title: 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.
abstract: 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.
authors.affiliation: Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. hymasw@aruplab.com
authors.forename: Weston
authors.initials: W
authors.lastname: Hymas
authors.forename: Jeffery
authors.initials: J
authors.lastname: Stevenson
authors.forename: E W
authors.initials: EW
authors.lastname: Taggart
authors.forename: David
authors.initials: D
authors.lastname: Hillyard
mesh_terms: D001769:Blood / Q000821:virology; D002138:Calibration / Q000592:standards; D002555:Cerebrospinal Fluid / Q000821:virology; D015342:DNA Probes; D005612:Freeze Drying; D014644:Genetic Variation; D015654:Herpesvirus 6, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D019349:Roseolovirus Infections / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes