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Nucleic acid probes and methods for detecting Enterobacter cloacae
| Details |
Inventors: Hogan, James John; Kop, Jo Ann; McDonough, Sherrol Hoffa;
Assignee: Gen-Probe Incorporated (San Diego, CA)
Primary Examiner: Marschel; Ardin H.
Assistant Examiner:
Attorney, Agent or Firm: Lyon & Lyon LLP
A method for preparing probes, as well as several probes for use in qualitative or quantitative hybridization assays are disclosed. The method comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a region of rRNA selected to be unique to a non-viral organism or group of non-viral organisms sought to be detected, said region of rRNA being selected by comparing one or more variable region rRNA sequences of said non-viral organism or group of non-viral organisms with one or more variable region rRNA sequences from one or more non-viral organisms sought to be distinguished. Hybridization assay probes for Mycobacterium avium, Mycobacterium intracellulare, the Mycobacterium tuberculosis-complex bacteria, Mycoplasma pneumoniae, Legionella, Salmonella, Chlamydia trachomatis, Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas group I, Neisseria gonorrhoeae, bacteria, and fungi also are disclosed. |
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DETAILED DESCRIPTION OF THE INVENTION Definitions The following terms, as used in this disclosure and claims, are defined as: nucleotide: a subunit of a nucleic acid consisting of a phosphate group, a 5' carbon sugar and a nitrogen containing base.
In RNA the 5' carbon sugar is ribose.
In DNA, it is a 2-deoxyribose.
The term also includes analogs of such subunits.
nucleotide polymer: at least two nucleotides linked by phosphodiester bonds.
oligonucleotide: a nucleotide polymer generally about 10 to about 100 nucleotides in length, but which may be greater than 100 nucleotides in length.
nucleic acid probe: a single stranded nucleic acid sequence that will combine with a complementary single stranded target nucleic acid sequence to form a double-stranded molecule (hybrid).
A nucleic acid probe may be an oligonucleotide or a nucleotide polymer.
hybrid: the complex formed between two single stranded nucleic acid sequences by Watson-Crick base pairings or non-canonical base pairings between the complementary bases.
hybridization: the process by which two complementary strands of nucleic acids combine to form double stranded molecules (hybrids).
complementarity: a property conferred by the base sequence of a single strand of DNA or RNA which may form a hybrid or double stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs on the respective strands.
Adenine (A) usually complements thymine (T) or Uracil (U), while guanine (G) usually complements cytosine (C).
stringency: term used to describe the temperature and solvent composition existing during hybridization and the subsequent processing steps.
Under high stringency conditions only highly homologous nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form.
Accordingly, the stringency of the assay conditions determine the amount of complementarity needed between two nucleic acid strands forming a hybrid.
Stringency is chosen to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid
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