US2004053320A1PendingUtilityA1
Hybridization probes derived from the spacer region between the 16S and 23S rRNA genes for the detection of non-viral microorganisms
Est. expiryApr 18, 2010(expired)· nominal 20-yr term from priority
C12Q 1/6888C12Q 1/689
66
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Claims
Abstract
The invention relates to a probe consisting of at least about 15 nucleotides from the spacer region between rRNA genes of a non-viral organism, particularly prokaryotic organism and more particularly bacteria, and preferably from about 15 nucleotides to about the maximum number of nucleotides of the spacer region and more preferably from about 15 to about 100 nucleotides to be used for the detection of non-viral microorganisms.
Claims
exact text as granted — not AI-modified1 . Probe consisting of at least about 15 nucleotides of the transcribed spacer region between the 16S and 23S rRNA genes of prokaryotic organisms, and more particularly bacteria, and preferably from about 15 nucleotides to about the maximum number of nucleotides of the spacer refion and more preferably from about 15 to about 100 nucleotides.
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, with 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 nucleotides, and preferably from about 15 to about the maximum number of nucleotides of the spacer region, and more preferably from about 15 to about 100 nucleotides 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 th 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 nucleotides, and preferably from about 15 to about the maximum number of nucleotides of the spacer region, and more preferably from about 15 to about 100 nucleotides, from the shortened spacer region, said sequence being able to hybridize specifically with the nucleic acids (DNA and/or RNAS) of the sought organism.
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 from 15 to the maximum number of nucleotides of the selected nucleic acid:
Group NGI1: CGATGCGTCG TTATTCTACT TCGC NGI1 GCGAAGTAGA ATAACGACGC ATCG NGI1IC GCGAAGUAGA AUAACGACGC AUCG NGI1ICR CGAUGCGUCG UUAUUCUACU UCGC NGI1R Group NGI2: TTCGTTTACC TACCCGTTGA CTAAGTAAGC AAAC NGI2 GTTTGCTTAC TTAGTCAACG GGTAGGTAAA CGAA NGI2IC GUUUGCUUAC UUAGUCAACG GGUAGGUAAA CGAA NGI2ICR UUGGUUUACC UACCCGUUGA CUAAGUAAGC AAAC NGI2R Group NMI1: GGTCAAGTGT GACGTCGCCC TG NMI1 CAGGGCGACG TCACACTTGA CC NMI1IC CAGGGCGACG UCACACUUGA CC NMI1ICR GGUCAAGUGU GACGUCGCCC UG NMI1R Group NMI2: GTTCTTGGTC AAGTGTGACG TC NMI2 GACGTCACAC TTGACCAAGA AC NMI2IC GACGUCACAC UUGACCAAGA AC NMI2ICR GUUCUUGGUC AAGUGUGACG UC NMI2R Group NMI3: