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We have developed a new research assay that combines sequence-specific sample preparation and isothermal amplification for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections. The assay targets both the omp gene and the cryptic plasmid of C. trachomatis and the multicopy opa gene of N. gonorrhoeae, which are amplified and detected in a single reaction. We evaluated the ability of the assay to detect C. trachomatis and N. gonorrhoeae infections in first-catch urine, swab, and liquid-based cytology samples. Total agreement between the new assay and APTIMA Combo 2 varied between 95.3% and 100%, depending on the sample type and target detected. Total agreement between the new assay and BD ProbeTec varied between 96.7% and 100%, depending on the sample type and target detected. The assay has a simple work flow, and endpoint results can be achieved in 3 h, including sample preparation. The assay described here was evaluated for research use and was compared to commercially available assays.Chlamydia trachomatis and Neisseria gonorrhoeae are among the most common causes of sexually transmitted infections (3). Infection with these organisms is often asymptomatic, which hinders diagnosis or treatment. Persistent infection can have serious negative consequences, including pelvic inflammatory disease and infertility. Untreated and undiagnosed infections also contribute to the further spread of the disease. Screening programs are recommended to prevent these consequences (17). Of the various testing methods currently available, nucleic acid amplification tests are among the most sensitive and specific methods for detection of C. trachomatis and N. gonorrhoeae organisms in clinical specimens (7, 9). The high sensitivity of nucleic acid amplification tests allows them to be used with less-invasive clinical samples, such as first-catch urine, which facilitates establishment of screening programs.The assay described here combines sequence-specific sample preparation and isothermal amplification for the detection of C. trachomatis and N. gonorrhoeae infections. Sequence-specific sample preparation is based on antibody capture of DNA-RNA hybrids. Ribo-oligonucleotide probes hybridize to specific target sequences, and the resulting hybrids are captured by antibodies specific for DNA-RNA hybrids. This process enriches the desired nucleic acid sequences in the sample. Antibodies are coupled to magnetic beads. Target amplification is accomplished by thermophilic helicase-dependent amplification (tHDA) (16). Amplification of captured strands of the DNA target occurs directly on magnetic beads through the combined action of primers, thermostable DNA polymerase, and helicase. Target DNA is detected by use of dually labeled probes in either endpoint or real-time detection modes. C. trachomatis and N. gonorrhoeae targets are amplified and detected simultaneously in a single closed reaction vessel.We investigated the ability of the assay to detect C. trachomatis and N. gonorrhoeae infections in clinical specimens. R...

Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.

In vitro carcinogenesis assays, in mammalian cells, are proving to be a necessary part of batteries of in vitro tests for detecting potential mutagens/carcinogens. The selection of which particular test to use requires an evaluation of test reliability, repeatability, sensitivity and degree of validation. Validation is particularly important because it is an integral part of the other criteria. Attachment independence is a common characteristic of transformed cells and is often used in follow-up testing to confirm transformation. In an earlier report we described a transformation assay, the Survival Assay, which measures the acquisition of attachment independence. The Rauscher leukemia virus-infected rat embryo target cells are treated with test compound for 72 h. Cells are then transferred to petri dishes containing a bottom layer of agar/medium. After 6 days the viable cell numbers of treated and control cultures are compared. We report here the results of testing 77 additional compounds in this assay; 73 of these were tested under code. Since definitive carcinogenicity data were not available for 18 of the test compounds, it is not possible to list specific correlations with in vivo test results. None the less the assay correctly identified 44 of the 49 carcinogens and 7 of the 10 noncarcinogens in the list. The noncarcinogen status of two of the latter is, however, open to question. Thus the Survival Assay appears to provide information rapidly as to the potential carcinogenicity of a test material, even when subjected to validation testing with coded compounds.