Rotor gene q mdx 5plex hrm

The Sentosa® SA EBV Quantitative PCR Test (Vela Diagnostics) was applied for quantification of EBV cfDNA with the aid of the integrated Sentosa® SX101 (Vela Diagnostics) and Rotor-Gene® Q MDx 5-plex HRM (Qiagen) instruments. 60 µL of DNA was automatically extracted from 200 µL of plasma using the Sentosa® SX Virus Total Nucleic Acid Kit v2.0 (Vela Diagnostics). 10 µL of purified DNA, equivalent to 33 µL of plasma was used for each reaction. The PCR master mix contained reagents and enzymes for the amplification of a 79-bp fragment of EBNA1, as well as a second set of primers/probes designed to detect EC3, a control for PCR inhibition and cfDNA extraction. The concentration of EBNA1 was automatically calculated based on the imported standard curve, with R² = 0.99, qPCR efficiency = 98%, m = (−3.367). The clinical sensitivity and specificity of the assay was reported as 100% and 98.8% respectively.

Extracted DNA from LBC samples (≤200 ng) was subjected to bisulfite treatment using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to protocol.
Approximately 50 ng of bisulfite-converted DNA was added into the methylation analysis. QIAsure (QIAGEN), a multiplex-methylation–specific rtPCR method, was used to evaluate the hypermethylation status of two human host cell genes (FAM19A4 and miR-124-2), including one internal control gene (ACTB). Cycle threshold values of the two targets in each sample were reported in relation to the internal control and a low copy number plasmid control, the calibrator. The assay was run on the Rotor-Gene Q MDx 5plex HRM (QIAGEN) system and results were automatically analyzed with the Rotor-Gene Assay Manager software.
In addition to an internal control validation for every sample, for each run a low copy number plasmid control, for example, the calibrator, and a non-template control, was included. Cycle threshold values of the two targets in the samples are reported in relation to the internal control and the calibrator. Detected hypermethylation in any of the two targets resulted in a positive test result.