GCGTTCGTTA TAGCTATCTA CTGTGC NMI3 GCACAGTAGA TAGCTATAAC GAACGC NMI3IC GCACAGUAGA UAGCUAUAAC GAACGC NMI3ICR GCGUUCGUUA UAGCUAUCUA CUGUGC NMI3R Group NMI4: TGCGTTCGAT ATTGCTATCT ACTGTGCA NMI4 TGCACAGTAG ATAGCAATAT CGAACGCA NMI4IC UGCACAGUAG AUAGCAAUAU CGAACGCA NMI4ICR UGCGUUCGAU AUUGCUAUCU ACUGUGCA NMI4R Group NMI5: TTTTGTTCTTGGTCAAGTGTGACGTCGCCCTGAATGGATTCTGTTCCATT NMI5 AATGGAACAGAATCCATTCAGGGCGACGTCACACTTGACCAAGAACAAAA NMI5C AAUGGAACAGAAUCCAUUCAGGGCGACGUCACACUUGACCAAGAACAAAA NMI5ICR UUUUGUUCUUGGUCAAGUGUGACGUCGCCCUGAAUGGAUUCUGUUCCAUU NMI5R Group NMI6 TTTGCCTAAC ATTCCGTIGA CTAGAACATC AGAC NMI6 GTCTGATGTT CTAGTCAACG GAATGTTAGG CAAA NMI6IC GUCUGAUGUU CUAGUCAACG GAAUGUUAGG CAAA NMI6ICR UUUGCCUAAC AUUCCGUUGA CUAGAACAUC AGAC NMI6R Group HDI1: TTATTATGCG CGAGGCATAT TG HDI1 CAATATGCCT CGCGCATAAT AA HDI1IC CAAUAUGCCU CGCGCAUAAU AA HDI1ICR UUAUUAUGCG CGAGGCAUAU UG HDI1R Group BCI1: TTAAACATCT TACCAAAG BCI1 CTTTGGTAAG ATGTTTAA BCI1IC CUUUGGUAAG AUGUUUAA BCI1ICR UUAAACAUCU UACCAAAG BCI1R Group BCI2: TTGATGTTTA AACTTGCTTG GTGGA BCI2 TCCACCAAGC AAGTTTAAAC ATCAA BCI2IC UCCACCAAGC AAGUUUAAAC AUCAA BCI2ICR UUGAUGUUUA AACUUGCUUG GUGGA BCI2R Group BPI1: CCACACCCAT CCTCTGGACA GGCTT BPI1 AAGCCTGTCC AGAGGATGGG TGTGG BPI1IC AAGCCUGUCC AGAGGAUGGG UGUGG BPI1ICR CCACACCCAU CCUCUGGACA GGCUU BPI1R Group HII1: ACGCATCAAA TTGACCGCAC TT HII1 AAGTGCGGTC AATTTGATGC GT HII1IC AAGUGCGGUC AAUUUGAUGC GU HII1ICR ACGCAUCAAA UUGACCGCAC UU HII1R Group HII2: ACTTTGAAGT GAAAACTTAA AG HII2 CTTIAAGTTT TCACTTCAAA GT HII2IC CUUUAAGUUU UCACUUCAAA GU HII2ICR ACUUUGAAGU GAAAACUUAA AG HII2R Group SAI1: AATCGAAAGG TTCAAATTGT T SAI1 AACAATTTGA ACCTTTCGAT T SAI1IC AACAAUUUGA ACCUUUCGAU U SAI1ICR AAUCGAAAGG UUCAAAUUGU U SAI1R Group SAI2: GGAAACCTGC CATTTGCGTC TT SAI2 AAGACGCAAA TGGCAGGTTT CC SAI2IC AAGACGCAAA UGGCAGGUUU CC SAI2ICR GGAAACCUGC CAUUUGCGUC UU SAI2R Group SAI3: TCCACGATCT AGAAATAGAT TGTAGAA SAI3 TTCTACAATC TATTTCTAGA TCGTGGA SAI3IC UUCUACAAUC UAUUUCUAGA UCGUGGA SAI3ICR UCCACGAUCU AGAAAUAGAU UGUAGAA SAI3R Group SAI4: TCTAGTTTTA AAGAAACTAG GTT SAI4 AACCTAGTTT CTTTAAAACT AGA SAI4IC AACCUAGUUU CUUUAAAACU AGA SAI4ICR UCUAGUUUUA AAGAAACUAG GUU SAI4R Group SPI1: GTGAGAGATC ACCAAGTAAT GCA SPI1 TGCATTACTT GGTGATCTCT CAC SPI1IC UGCAUUACUU GGUGAUCUCU CAC SPI1ICR GUGAGAGAUC ACCAAGUAAU GCA SPI1R Group SPI2 AGGAACTGCG CATTGGTCTT SPI2 AAGACCAATG CGCAGTTCCT SPI2IC AAGACCAAUG CGCAGUUCCU SPI2ICR AGGAACUGCG CAUUGGUCUU SPI2R Group SPI3 GAGTTTATGA CTGAAAGGTC AGAA SPI3 TTCTGACCTT TCAGTCATAA ACTC SPI3IC UUCUGACCUU UCAGUCAUAA ACUC SPI3ICR GAGUUUAUGA CUGAAAGGUC AGAA SPI3R
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.
