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

• Published in: Journal of Clinical Microbiology Vol. 43; no. 11; pp. 5541 - 5546
• Main Authors: Beuselinck, Kurt, Ranst, Marc van, Eldere, J. van
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.11.2005
• Subjects: Automation; Biological and medical sciences; Cytomegalovirus; Cytomegalovirus - genetics; Cytomegalovirus - isolation & purification; DNA Virus Infections - diagnosis; DNA Virus Infections - virology; DNA, Viral - blood; DNA, Viral - isolation & purification; Edetic Acid; Epstein-Barr virus; Fundamental and applied biological sciences. Psychology; Hepacivirus - genetics; Hepacivirus - isolation & purification; Hepatitis B virus; Hepatitis B virus - genetics; Hepatitis B virus - isolation & purification; Hepatitis C virus; Herpesvirus 4, Human - genetics; Herpesvirus 4, Human - isolation & purification; Humans; Indicators and Reagents; Infectious diseases; Medical sciences; Microbiology; Polymerase Chain Reaction - methods; Polyomavirus; RNA Virus Infections - diagnosis; RNA Virus Infections - virology; RNA, Viral - blood; RNA, Viral - isolation & purification; Sensitivity and Specificity; Virology; High throughput screening; Polymerase chain reaction; Microbiology; Real time; Quantitative analysis
• ISSN: 0095-1137; 1098-660X; 1098-5530
• DOI: 10.1128/JCM.43.11.5541-5546.2005

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 EDTA-blood 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 x 10⁵ HCV IU/ml and from 100 to 1.0 x 10⁶ HBV IU/ml. Intra- and interrun variability was below 0.23 log₁₀ IU/ml for 2.98 to 5.28 log₁₀ HCV IU/ml and 2.70 to 5.20 log₁₀ 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 log₁₀ HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log₁₀ HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log₁₀ 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 log₁₀ 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.