of nucleotides of the selected nucleic acid : - or a variant sequence which distinguishes of any of the preceding sequences : * either by addition to or removal from any of their respective extremities of one or several nucleotides; * or changing within any of said sequences of one or more nucleotides; * or both; yet provided that in any of the above circumstances said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.

Claim 25

Process for detecting Bordetella pertussis strains in a biological sample, wherein said process comprises contacting said biological sample in which the nucleic acids (DNAs and/or RNAs) of the strains have been made accessible to hybridization, if need be, under suitable denaturation conditions, said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, particularly in which at least one of the probes of claim 24 is used as amplification primer, with a probe according to any one of claim 24 under conditions enabling hybridization between the probe and complementary nucleic acids of the Bordetella pertussis strains, which may be present in the sample, and detecting the hybrids possibly formed, particularly with a probe hybridizing both DNA and RNA of Bordetella pertussis strain which may be present in the biological sample.

Claim 26

Process for detecting Bordetella pertussis , in a biological sample, according to claim 24, wherein: the hybridization medium contains about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) about 25 mM of phosphate buffer pH 7.1, 20% deionized formamide, 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and about 0.1 mg/ml sheared, denatured salmon sperm DNA, and/or wash medium contains about 3 x SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 40, the hybridization temperature being suitable adjusted to the range of about 55°C and/or the wash temperature to the range of about 55°C, and particularly, wherein said target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT) respectively are as follows: AAGCCTGTCC AGAGGATGGG TGTGG HT and/or WT : 55°C.

Claim 27

Process for detecting Bordetella pertussis strains from other organisms, wherein said process comprises contacting said biological sample, in which the nucleic acids (DNAs and/or RNAs) have been made accessible to hybridization, if need be, under suitable denaturation conditions, with two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Bordetella pertussis and which is selected from any one of the probes of claim 24, whenever required under hybridization and washing conditions adjusted such as to ensure specific hybridization with complementary nucleic acids of the Bordetella pertussis strains, which may be present in the sample, yet not with complementary DNA or RNA of other organisms and detecting the hybrids possibly formed.

Claim 28

Kit for the detection in vitro of a large number, preferably all Bordatella pertussis strains in a biological sample, said kit containing : either - at least one probe selected among any of those according to claim 24; - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a large number, preferably all strains of Bordetella pertussis to be carried out; - when appropriate means for detecting the hybrids resulting from the preceding hybridization, or - at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Bordetella pertussis and which is selected from any one of the probes of claim 24, - the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Bordetella pertussis to be carried out, - when appropriate means for detecting the hybrids resulting from the preceding hybridization.