AEGiS-11IAC: Novel ribonuclease resistant RNA standards for HIV diagnostics.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Novel ribonuclease resistant RNA standards for HIV diagnostics.

Int Conf AIDS 1996 Jul 7-12; 11:22 (abstract no. LB.A.6006)
Pasloske B, DuBois D, Winkler M; Ambion, Inc., Austin, TX. Fax: (512) 445-7139.

INTRODUCTION: Quantitative assays (PCR, bDNA, NASBA) for RNA viruses are rapidly increasing in use for both patient management and as markers for the efficacy of antiviral agents in clinical trials. These developments have created a need for well-defined RNA standards for calibrating individual assays for comparing data generated from competing technologies. A major drawback of current standards is that many consist of naked RNA standards that cannot be directly added to clinical samples, but must be added at or after the RNA extraction procedure. Events occurring prior to RNA extraction are not controlled for in this format. In addition, the standards themselves are vulnerable to inadvertent degradation during storage and handling.

METHODS/RESULTS: We have developed a novel strategy for preparing Protected RNA (ProRNA) standards which are resistant to environmental nucleases, and mimic some of the physical properties of HIV. ProRNA standards are macromolecular structures based on the MS2 RNA bacteriophage. ProRNA standards may contain an insert of a defined RNA sequence of HIV, hepatitis C virus (HCV), or other RNA viruses of interest. Specifically, we have constructed a ProRNA standard encoding the 142 base sequence that serves as the internal quantitative standard (QS) in the Roche Amplicor HIV Monitor assay. We added the ProRNA-QS standard directly to human plasma and were able to obtain the same signal in the Monitor Assay after a 4 hour incubation, whereas the naked RNA was degraded instantaneously. Additionally, ProRNA-QS was substituted for naked QS-RNA internal controls in a series of 20 consecutive runs (over a 6 week period) of the Amplicor HIV assay. No change in the signal was observed. It is remarkable that this RNA standard was stored at 4 degrees C during this time. Data will be presented showing the impact of this substitution on the resulting coefficient of variation among assay runs. ProRNA is produced in E. Coli using a simple procedure and does not require the usual precautions for handling RNA.

CONCLUSIONS: We have developed a general method for producing RNA, and making viral standards in particular, which are resistant to ribonucleases, stable at 4 degrees C, non-infectious, relatively inexpensive, and straightforward to produce.

960707
LBA6006