4 . Probe for detecting one or more Neisseria gonorrhoeae strains, containing: either a sequence belonging to a nucleic acid selected from the following groups of nucleic acids and which includes from 15 to the maximum number of nucleotides of the selected nucleic acid: Group NGI1: CGATGCGTCG TTATTCTACT TCGC NGI1 GCGAAGTAGA ATAACGACGC ATCG NGI1IC GCGAAGUAGA AUAACGACGC AUCG NGI1ICR CGAUGCGUCG UUAUUCUACU UCGC NGI1R Group NGI2: TTCGTTTACC TACCCGTTGA CTAAGTAAGC AAAC NGI2 GTTTGCTTAC TTAGTCAACG GGTAGGTAAA CGAA NGI2IC GUUUGCUUAC UUAGUCAACG GGUAGGUAAA CGAA NGI2ICR UUGGUUUACC UACCCGUUGA CUAAGUAAGC AAAC NGI2R 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.
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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to any of the probes 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 to both DNA and RNA of a Neisseria gonorrhoeae strain which may be present in the biological sample.
6 . Process for detecting Neisseria gonorrhoeae , in a biological sample, according to claim 5 , wherein: the hybridization medium contains about 3×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
the wash medium contains about 3×SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is any of the probes of claim 4 , the hybridization temperatur being suitably adjusted to the range of ab ut 50° C. and/or the wash temperature to the rang 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.
GUUUGCUUAC UUAGUCAACG GGUAGGUAAA CGAA
HT and/or WT: 50° C.
7 . Kit for the detection in vitro of a large number, preferably all Neisseria gonorrhoeae strains in a biological sample, with said kit containing: either
at least one probe selected among any of those according to claim 4; the butter 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; the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, 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, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least one probe select d among any of those according to claim 4 , which is fixed to a solid support, the primers needed for performing enzymatic amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffers or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Neisseria gonorrhoeae to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
8 . 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 from 15 to the maximum number of nucleotides of the selected nucleic acid: Group NMI1: GGTCAAGTGT GACGTCGCCC TG NMI1 CAGGGCGACG TCACACTTGA CC NMI1IC CAGGGCGACG UCACACUUGA CC NMI1ICR GGUCAAGUGU GACGUCGCCC UG NMI1R Group NMI2: GTTCTTGGTC AAGTGTGACG TC NMI2 GACGTCACAC TTGACCAAGA AC NMI2IC GACGUCACAC UUGACCAAGA AC NMI2ICR GUUCUUGGUC AAGUGUGACG UC NMI2R Group NMI3: GCGTTCGTTA TAGCTATCTA CTGTGC NMI3 GCACAGTAGA TAGCTATAAC GAACGC NMI3IC GCACAGUAGA UAGCUAUAAC GAACGC NMI3ICR GCGUUCGUUA UAGCUAUCUA CUGUGC NMI3R Group NMI4: TGCGTTCGAT ATTGCTATCT ACTGTGCA NMI4 TGCACAGTAG ATAGCAATAT CGAACGCA NMI4IC UGCACAGUAG AUAGCAAUAU CGAACGCA NMI4ICR UGCGUUCGAU AUUGCUAUCU ACUGUGCA NMI4R Group NMI5: TTTTGTTCTTGGTCAAGTGTGACGTCGCCCTGAATGGATTCTGTTCCATT NMI5 AATGGAACAGAATCCATTCAGGGCGACGTCACACTTGACCAAGAACAAAA NMI5C AAUGGAACAGAAUCCAUUCAGGGCGACGUCACACUUGACCAAGAACAAAA NMI5ICR UUUUGUUCUUGGUCAAGUGUGACGUCGCCCUGAAUGGAUUCUGUUCCAUU NMI5R Group NMI6: TTTGCCTAAC ATTCCGTTGA CTAGAACATC AGAC NMI6 GTCTGATGTT CTAGTCAACG GAATGTTAGG CAAA NMI6IC GUCUGAUGUU CUAGUCAACG GAAUGUUAGG CAAA NMI6ICR UUUGCCUAAC AUUCCGUUGA CUAGAACAUC AGAC NMI6R 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.
