Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR

Abstract

The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples ( hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTAblood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 ؎ 0.06 for HCV and 0.97 ؎ 0.03 for HBV, indicating a linear extraction from 100 to 1.0 ؋ 10 5 HCV IU/ml and from 100 to 1.0 ؋ 10 6 HBV IU/ml. Intra-and interrun variability was below 0.23 log 10 IU/ml for 2.98 to 5.28 log 10 HCV IU/ml and 2.70 to 5.20 log 10 HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log 10 HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log 10 HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log 10 copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log 10 copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.Real-time thermocyclers have greatly decreased the amount of hands-on time in nucleic acid (NA)-based diagnostics for pathogens. The use of commercially available universal master mixes that contain all reagents except the pathogen-specific primer-probe combination has further decreased the number of pipetting steps and thus reduced labor and the possibility of errors.NA extraction has now become the most critical and laborintensive step in NA-based diagnostic assays. The overall sensitivity of the assay is determined by the NA yield, its purity, and the amount of sample equivalents that can be transferred into the amplification reaction. Conventional manual sample preparation methods are labor intensive and susceptible to contamination, handling variations, or errors (3, 9, 12).Since both the pathogen range and the number of different sample types are expanding and since multiplex downstream testing is becoming standard practice, there is a need for a generic extraction method (2, 5, 10). Ideally, the NA extraction procedure should yield pure NA from different pathogens and from a broad range of sample types.Recently, Abbott introduced m1000, an automated generic RNA and DNA extraction system using magnetic microparticle processing (17, 18). The m1000 system provides the necessary features for full automation of NA extraction of up to...

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