Quantification of HEV RNA by Droplet Digital PCR.

Florence Nicot,Julie Chiabrando,Michelle Cazabat,Nassim Kamar,Olivier Marion,Karine Sauné,Sébastien Lhomme,Martine Dubois,Jacques Izopet,Florence Abravanel

doi:10.3390/v8080233

Abstract

The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR) assay (Spearman rs = 0.89, p < 0.0001). The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types.

Highlights

Hepatitis E virus (HEV) is a non-enveloped single-stranded, positive sense RNA virus [1].Four main genotypes have been identified, of which genotype 3 predominates in industrialized countries [2,3,4,5]
We have evaluated a new method for quantification of hepatitis E virus (HEV) RNA, reverse transcriptase droplet digital PCR (RT-ddPCR), that gives absolute quantification of the target gene [11,12]
Reverse transcription was carried out at 61 ◦ C for 30 min followed by denaturation at 95 ◦ C for 5 min, and DNA was amplified with 40 PCR cycles at 95 ◦ C (30 s) and 56 ◦ C (1 min) on a QX200 droplet digital PCR system (BioRad)

Summary

Introduction

Hepatitis E virus (HEV) is a non-enveloped single-stranded, positive sense RNA virus [1]. Four main genotypes have been identified, of which genotype 3 predominates in industrialized countries [2,3,4,5]. HEV genotype 3 is of increasing concern for immunocompromised patients as it can lead to chronic infection and cirrhosis [6,7]. An HEV infection is best diagnosed by a combination of serological tests and nucleic acid amplification techniques [1]. RNA is needed for pathophysiological studies and for monitoring the HEV viral loads of chronically infected patients on antiviral therapy [8,9]. An evaluation of several reverse transcriptase quantitative

Methods

Results

Conclusion