Progress 05/01/03 to 04/30/07
Outputs
OUTPUTS: The project was completed at the end of the previous review period. The results of the study were formated into a Assay Validation Packet and provided to the primary user of the assay, USDA APHIS, for review.
PARTICIPANTS: Luis Rodriguez. PI. USDA APHIS Sharon Hietala. CAHFS, University of California, Davis. Beate Crossley. CAHFS, Univsersity of California, Davis. Rupanshu Verma. CAHFS Technical support. Laura Case. CAHFS Technical Support. The technical support position at CAHFS included student employees that obtained training in molecular detection techniques and assay validation reqirements. The assay prototypes and armored RNA studies were used as professional training opportunites for 4 CAHFS molecular diagnostic technicians.
Impacts
The CAHFS provided bench-level assay and field validation during development and verification of a novel realtime PCR for Vesicular Stomatitis viruses (VSV). The assay was tested in two formats; a dried-down reagent format designed for effective distribution and inter-laboratory standardization, and compared to a wet-reagent PCR format. The CAHFS provided serum-virus neutralization testing for VSV-New Jersey and Indiana on cattle obtained from 30 herds in order to assess exposure status based on VSV serologic responses. The CAHFS provided realtime RT PCR testing on oral and hoof swabs from the same animals using a prototype dried-down version of the VSV assay designed by the principal investigator. A sub-set of 500 samples for PCR analysis were selected from 18 operations where VSV antibody titers between 1:4 and 1:256 were detected, and 12 operations with no serologic evidence of VSV exposure. The specimens were evaluated by realtime RT-PCR in a 96-well format for
side-by-side comparison of the assay in dried-down format and a modified version with an expanded target range in a wet-reagent format. Equivalency testing of the two formats was performed in order to demonstrate flexibility of the assay for use in both surveillance and diagnostic applications. Reference virus VSV-New Jersey and VSV-Indiana yielded similar performance measures, including limits of detection and reproducibility between the 2 formats. The 500 negative cohort field samples demonstrated perfect assay specificity regardless of format. Vesicular Stomatitis New Jersey and Indiana reference viruses tested over the course of the study demonstrated excellent within and between- assay reproducibility for both formats. The wet reagent format demonstrated better reproducibility (Indiana mean Ct = 28.98, SD = 0.35, New Jersey mean Ct = 30.01, SD = 0.66) compared to the dried-down reagents, with an average 1.13 Ct variability between runs. Additionally, the dried-down format had 4
assay failures tracked to desiccant failure or missing probe, compared to no assay failures with the wet-reagent format. An armored RNA construct containing the VSV PCR assay targets was developed by the CAHFS participants, and was evaluated by spiking into clinical material for time and temperature stability. The armored RNA was reproducible (between run standard deviation 0.06 for VSV-NJ, 1.00 for VSV-Indiana) at a concentration of 10-5 VSV-specific target sequences. Armored RNA spiked into clinical material was stabile under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling samples in a diagnostic laboratory setting, including 24 hours at 37C, 2 weeks at ambient room temperature,-20C, and -70C. The assay analytic performance mimicked the linearity and dynamic range of the realtime RT-PCR assay using reference VSV, indicating that the armored RNA can be used as a non-infectious, quantifiable, and stabile PCR reagent
for an assay positive control, for training panels, and for proficiency test purposes.
Publications
- Hietala SK and Crossley BM. (2006) Armored RNA as virus surrogate in a real-time reverse transcriptase PCR assay proficiency panel. J Clin Microbiol.: 44(1):67-70.
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