Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase-based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real-time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)-1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase-based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results.
Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter-and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within ؎0.5 log 10 unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.
The QIAsymphony sample preparation and artus EBV RG PCR test system is suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory.
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