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William Hauser1, Michael Mira1, Guiqing Wang, MD PhD1, Dieter Schapfel, MD1
1Enzo Life Sciences, Farmingdale, NY USA
INTRODUCTION
More than 170 million individuals are chronically infected with Hepatitis C Virus (HCV), and it is a major cause of hepatocellular carcinoma, liver cirrhosis, and liver-related mortality. Successful HCV treatment results in sustained virologic response (SVR), defined as undetectable levels of HCV RNA in the blood 12 or more weeks after completing treatment. A sensitive method to quantify HCV RNA is paramount to the management of patients undergoing antiviral therapy.
A quantitative HCV viral load assay using novel probe technology was validated for use in the quantitative detection of HCV RNA. The AMPIPROBE® HCV Assay is a real-time reverse transcription quantitative polymerase chain reaction assay that incorporates probe detection technology in primer design. It is intended for the quantitative detection of HCV RNA, genotypes 1 through 6, in plasma or serum.
The limit of detection (LOD) of the assay was determined using spiked plasma or serum specimens with Acrometrix® reference materials calibrated by using the World Health Organization HCV RNA standard per guidelines described in CLSI EP17-A. The LOD of HCV RNA in EDTA plasma was determined to be as low as 0.90 log IU/mL (7.9 IU/mL) and in serum as low as 0.74 log IU/mL (5.5 IU/mL), both with a positive rate greater than 95%. For both plasma and serum, the lower limit of quantification was 1.0 log IU/mL (10 IU/mL) via 95% hit rate analysis. A reference panel was utilized to confirm that the AMPIPROBE® HCV Assay was able to detect genotypes 1a, 1b, 2a, 3a, 4acd, 5a, and 6 at 15 IU/mL or greater at a 95% hit rate. The linear range was evaluated in accordance with the methods recommended in the CLSI guideline EP06-A. The linear range was determined to be from 0.7 to 7.4 log IU/mL (5 to 25,000,000 IU/mL).
The LOD and LOQ determined for the AMPIPROBE® HCV Assay are lower than that of comparable HCV viral load assays, making it a more sensitive assay suitable for monitoring viral load during antiviral therapy.
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| Application | qPCR |
|---|---|
| Sensitivity | In a validation study approved by the New York State Department of Health, the AMPIPROBE® HCV assay was determined to have the following sensitivity:LOD serum – 5.46 IU/mL via 95% Probit analysisLOD plasma – 7.91 IU/mL via 95% Probit analysisLOQ serum – 10 IU/mL via 95% hit rate analysisLOQ plasma – 10 IU/mL via 95% hit rate analysisIn the same validation study, the linear range of both serum and plasma was determined to be 5 to 25,000,000 IU/mL. |
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