9 . Process for detecting Neisseria menincritidis 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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to any one of claim 8 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 to both DNA and RNA of a Neisseria meningitidis strain which may be present in the biological sample.
10 . Process for detecting Neisseria meningitidis , in a biological sample, according to claim 9 , wherein: the hybridization medium contains about 3×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
the wash medium contains about 3×SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 8 , the hybridization temperature being suitably 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:
CAGGGCGACG TCACACTTGA CC
HT and/or WT: 45° C.
GACGTCACAC TTGACCAAGA AC
HT and/or WT: 45° C.
GCACAGTAGA TAGCTATAAC GAACGC
HT and/or WT: 40° C.
TGCACAGTAG ATAGCAATAT CGAACGCA
HT and/or WT: 48° C.
TTTTGTTCTTGGTCAAGGTGTGACGTCGCCCTGAATGGATTCTGTTCCATT
H and/or WT: 58° C.
GTCTGATGTT CTAGTCAACG GAATGTTAGG CAAA
HT and/or WT: 50° C.
11 . Kit for the detection in vitro of a large number, preferably all Neisseria meningitidis strains in a biological sample, with said kit containing: either
at least one probe selected among any of those according to claim 8; 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; the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, 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 8 , 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, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least one probe selected among any of those according to claim 4 , which is fixed to a solid support, the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffer or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Neisseria meningitidis to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
12 . 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 from 15 to the maximum number of nucleotides of the selected nucleic acid: Group HDI1: TTATTATGCG CGAGGCATAT TG HDI1 CAATATGCCT CGCGCATAAT AA HDI1IC CAAUAUGCCU CGCGCAUAAU AA HDI1ICR UUAUUAUGCG CGAGGCAUAU UG HDI1R 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.
13 . 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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to any one of claim 12 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 to both DNA and RNA of a Haemophilus ducreyi strain which may be present in the biological sample.
14 . Process for detecting Haemophilus ducreyi , in a biological sample, according to anyone of claim 13 , wherein:
the hybridization medium contains about 3×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
the wash medium contains about 3×SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 12 , the hybridization temperature being suitable adjusted to th range of about 40° C. and/or the wash temperatur 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.
15 . Kit for the detection in vitro of a large number, preferably all Haemophilus ducreyi strains in a biological sample, with said kit containing: either
at least one probe selected among any of those according to claim 12; 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; the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, 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 12 , 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, means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least one probe selected among any of those according to claim 12 , which is fixed to a solid support, the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffer or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Haemophilus ducreyi to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
16 . Probe for detecting one or more Branhameiia catarrhalis strains, containing:
either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes from 15 to the maximum number of nucleotides of the selected nucleic acid: Group BCI1: TTAAACATCT TACCAAAG BCI1 CTTTGGTAAG ATGTTTAA BCI1IC CUUUGGUAAG AUGUUUAA BCI1ICR UUAAACAUCU UACCAAAG BCI1R Group BCI2: TTGATGTTTA AACTTGCTTG GTGGA BCI2 TCCACCAAGC AAGTTTAAAC ATCAA BCI2IC UCCACCAAGC AAGUUUAAAC AUCAA BCI2ICR UUGAUGUUUA AACUUGCUUG GUGGA BCI2R 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 provid d that in any of the above circumstances said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.
17 . 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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to any one of claim 16 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 to both DNA and RNA of a Branhamella catarrhalis strain which may be present in the biological sample.
18 . Process for detecting Branhamella catarrhalis , in a biological sample, according to claim 17 , wherein: the hybridization medium contains about 3×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
the wash medium contains about 3×SSC, 25=M phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 16 , the hybridization temperature being suitable adjusted to the range of about 30° C. to 42° C. and/or the wash temperature to the range of about 30° C. to 42° 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.
TCCACCAAGC AAGTTTAAAC ATCAA
HT and/or WT: 42° C.
