Use of lyophilized standards for the calibration of a newly developed real time PCR assay for human herpes type six (HHV6) variants A and B

• Published in: Journal of virological methods 2005-09, Vol.128 (1), p.143-150
• Main Authors: Hymas, Weston, Stevenson, Jeffery, Taggart, E.W., Hillyard, David
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Blood - virology; Calibration - standards; Cerebrospinal Fluid - virology; DNA Probes; Freeze Drying; Fundamental and applied biological sciences. Psychology; Genetic Variation; Herpesvirus 6, Human - classification; Herpesvirus 6, Human - genetics; Herpesvirus 6, Human - isolation & purification; HHV6; Human herpes 6; Human herpesvirus 6; Humans; Lyophilized; Microbiology; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Quantitation; Real time PCR; Reference Standards; Roseolovirus Infections - diagnosis; Roseolovirus Infections - virology; Sensitivity and Specificity; Techniques used in virology; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2005.05.003

A quantitative real time PCR assay utilizing an Eclipse minor groove binding hybridization probe was developed to detect and type human herpes 6. A 115 base pair product from the U67 gene was selected for amplification and the assay included a noncompetitive internal control. The probe's melting temperature from the amplified sequence differentiated between HHV6 variants (A and B). In this study, 120 samples (60 spiked and 60 negative) comprising CSF, plasma, and serum were tested at high and medium levels, and near the limit of quantitation. The use of stored standard curves for assay calibration was compared to curves run on each assay, and the stability of liquid frozen versus lyophilized frozen stocks for calibrators and controls was assessed. After 9 months of clinical testing, assay performance was examined to determine the percent positive rate and positive sample reproducibility, as well as to evaluate standard curve stability. We obtained 100% correlation to expected results for positive and negative samples. A stored curve proved easier, more cost effective, and more reliable than running a standard curve on each assay. The use of lyophilized standards contributes substantially to the maintenance of reproducible testing over an extended period of time.