Detecting Mycobacterium tuberculosis by nucleic acid sequence amplification
First Claim
1. A method for amplifying a nucleic acid having a Mycobacterium nucleotide base sequence in a sample, comprising the step of contacting said nucleic acid under nucleic acid amplification conditions witha) at least one oligonucleotide of about 20 to about 100 bases in length comprising a nucleic acid sequence selected from the group consisting of xGCCGTCACCCCACCAACAAGCT (SEQ ID NO:
- 22), xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
2), xCCAGGCCACTTCCGCTAACC (SEO ID NO;
23), xCGCGGAACAGGCTAAACCGCACGC (SEQ ID NO;
7), and their fully complementary sequences of the same length, wherein x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) at least one nucleic acid polymerase.
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Accused Products
Abstract
A method, composition and kit for synthesizing multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH are provided in which multiple RNA copies of the target sequence autocatalytically generate additional copies using a mixture of blocked and unblocked primers and/or promoter-primers to initiate DNA and RNA synthesis, preferably with reduced non-specific product formation. The invention is useful for generating copies of a nucleic acid target sequence for purposes that include assays to quantitate specific nucleic acid sequences in clinical, environmental, forensic and similar samples, cloning and generating probes.
38 Citations
57 Claims
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1. A method for amplifying a nucleic acid having a Mycobacterium nucleotide base sequence in a sample, comprising the step of contacting said nucleic acid under nucleic acid amplification conditions with
a) at least one oligonucleotide of about 20 to about 100 bases in length comprising a nucleic acid sequence selected from the group consisting of xGCCGTCACCCCACCAACAAGCT (SEQ ID NO: - 22), xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
2), xCCAGGCCACTTCCGCTAACC (SEO ID NO;
23), xCGCGGAACAGGCTAAACCGCACGC (SEQ ID NO;
7), and their fully complementary sequences of the same length, wherein x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) at least one nucleic acid polymerase.
- 22), xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
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2. A method for detecting whether a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence is present in a sample, comprising the steps of
a) contacting said nucleic acid with an oligonucleotide of about 23 to about 100 bases in length comprising a nucleic acid sequence selected from the group consisting of GTCTTGTGGTGGAAAGCGCTTTAG (SEO ID NO: - 3, GGAGGATATGTCTCAGCGCTACC (SEO ID NO;
8), and their fully complementary sequences of the same length, wherein said oligonucleotide will form a detectable hybrid under hybridization assay conditions with a Mycobacterium tuberculosis nucleic acid, andb) detecting the presence or absence of said detectable hybrid as an indication of, respectively, the presence or absence of said nucleic acid in said sample.
- 3, GGAGGATATGTCTCAGCGCTACC (SEO ID NO;
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3. A method for detecting whether a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence is present in a sample, comprising the steps of
a) amplifying said nucleic acid with at least one oligonucleotide of about 22 to about 100 bases in length comprising a nucleotide base sequence selected from the group consisting of the sequences xGCCGTCACCCCACCAACAAGCT (SEO ID NO: - 22), and xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
2), wherein x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) detecting the amplified nucleic acid, if present, with a nucleotide polymer of about 24 to about 100 bases in length comprising a nucleotide base sequence consisting of the sequence GTCTTGTGGTGGAAAGCGCTTTAG (SEO ID NO;
3).
- 22), and xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
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4. A method for detecting whether a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence is present in a sample, comprising the steps of
a) amplifying said nucleic acid with at least one oligonucleotide of about 20 to about 100 bases in length comprising a nucleotide base sequence selected from the group consisting of the sequences xCCAGGCCACTTCCGCTAACC (SEO ID NO: - 7), and xCGCGGAACAGGCTAAACCGCACGC (SEO ID NO;
23), wherein x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) detecting the amplified nucleic acid, if present, with a nucleotide polymer of about 23 to about 100 bases in length comprising a nucleotide base sequence consisting of the sequence GGAGGATATGTCTCAGCGCTACC (SEO ID NO;
8).
- 7), and xCGCGGAACAGGCTAAACCGCACGC (SEO ID NO;
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5. A method for amplifying a nucleic acid having a Mycobacterium nucleotide base sequence in a sample, comprising the step of contacting said nucleic acid under nucleic acid amplification conditions with
a) a mixture oligonucleotides of about 20 to about 100 bases in length each comprising a nucleic acid sequence selected from the group consisting of xGCCGTCACCCCACCAACAAGCT (SEO ID NO: - 22), xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
2). xCCAGGCCACTTCCGCTAACC (SEO ID NO;
23), xCGCGGAACAGGCTAAACCGCACGC (SEO ID NO;
7), and their fully complementary sequences of the same length, wherein at least one and less than all of said oligonucleotides are modified at the 3'"'"' end to reduce or block chain extension, and x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) at least one nucleic acid polymerase.
- 22), xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
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6. A method for amplifying a nucleic acid having a Mycobacterium nucleotide base sequence in a sample, comprising the step of contacting said nucleic acid under nucleic acid amplification conditions with
a) a mixture oligonucleotides of about 20 to about 100 bases in length each comprising a nucleic acid sequence selected from the group consisting of xGCCGTCACCCCACCAACAAGCT (SEO ID NO: - 22), xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
2). xCCAGGCCACTTCCGCTAACC (SEO ID NO;
23), xCGCGGAACAGGCTAAACCGCACGC (SEO ID NO;
7), and their fully complementary sequences of the same length, wherein one of said oligonucleotides is modified at the 3'"'"' end to reduce or block chain extension, another of said oligonucleotides is either unmodified or differently modified at the 3'"'"' end to reduce or block chain extension, and x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) at least one nucleic acid polymerase.
