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

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.

Hybridization Assays Using an Expressible DNA Fragment Encoding Firefly Luciferase as a Label

“…This has been accomplished by using sequential hybridization with three types of probes. 16 The primary probe contains two parts, a region complementary to the target and a segment that allows binding of a polymerized secondary probe (amplification multimer). Then, each secondary probe reacts with an enzyme-labeled probe.…”

Forks and combs and DNA: the synthesis of branched oligodeoxyribonucleotides

“…Using several copies of the comb-type amplification multimers containing five secondary sequences, we have been able to detect as few as 1,000 organisms in hybridization assays for Chlamydia trachomatis, Neisseria gonorrhoeae, and the tetracyclineand penicillinresistance genes of several bacteria (27).…”

Advances in nucleic acid-based detection methods.

“…When this method was combined with the sensitivity of chemiluminescence, as few as 1,000 viral hepatitis genomes were detected directly in human serum or plasma (91). The method has also been applied to the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and penicillin and tetracycline resistance plasmids (92).…”