Method Description

The RealStar HHV-6 (human herpesvirus-6 virus) PCR (polymerase chain reaction) Kit 1.0 (Altona Diagnostics) and the Exact Diagnostics HHV6-A/B Verification Panels and controls (Bio-Rad) are utilized for this assay. The assay employs TaqMan hydrolysis probe chemistry, with production of visible amplification curves and associated crossing point (Cp) values; no melting temperature curve is produced. A probe specific for HHV-6A DNA is labeled with the fluorophore FAM and is detected on the 465-510 channel. A probe specific for HHV-6B DNA is labeled with the fluorophore Cy5 and is detected on the 533-580 channel. The probe specific for the Internal Control (IC) is labeled with the fluorophore JOE, is detected on the 618-660 channel, and will be used within the mastermix. Using probes linked to distinguishable dyes enables the parallel detection and differentiation of HHV-6A and HHV-6B DNA as well as the detection of the IC in corresponding detector channels of the LC480 II instrument. The MagNA Pure 96 instrument (Roche Diagnostics) using the MP96 DNA and Viral NA Small Volume Kit with an input of 200 mcL and a final elution volume of 100 mcL is utilized for viral nucleic acid extraction. The Pathogen Universal 200 protocol is used.(Package insert: RealStar HHV-6 Virus PCR Kit 1.0. Altona Diagnostics; 09/2018)