19 . Kit for the detection in vitro of a large number, preferably all Branhamella catarrhalis strains in a biological sample, with said kit containing: either
at least one probe selected among any of those according to claim 16; 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; the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, 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 16 , 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, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least one probe selected among any of those according to claim 16 , which is fixed to a solid support, the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffer or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Branhamella catarrhalis to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
20 . 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 from 15 to the maximum number of nucleotides of the selected nucleic acid: Group BPI1: CCACACCCAT CCTCTGGACA GGCTT BPI1 AAGCCTGTCC AGAGGATGGG TGTGG BPI1IC AAGCCUGUCC AGAGGAUGGG UGUGG BPI1ICR CCACACCCAU CCUCUGGACA GGCUU BPI1R 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.
21 . 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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to any one of claim 20 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 to both DNA and RNA of a Bordetella pertussis strain which may be present in the biological sample.
22 . Process for detecting Bordetella pertussis , in a biological sample, according to claim 21 , wherein: the hybridization medium contains about 3×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
the wash medium contains about 3×SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 20 , 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.
23 . Kit for the detection in vitro of a large number, preferably all Bordetella pertussis strains in a biological sample, with said kit containing: either
at least one probe selected among any of those according to claim 20; 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; the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, 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 20 , the buffer or components necessary for producing the buffer enabling hybridization reaction betw en these probes and the DNAs and/or RNAs of a strain of Bordetella pertussis to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least one probe selected among any of those according to claim 20 , which is fixed to a solid support, the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffer or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Bordetella pertussis to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
24 . Probe for detecting one or more Haemophilus influenzae strains, containing:
either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes from 15 to the maximum number of nucleotides of the selected nucleic acid: Group HII1: ACGCATCAAA TTGACCGCAC TT HII1 AAGTGCGGTC AATTTGATGC GT HII1IC AAGUGCGGUC AAUUUGAUGC GU HII1ICR ACGCAUCAAA UUCACCGCAC UU HII1R Group HII2: ACTTTGAAGT GAAAACTTAA AG HII2 CTTTAAGTTT TCACTTCAAA GT HII2IC CUUUAAGUUU UCACUUCAAA GU HII2ICR ACUUUGAAGU GAAAACUUAA AG HII2R 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.
25 . Process for detecting Haemophilus influenzae 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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to anyone of claim 24 under conditions enabling hybridization between the probe and complementary nucleic acids of the Haemophilus influenzae strains, which may be present in the sample, and detecting the hybrids possibly formed, particularly with a probe hybridizing to both DNA and RNA of a Haemophilus influenzae strain which may be present in the biological sample.
26 . Process for detecting Haemophilus influenzae , in a biological sample, according to claim 25 , wherein: the hybridization medium contains about 3×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
the wash medium contains about 3×SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 24 , the hybridization temperature being suitable adjusted to the range of about 35° C. to 55° C. and/or the wash temperature to the range of about 35° C. to 55° C., and particularly wherein said target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT), respectively, are as follows:
AAGTGCGGTC AATTTGATGC GT
HT and/or WT: 55° C.
CTTTAAGTTT TCACTTCAAA GT
HT and/or WT: 35° C.
27 . Kit for the detection in vitro of a large number, preferably all Haemophilus influenzae strains in a biological sample, with 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 Haemophilus influenzae to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Haemophilus influenzae 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 Haemophilus influenzae to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least one probe selected among any of those according to claim 24 , which is fixed to a solid support, the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffer or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Haemophilus influenzae to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
28 . Probe for detecting one or more Streptococcus pneumoniae strains, containing:
either a sequence belonging to a nucleic acid selected from the following groups of nucleic acids and which includes from 15 to the maximum number of nucleotides of the selected nucleic acid: Group SPI1: GTGAGAGATC ACCAAGTAAT GCA SPI1 TGCATTACTT GGTGATCTCT CAC SPI1IC UGCAUUACUU GGUGAUCUCU CAC SPI1ICR GUGAGAGAUC ACCAAGUAAU GCA SPI1R Group SPI2 AGGAACTGCG CATTGGTCTT SPI2 AAGACCAATG CGCAGTTCCT SPI2IC AAGACCAAUG CGCAGUUCCU SPI2ICR AGGAACUGCG CAUUGGUCUU SPI2R Group SPI3 GAGTTTATGA CTGAAAGGTC AGAA SPI3 TTCTGACCTT TCAGTCATAA ACTC SPI3IC UUCUGACCUU UCAGUCAUAA ACUC SPI3ICR GAGUUUAUGA CUGAAAGGUC AGAA SPI3R 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.
