The Government of the USA as represented by the Secretary of the Dept. of Health and Human Services
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초록▼
Methods for detecting presence of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma par
Methods for detecting presence of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and Neisseria meningitidis nucleic acids in a sample, such as a biological sample obtained from a subject, or an environmental sample, are provided. This disclosure also provides probes, primers, and kits for detecting one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and Neisseria meningitidis in a sample.
대표청구항▼
1. A method for detecting presence of Klebsiella pneumoniae in a sample, comprising: contacting the sample with a nucleic acid probe comprising the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 60, or the reverse complement thereof, and a donor fluorophore, an acceptor fluorophore, or a combi
1. A method for detecting presence of Klebsiella pneumoniae in a sample, comprising: contacting the sample with a nucleic acid probe comprising the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 60, or the reverse complement thereof, and a donor fluorophore, an acceptor fluorophore, or a combination thereof;amplifying a nucleic acid from Klebsiella pneumoniae, wherein the amplifying comprises contacting the sample with a pair of primers comprising the nucleic acid sequences of SEQ ID NOs: 18 and 19 or SEQ ID NOs: 58 and 59; anddetecting hybridization between the nucleic acid probe and a nucleic acid in the sample, wherein detection of hybridization indicates the presence of Klebsiella pneumoniae in the sample. 2. The method of claim 1, further comprising detecting one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Streptococcus agalactiae, and Bartonella spp. in the sample by: (a) contacting the sample with one or more nucleic acid probes, wherein:the pathogen is Pseudomonas aeruginosa and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 17;the pathogen is Bartonella spp. and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 50;the pathogen is Acinetobacter baumannii and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 14;the pathogen is Staphylococcus aureus and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 32;the pathogen is Toxoplasma gondii and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 23;the pathogen is Moraxella catarrhalis and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 26;the pathogen is Escherichia coli and/or Shigella and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 29;the pathogen is Chlamydia trachomatis and the probes comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 38;the pathogen is Ureaplasma urealyticum and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 41;the pathogen is Ureaplasma parvum and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 44;the pathogen is Ureaplasma spp. and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 47; and/orthe pathogen is Streptococcus agalactiae and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 56; and(b) detecting hybridization between the one or more nucleic acid probes and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of one or more of said pathogens in the sample. 3. The method of claim 2, further comprising amplifying a nucleic acid from one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Streptococcus agalactiae, and Bartonella spp. by contacting the nucleic acid with at least one primer, wherein: the pathogen is Pseudomonas aeruginosa and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16;the pathogen is Bartonella spp. and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 51;the pathogen is Acinetobacter baumannii and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13;the pathogen is Staphylococcus aureus and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31;the pathogen is Toxoplasma gondii and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22;the pathogen is Moraxella catarrhalis and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 25;the pathogen is Escherichia coli and/or Shigella and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 28;the pathogen is Chlamydia trachomatis and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37;the pathogen is Ureaplasma urealyticum and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 39 or SEQ ID NO: 40;the pathogen is Ureaplasma parvum and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43;the pathogen is Ureaplasma spp. and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46; and/orthe pathogen is Streptococcus agalactiae and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 54 or SEQ ID NO: 55. 4. The method of claim 1, wherein the sample comprises a biological sample or environmental sample. 5. The method of claim 4, wherein the sample is a biological sample comprising tissue, blood, serum, cerebral spinal fluid, middle ear fluid, bronchoalveolar lavage, tracheal aspirate, sputum, nasopharyngeal aspirate, oropharyngeal aspirate, or saliva. 6. The method of claim 4, wherein the sample is an environmental sample comprising a food sample, a water sample, or a surface swab. 7. An isolated nucleic acid probe consisting of the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 60 and a donor fluorophore, an acceptor fluorophore, or a combination thereof. 8. A kit for detection of Klebsiella, comprising the isolated nucleic acid probe of claim 7 and a pair of primers comprising the nucleic acid sequences of SEQ ID NOs: 18 and 19 or SEQ ID NOs: 58 and 59. 9. The kit of claim 8, further comprising one or more primers comprising the nucleic acid sequence of any one of SEQ ID NOs: 12, 13, 15, 16, 21, 22, 24, 25, 27, 28, 30, 31, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 54, and 55. 10. The kit of claim 9, wherein the one or more primers consist of the nucleic acid sequence of any one of SEQ ID NOs: 12, 13, 15, 16, 21, 22, 24, 25, 27, 28, 30, 31, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 54, and 55. 11. The kit of claim 8, further comprising one or more isolated nucleic acid probes up to 40 nucleotides in length, comprising the nucleic acid sequence of any one of SEQ ID NOs: 14, 17, 23, 26, 29, 32, 38, 41, 44, 47, 50, and 56, and a donor fluorophore, an acceptor fluorophore, or a combination thereof.
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