Nucleic acid hybridization assay probes, helper probes and amplification oligonucleotides targeted to mycoplasma pneumoniae nucleic acid

A probe for detecting whether Mycoplasma pneumoniae may be present in a sample comprising an oligonucleotide which under stringent hybridization assay conditions hybridizes to a Mycoplasma pneumoniae target nucleic acid sequence region to form a hybrid stable for detection, said oligonucleotide comprising a nucleic acid base sequence having no more than a 20% nucleotide base difference, excluding RNA or DNA equivalent nucleotides, than a nucleotide base sequence selected from the group consisting of:

• SEQ ID NO:
• 2:
• CACACTCTAG ATTAATAGTT TCCAATGC,
• SEQ ID NO:
• 3:
• CATGCGCTTC CTAATGGTTA GC,
• SEQ ID NO:
• 4:
• GCTGTTTCCA ACTACCGGAT TGCTC,
• SEQ ID NO:
• 7:
• CCTTTTGCGC GCTGCTTTCC,
• SEQ ID NO:
• 8:
• CGTCTACCAC AAGATATAAA TCTTATCC,
• SEQ ID NO:
• 24:
• GCATTGGAAA CTATTAATCT AGAGTGTG,
• SEQ ID NO:
• 27:
• GCTAACCATT AGGAAGCGCA TG,
• SEQ ID NO:
• 30:
• GAGCAATCCG GTAGTTGGAA ACAGC,
• SEQ ID NO:
• 39:
• GGAAAGCAGC GCGCAAAAGG, and
• SEQ ID NO:
• 42:
• GGATAAGATT TATATCTTGT GGTAGACG,
• wherein under said conditions said oligonucleotide preferentially hybridizes to said Mycoplasma pneumoniae nucleic acid over nucleic acids present in Mycoplasma genitalium, Mycoplasma orale, Mycoplasma faucium, Mycoplasma buccale, and Mycoplasma salivarium.

A probe mix for detecting the presence of Mycoplasma pneumoniae nucleic acid comprising,

• a) a hybridization assay probe which forms a hybrid stable for detection under stringent hybridization conditions with Mycoplasma pneumoniae nucleic acid, wherein said hybridization assay probe comprises a nucleotide base sequence which is at least 90% complementary to at least 10 contiguous nucleotides present in a hybridization assay probe target sequence selected from the group consisting of:
• SEQ ID NO:
• 2:
• CACACTCTAG ATTAATAGTT TCCAATGC,
• SEQ ID NO:
• 3:
• CATGCGCTTC CTAATGGTTA GC,
• SEQ ID NO:
• 4:
• GCTGTTTCCA ACTACCGGAT TGCTC,
• SEQ ID NO:
• 7:
• CCTTTTGCGC GCTGCTTTCC,
• SEQ ID NO:
• 8:
• CGTCTACCAC AAGATATAAA TCTTATCC,
• SEQ ID NO:
• 26:
• GCAUUGGAAA CUAUUAAUCU AGAGUGUG,
• SEQ ID NO:
• 29:
• GCUAACCAUU AGGAAGCGCA UG,
• SEQ ID NO:
• 32:
• GAGCAAUCCG GUAGUUGGAA ACAGC,
• SEQ ID NO:
• 41:
• GGAAAGCAGC GCGCAAAAGG, and
• SEQ ID NO:
• 44:
• GGAUAAGAUU UAUAUCUUGU GGUAGACG,
• wherein under said conditions said hybridization assay probe preferentially hybridizes to said Mycoplasma pneumoniae nucleic acid over nucleic acids present in Mycoplasma genitalium, Mycoplasma orale, Mycoplasma faucium, Mycoplasma buccale, and Mycoplasma salivarium;
• and
• b) one or more helper oligonucleotides which hybridize to Mycoplasma pneumoniae nucleic acid under said conditions and enhance hybridization between said hybridization assay probe and a Mycoplasma pneumoniae nucleic acid sequence region.

