title: Quantification of HEV RNA by Droplet Digital PCR.
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.
authors.affiliation: CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie, Toulouse F-31300, France. nicot.f@chu-toulouse.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Nicot
authors.affiliation: CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie, Toulouse F-31300, France. cazabat.m@chu-toulouse.fr.|INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France. cazabat.m@chu-toulouse.fr.
authors.forename: Michelle
authors.initials: M
authors.lastname: Cazabat
authors.affiliation: CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie, Toulouse F-31300, France. lhomme.s@chu-toulouse.fr.|INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France. lhomme.s@chu-toulouse.fr.|Université Toulouse III Paul-Sabatier, Faculté de Médecine Toulouse, Toulouse F-31300, France. lhomme.s@chu-toulouse.fr.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Lhomme
authors.affiliation: INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France. marion-olivier@hotmail.fr.|CHU Toulouse, Hôpital Rangueil, Département de Néphrologie, Dialyse et Transplantation multi-organe, Toulouse F-31300, France. marion-olivier@hotmail.fr.
authors.forename: Olivier
authors.initials: O
authors.lastname: Marion
authors.affiliation: CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie, Toulouse F-31300, France. saune.k@chu-toulouse.fr.|INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France. saune.k@chu-toulouse.fr.|Université Toulouse III Paul-Sabatier, Faculté de Médecine Toulouse, Toulouse F-31300, France. saune.k@chu-toulouse.fr.
authors.forename: Karine
authors.initials: K
authors.lastname: Sauné
authors.affiliation: CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie, Toulouse F-31300, France. chiabrando.j@chu-toulouse.fr.
authors.forename: Julie
authors.initials: J
authors.lastname: Chiabrando
authors.affiliation: CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie, Toulouse F-31300, France. dubois.m@chu-toulouse.fr.|INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France. dubois.m@chu-toulouse.fr.
authors.forename: Martine
authors.initials: M
authors.lastname: Dubois
authors.affiliation: INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France. kamar.n@chu-toulouse.fr.|CHU Toulouse, Hôpital Rangueil, Département de Néphrologie, Dialyse et Transplantation multi-organe, Toulouse F-31300, France. kamar.n@chu-toulouse.fr.
authors.forename: Nassim
authors.initials: N
authors.lastname: Kamar
authors.affiliation: CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie, Toulouse F-31300, France. abravanel.f@chu-toulouse.fr.|INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France. abravanel.f@chu-toulouse.fr.|Université Toulouse III Paul-Sabatier, Faculté de Médecine Toulouse, Toulouse F-31300, France. abravanel.f@chu-toulouse.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Abravanel
authors.affiliation: CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie, Toulouse F-31300, France. izopet.j@chu-toulouse.fr.|INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France. izopet.j@chu-toulouse.fr.|Université Toulouse III Paul-Sabatier, Faculté de Médecine Toulouse, Toulouse F-31300, France. izopet.j@chu-toulouse.fr.
authors.forename: Jacques
authors.initials: J
authors.lastname: Izopet
mesh_terms: D016752:Hepatitis E virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods* / Q000592:standards
chemical_list: D012367:RNA, Viral
keywords: HEV quantification; absolute quantification; ddPCR