29 . Process for detecting Streptococcus pneumoniae 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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to anyone of claim 28 under conditions enabling hybridization between the probe and complementary nucleic acids of the Streptococcus pneumoniae strains, which may be present in the sample, and detecting the hybrids possibly formed, particularly with a probe hybridizing to both DNA and RNA of a Streptococcus pneumoniae strain which may be present in the biological sample.
30 . Process for detecting Streptococcus pneumoniae , in a biological sample, according to claim 29 , wherein: the hybridization medium contains about 3×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
the wash medium contains about 3×SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 28 , the hybridization temperature being suitable adjusted to the range of about 45° C. and/or the wash temperature to the range of about 45° C., and particularly wherein said target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT), respectively, are as follows:
TGCATTACTT GGTGATCTCT CAC
HT and/or WT: 45° C.
AAGACCAATG CGCAGTTCCT
HT and/or WT: 45° C.
TTCTGACCTT TCAGTCATAA ACTG
HT and/or WT: 45° C.
31 . Kit for the detection in vitro of a large number, preferably all Streptococcus pneumoniae strains in a biological sample, with said kit containing: either
at least one probe selected among any of those according to claim 28; 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 Streptococcus pneumoniae to be carried out; the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Streptococcus pneumoniae and which is selected from any one of the probes of claim 28 , 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 Streptococcus pn umoniae to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least one probe selected among any of thos according to claim 28 , which is fixed to a solid support, the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffers or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Streptococus pneumoniae to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
32 . Probe for detecting one or more Streptococcus agalactiae strains, containing:
either a sequence belonging to a nucleic acid selected from the following groups of nucleic acids and which includes from 15 to the maximum number of nucleotides of the selected nucleic acid: Group SAI1: AATCGAAAGG TTCAAATTGT T SAI1 AACAATTTGA ACCTTTCGAT T SAI1IC AACAAUUUGA ACCUUUCGAU U SAI1ICR AAUCGAAAGG UUCAAAUUGU U SAI1R Group SAI2: GGAAACCTGC CATTTGCGTC TT SAI2 AAGACGCAAA TGGCAGGTTT CC SAI2IC AAGACGCAAA UGGCAGGUUU CC SAI2ICR GGAAACCUGC CAUUUGCGUC UU SAI2R Group SAI3: TCCACGATCT AGAAATAGAT TGTAGAA SAI3 TTCTACAATC TATTTCTAGA TCGTGGA SAI3IC UUCUACAAUC UAUUUCUAGA UCGUGGA SAI3ICR UCCACGAUCU AGAAAUAGAU UGUAGAA SAI3R Group SAI4: TCTAGTTTTA AAGAAACTAG GTT SAI4 AACCTAGTTT CTTTAAAACT AGA SAI4IC AACCUAGUUU CUUUAAAACU AGA SAI4ICR UCUAGUUUUA AAGAAACUAG GUU SAI4R 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.
33 . Process for detecting Streptococcus agalactiae 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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to anyone f claim 32 under conditions enabling hybridization between the probe and complementary nucleic acids of the Streptococcus agalactiae strains, which may be present in the sample, and detecting the hybrids possibly formed, particularly with a probe hybridizing to both DNA and RNA of a Streptococcus agalactiae strain which may be present in the biological sample.
34 . Process for detecting Streptococcus aqalactiae , in a biological sample, according to claim 33 , wherein:
the hybridization medium contains about 3×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
the wash medium contains about 3×SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide and wherein the probe used is anyone of the probes of claim 32 , the hybridization temperature being suitable adjusted to the range of about 35° C. to 45° C. and/or the wash temperature to the range of about 35° C. to 45° C., and particularly wherein said target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT), respectively, are as follows:
AACAATTTGA ACCTTTCGAT T
HT and/or WT: 35° C.