A probe mix for detecting the presence of Mycoplasma pneumoniae nucleic acid comprising,

• a) a hybridization assay probe which forms a hybrid stable for detection under stringent hybridization conditions with Mycoplasma pneumoniae nucleic acid, wherein said hybridization assay probe comprises a nucleotide base sequence which is at least 90% complementary to at least 10 contiguous nucleotides present in a hybridization assay probe target sequence of SEQ ID NO:
• 35:
• CCAAACUUAU CAUUAGAUAG GGGUUGUAGG, wherein under said conditions said hybridization assay probe preferentially hybridizes to said Mycoplasma pneumoniae nucleic acid over nucleic acids present in Mycoplasma genitalium, Mycoplasma orale, Mycoplasma faucium, Mycoplasma buccale, and Mycoplasma salivarium;
• and
• b) one or more helper oligonucleotides, wherein said one or more helper oligonucleotides hybridizes to Mycoplasma pneumoniae nucleic acid under said conditions and enhances hybridization between said probe and a Mycoplasma pneumoniae nucleic acid sequence region, provided that at least one of said one or more helper oligonucleotides comprises a nucleotide base sequence which is at least 90% complementary to at least 10 contiguous nucleotides present in a helper target sequence selected from the group consisting of:
• SEQ ID NO:
• 38:
• GUGGACUUGA AAACGAUAGA AGAAGC, and
• SEQ ID NO:
• 72:
• CGUGUGUAGU GGCGAGCGAA AGCGGAACA.

A probe mix for detecting the presence of Mycoplasma pneumoniae nucleic acid comprising,

• a) a hybridization assay probe which forms a hybrid stable for detection under stringent hybridization conditions with Mycoplasma pneumoniae nucleic acid, wherein said hybridization assay probe comprises a nucleotide base sequence which is at least 90% complementary to at least 10 contiguous nucleotides present in a hybridization assay probe target sequence of SEQ ID NO:
• 38:
• GUGGACUUGA AAACGAUAGA AGAAGC, wherein under said conditions said hybridization assay probe preferentially hybridizes to said Mycoplasma pneumoniae nucleic acid over nucleic acids present in Mycoplasma genitalium, Mycoplasma orale, Mycoplasma faucium, Mycoplasma buccale, and Mycoplasma salivarium;
• and
• b) one or more helper oligonucleotides which hybridizes to Mycoplasma pneumoniae nucleic acid under said conditions and enhances hybridization between said hybridization assay probe and a Mycoplasma pneumoniae nucleic acid sequence region, provided that at least one of said one or more helper oligonucleotides comprises a nucleotide base sequence which is at least 90% complementary to at least 10 contiguous nucleotides present in a helper target sequence selected from the group consisting of:
• SEQ ID NO:
• 35:
• CCAAACUUAU CAUUAGAUAG GGGUUGUAGG, and
• SEQ ID NO:
• 41:
• GGAAAGCAGC GCGCAAAAGG.

A helper oligonucleotide of from 12-100 nucleotides in length and having a nucleotide base sequence perfectly complementary to at least 10 contiguous nucleotide bases present in a helper target nucleotide base sequence selected from the group consisting of:

• SEQ ID NO:
• 56:
• AGUUUUGGAA UUUCAUGUGG AGCGGUGAAA UGCGUAG, SEQ ID NO:
• 59:
• CCGCCCGUCA AACUAUGAAA GCUGGUAAUA UUUAAAAACG UGUU, SEQ ID NO:
• 63:
• AAGGAUAGCA CCGGUGAUUG GAGUGAAGUC G, SEQ ID NO:
• 66:
• GGUGGUAAGA ACCUCAGAUC CGGAGAUUUC CGAAUG, SEQ ID NO:
• 72:
• CGUGUGUAGU GGCGAGCGAA AGCGGAACA, SEQ ID NO:
• 75:
• GUGAUAGCCC CGUAUUUGAA AUUGUUUUCA UACCUAGCGA G, and SEQ ID NO:
• 81:
• AGCGUUGUAU UGGAGUUGAA GUCAAAGCGU GAGC.

