Application of a rapid non-radioisotopic nucleic acid analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistances.

Abstract

Abstract We devised a versatile method for detecting nucleic acids in crude lysates of biological samples. A controlled network of nucleic acid hybrids composed of the target fragment, several oligonucleotide probes, branched DNA amplifiers, and labeled oligonucleotides is produced on a solid phase to ultimately incorporate 60 to 300 molecules of alkaline phosphatase, which are detected with a chemiluminescent substrate. The visible light output can be recorded on a luminometer or on instant black-and-white film. Assays have been developed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and for genes conferring penicillin and tetracycline resistance. Conducted much like ELISAS, the assays are performed in about 4 h (for 96 samples) in microliter dishes. The molecular detection limit of approximately 50,000 molecules of double-stranded DNA has permitted us to detect 1 to 10 x 10(3) of C. trachomatis and N. gonorrhoeae with specific probe sequences. Both plasmid and genomic target sequences can be detected by the same procedure. All of the assay components, except for a set of unmodified oligonucleotide probes, are universally applicable for all targets.