AAGACGCAAA TGGCAGGTTT CC
HT and/or WT: 45° C.
TTCTACAATC TATTTCTAGA TCGTGGA
HT and/or WT: 45° C.
AACCTAGTTT CTTTAAAACT AGA
HT and/or WT: 37° C.
35 . Kit for the detection in vitro of a large number, preferably all Streptococcus agalactiae strains in a biological sample, with said kit containing: either
at least one probe selected among any of those according to claim 32; 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 Streptococcus agalactiae to be carried out; the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Streptococcus agalactiae and which is selected from any one of the probes of claim 32 , 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 Streptococcus agalactiae to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or at least one probe selected among any of those according to claim 32 , which is fixed to a solid support, the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffers or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and th DNAs and/or RNAs of a strain of Streptococcus agalactiae to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
36 . Probe for detecting one or more Campylobacter jejuni and Campylobacter coli strains, containing a sequence from 15 to the maximum number of nucleotides derived from the 16S-23S rRNA spacer sequence shown in FIG. 10 or its complement provided that the probe, at the appropriate conditions, hybridizes exclusively with DNA and/or RNA from Campylobacter jejuni and Campylobacter coli strains and not with DNA and/or RNA from other organisms.
37 . Process for detecting Campylobacter jejuni and Campylobacter coli 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, with said nucleic acid to be detected being possibly amplified using the polymerase chain reaction, with two primers, more preferably two more evolutionarily conserved primers, flanking the target sequence of the probe—with a probe according to anyone of claim 36 under conditions enabling hybridization between the probe and complementary nucleic acids of the Campylobacter jejuni and Campylobacter coli strains, which may be present in the sample, and detecting the hybrids possibly formed, particularly with a probe hybridizing to both DNA and RNA of a Campylobacter jejuni or Campylobacter coli strain which may be present in the biological sample.
38 . Kit for the detection in vitro of a large number, preferably all Campylobacter jejuni and Campylobacter coli strains in a biol gical sample, with said kit containing:
either
at least one probe selected among any of those according to claim 36;
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 Campylobacter jejuni or Campylobacter coli to be carried out;
the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or
at least two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Campylobacter jejuni and Campylobacter coli and which is selected from any one of the probes of claim 36 ,
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 Campylobacter jejuni or Campylobacter coli to be carried out,
the means for detecting the hybrids resulting from the preceding hybridization, when appropriate, or
at least one probe selected among any of thos according to claim 36 , which is fixed to a solid support,
the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate,
the buffers or components necessary for producing the buffers enabling enzymatical amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Campylobacter jejuni or Campylobacter coli to be carri d out,
the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.
39 . Process for the in vitro detection of one microorganism or to the simultaneous in vitro detection of several microorganisms contained in a biological sample using anyone of the probes according to claims 1 to 4, 8, 12, 16, 20, 24, 28, 32 and 36, and specific for the Microorganism(s) to be detected wherein the DNA and/or RNA present in the biological sample (and comprising the target sequence) is labeled, preferably using enzymatic amplification with at least one set of primers flanking the probe region, and wherein said biological sample is contacted with a membrane on which one or more oligonucleotide probes are dot spotted on a known location, in a medium enabling specific hybridization of the amplified target sequence and the probes on the membrane and wherein the hybrids resulting from the hybridizations are detected by appropriate means.
40 . Kit for the in vitro detection of one microorganism or for the simultaneous in vitro detection of several microorganisms contained in a biological sample, with said kit containing:
at least one of the probes according to claims 1 to 4 , 8 , 12 , 16 , 20 , 24 , 28 , 32 and 36 , and specific for the microorganism(s) to be detected, which is dot spotted to a membrane, the primers needed for performing enzymatical amplification of the DNA and/or RNA containing the target sequence of the above-mentioned probe, when appropriate, the buffers or components necessary for producing the buffers enabling enzymatic amplification and/or enabling hybridization reaction between these probes and the DNAs and/or RNAs of a microorganism or microorganisms which are to be detected to to be carried out, the means for detecting the hybrids resulting from the preceding hybridization, when appropriate.Join the waitlist — get patent alerts
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