A helper oligonucleotide up to 50 nucleotides in length comprising a nucleotide base sequence selected from the group consisting of:

• SEQ ID NO:
• 11:
• GCAGCTGCCT TTAACACCAG ACTTTTCAAT CC, SEQ ID NO:
• 12:
• CTACGCATTT CACCGCTCCA CATGAAATTC CAAAACT, SEQ ID NO:
• 13:
• AACACGTTTT TAAATATTAC CAGCTTTCAT AGTTTGACGG GCGG, SEQ ID NO:
• 14:
• CGACTTCACT CCAATCACCG GTGCTATCCT T, SEQ ID NO:
• 15:
• CATTCGGAAA TCTCCGGATC TGAGGTTCTT ACCACC, SEQ ID NO:
• 16:
• CACGTATCGC TTTAATATGA CTATTTATTC ATC, SEQ ID NO:
• 17:
• TGTTCCGCTT TCGCTCGCCA CTACACACG, SEQ ID NO:
• 18:
• CTCGCTAGGT ATGAAAACAA TTTCAAATAC GGGGCTATCA C, SEQ ID NO:
• 19:
• ATAAATTCGG TAATATACTT AGCCCCGTTA CATCTTCGGC GC, SEQ ID NO:
• 20:
• GCTCACGCTT TGACTTCAAC TCCAATACAA CGCT,
• and RNA equivalents thereto.

A composition for amplifying and detecting Mycoplasma pneumoniae nucleic acid comprising:

• a) an amplification oligonucleotide, said amplification oligonucleotide being up to four nucleotides longer, or having up to two deleted nucleotides, and having no more than a 20% nucleotide base difference excluding RNA or DNA equivalent nucleotides, than an amplification sequence selected from the group consisting of:
• SEQ ID NO:
• 51:
• GGATTGAAAA GTCTGGTGTT AAAGGCAGCT GC,
• SEQ ID NO:
• 82:
• CGCCACTGGT GTTCCTTCAT ATATCTACGC,
• SEQ ID NO:
• 83:
• ATCAAAGTTG AAAGGACCTG CAAGGGTTCG T, and
• SEQ ID NO:
• 84:
• CTGCTGGCAC ATAGTTAGTC GTC;
• and
• b) an oligonucleotide hybridization assay probe able to preferentially hybridize to said Mycoplasma pneumoniae nucleic acid over Mycoplasma genitalium nucleic acid, wherein said hybridization assay probe comprises a nucleic acid sequence having no more than a 20% nucleotide base difference, excluding RNA or DNA equivalent nucleotides, than a detection sequence selected from the group consisting of:
• SEQ ID NO:
• 2:
• CACACTCTAG ATTAATAGTT TCCAATGC,
• SEQ ID NO:
• 24:
• GCATTGGAAA CTATTAATCT AGAGTGTG,
• SEQ ID NO:
• 85:
• CTCTAGCCAT TACCTGCTAA AGTC, and
• SEQ ID NO:
• 87:
• GACTTTAGCA GGTAATGGCT AGAG.

An oligonucleotide for amplifying Mycoplasma pneumoniae or Mycoplasma genitalium which is up to 50 nucleotides in length comprising a nucleotide base sequence selected from the group consisting of:

• SEQ ID NO:
• 51:
• GGATTGAAAA GTCTGGTGTT AAAGGCAGCT GC,
• SEQ ID NO:
• 84:
• CTGCTGGCAC ATAGTTAGTC GTC,
• SEQ ID NO:
• 82:
• CGCCACTGGT GTTCCTTCAT ATATCTACGC, and RNA equivalents thereto.

A probe for detecting whether Mycoplasma pneumoniae may be present in a sample consisting of one or more labels and a nucleotide base sequence selected from the group consisting of:

• SEQ ID NO:
• 2:
• CACACTCTAG ATTAATAGTT TCCAATGC,
• SEQ ID NO:
• 3:
• CATGCGCTTC CTAATGGTTA GC,
• SEQ ID NO:
• 4:
• GCTGTTTCCA ACTACCGGAT TGCTC,
• SEQ ID NO:
• 7:
• CCTTTTGCGC GCTGCTTTCC,
• SEQ ID NO:
• 8:
• CGTCTACCAC AAGATATAAA TCTTATCC,
• SEQ ID NO:
• 24:
• GCATTGGAAA CTATTAATCT AGAGTGTG,
• SEQ ID NO:
• 27:
• GCTAACCATT AGGAAGCGCA TG,
• SEQ ID NO:
• 30:
• GAGCAATCCG GTAGTTGGAA ACAGC,
• SEQ ID NO:
• 39:
• GGAAAGCAGC GCGCAAAAGG,
• SEQ ID NO:
• 42:
• GGATAAGATT TATATCTTGT GGTAGACG, and RNA equivalents thereof.