- 22), xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
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7. A method for detecting whether a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence is present in a sample, comprising the steps of
a) amplifying said nucleic acid with a mixture of oligonucleotides of about 22 to about 100 bases in length comprising a nucleotide base sequence selected from the group consisting of the sequences xGCCGTCACCCCACCAACAAGCT (SEO ID NO: - 22), and xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEC ID NO;
2), wherein at least one and less than all of said oligonucleotides are modified at the 3'"'"' end to reduce or block chain extension, and x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) detecting the amplified nucleic acid, if present, with a nucleotide polymer of about 24 to about 100 bases in length comprising a nucleotide base sequence consisting of the sequence GTCTTGTGGTGGAAAGCGCTTTAG (SEO ID NO;
3).
- 22), and xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEC ID NO;
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8. A method for detecting whether a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence is present in a sample, comprising the steps of
a) amplifying said nucleic acid with a mixture of oligonucleotides of about 20 to about 100 bases in length each comprising a nucleotide base sequence selected from the group consisting of the sequences xCCAGGCCACTTCCGCTAACC (SEO ID NO: - 7), and xCGCGGAACAGGCTAAACCGCACGC (SEO ID NO;
23), wherein at least one and less than all of said oligonucleotides are modified at the 3'"'"' end to reduce or block chain extension, and x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) detecting the amplified nucleic acid, if present, with a nucleotide polymer of about 23 to about 100 bases in length comprising a nucleotide base sequence consisting of the sequence GGAGGATATGTCTCAGCGCTACC (SEO ID NO;
8).
- 7), and xCGCGGAACAGGCTAAACCGCACGC (SEO ID NO;
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9. A method for detecting whether a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence is present in a sample, comprising the steps of
a) amplifying said nucleic acid with a mixture of oligonucleotides of about 22 to about 100 bases in length comprising a nucleotide base sequence selected from the group consisting of the sequences xGCCGTCACCCCACCAACAAGCT (SEO ID NO: - 22), and xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
2), wherein one of said oligonucleotides is modified at the 3'"'"' end to reduce or block chain extension, another of said oligonucleotides is either unmodified or differently modified at the 3'"'"' end to reduce or block chain extension, and x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) detecting the amplified nucleic acid, if present, with a nucleotide polymer of about 24 to about 100 bases in length comprising a nucleotide base sequence consisting of the sequence GTCTTGTGGTGGAAAGCGCTTTAG (SEO ID NO;
3).
- 22), and xGGGATAAGCCTGGGAAACTGGGTCTAATACC (SEO ID NO;
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10. A method for detecting whether a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence is present in a sample, comprising the steps of
a) amplifying said nucleic acid with a mixture of oligonucleotides of about 20 to about 100 bases in length each comprising a nucleotide base sequence selected from the group consisting of the sequences xCCAGGCCACTTCCGCTAACC (SEO ID NO: - 7), and xCGCGGAACAGGCTAAACCGCACGC (SEQ ID NO;
23), wherein one of said oligonucleotides is modified at the 3'"'"' end to reduce or block chain extension, another of said oligonucleotides is either unmodified or differently modified at the 3'"'"' end to reduce or block chain extension, and x is selected from the group consisting of nothing and a sequence recognized by an RNA polymerase, andb) detecting the amplified nucleic acid, if present, with a nucleotide polymer of about 23 to about 100 bases in length comprising a nucleotide base sequence consisting of the sequence GGAGGATATGTCTCAGCGCTACC (SEO ID NO;
8).
- 7), and xCGCGGAACAGGCTAAACCGCACGC (SEQ ID NO;
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11. A method for detecting a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence in a sample, comprising the steps of:
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a) amplifying said nucleic acid with one or more oligonucleotides from about 10 to about 100 nucleotide bases in length able to bind to a region of said nucleic acid, wherein said region consists of a 16S rRNA Mycobacterium tuberculosis nucleotide base sequence selected from the group consisting of SEQ ID NO;
2, SEQ ID NO;
22, and their fully complementary sequences of the same length; andb) detecting the presence or absence of amplified nucleic acid with a nucleic acid hybridization assay as an indication of, respectively, the presence or absence of Mycobacterium tuberculosis in said sample. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. A method for detecting a nucleic acid having a Mycobacterium tuberculosis nucleotide base sequence in a sample, comprising the steps of:
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a) amplifying said nucleic acid with one or more oligonucleotides from about 10 to about 100 nucleotide bases in length which bind to a region of said nucleic acid in whole or in part to initiate nucleic acid synthesis, wherein said region consists of a 23S rRNA Mycobacterium tuberculosis nucleotide base sequence selected from the group consisting of SEQ ID NO;
7, SEQ ID NO;
23, and their fully complementary sequences of the same length; andb) detecting the presence or absence of amplified nucleic acid as an indication of, respectively, the presence or absence of Mycobacterium tuberculosis in said sample. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57)
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45. A method for detecting a nucleic acid having a Mycobacterium tuberculosis target sequence in a sample comprising the steps of:
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a) contacting said sample under stringent hybridization conditions with a nucleic acid hybridization assay probe comprising an oligonucleotide of between 10 and 100 nucleotide bases in length able to form a detectable hybrid under said conditions with at least 10 adjacent nucleotide bases of said Mycobacterium tuberculosis target sequence, wherein the nucleotide base sequence of said target sequence is selected from the group consisting of SEQ ID NO;
8, SEQ ID NO;
3, RNA equivalents of SEQ ID NO;
8 and SEQ ID NO;
3, and the nucleotide base sequences fully complementary and of the same length to these sequences, andb) detecting the presence or absence said hybrid as an indication of, respectively, the presence or absence of said nucleic acid in said sample. - View Dependent Claims (52)
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Specification