Universal detection of hepatitis E virus by two real-time PCR assays: TaqMan® and Primer-Probe Energy Transfer

Gyarmati, Péter; Mohammed, Nahla; Nordér, Helené; Blomberg, Jonas; Belák, Sándór; Widén, Frederik

doi:10.1016/j.jviromet.2007.07.014

Cited by 89 publications

(66 citation statements)

References 26 publications

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“…Several protocols for real-time PCR targeting the ORF3 region (13,17,40) or the ORF2 region (1,16,34,47) That study demonstrated that real-time RT-PCRs are more sensitive than nested PCRs, but the sensitivities of the majority of the assays differed enormously (100-fold to 1,000-fold), independent of the virus strains (3). Among protocols targeting ORF3, we selected one developed by Jothikumar et al, because it was the most frequently used protocol in a recent evaluation performed by Baylis et al (17).…”

Section: Discussionmentioning

confidence: 99%

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

et al. 2012

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Genotype 3 hepatitis E viruses (HEVs) are distributed across the world and are now considered to be an emerging public health concern in industrialized countries. At least 10 genotype 3 subtypes have been identified in humans and animals worldwide. It was recently reported that the sensitivities of HEV RNA assays differ greatly. We have assessed the influence of genotype 3 diversity on the performances of two HEV RNA assays: one targeting the ORF3 gene and the other targeting the ORF2 gene. We tested a panel of 5 HEV-positive reference samples of genotypes 3a, 3b, 3c, 3e, and 3f at 10-fold serial dilutions. The HEV RNA concentrations obtained with both reverse transcription (RT)-PCRs were correlated, but the RT-PCR based on ORF2 underestimated the HEV RNA concentrations. The mean [ORF3 ؊ ORF2] difference was 1.41 log copies/ml. We also tested 34 clinical specimens of genotypes 3c (n ‫؍‬ 15), 3e (n ‫؍‬ 4), and 3f (n ‫؍‬ 15), representing the most prevalent subtypes in Europe. The mean [ORF3 ؊ ORF2] differences were 1.41 log copies/ml for genotype 3c, 0.96 log copies/ml for genotype 3e, and 0.70 log copies/ml for genotype 3f. The bias between the 2 RT-PCR assays was significantly greater for genotype 3c than for genotype 3f (P ‫؍‬ 0.007). We therefore recommend the use of an RT-PCR protocol based on ORF3 to quantify HEV RNA of genotype 3 strains.

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Section: Discussionmentioning

confidence: 99%

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

et al. 2012

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“…The first round PCR was conducted on 5 μL of cDNA in a final volume of 25 μL containing 2X GoTaq Hot Start Mastermix, and 200 nM of primers JVHEV-F, 31 and TqRev. 32 Cycling conditions were as follows: 95 C for 2 min, and then 35 cycles of 95 C for 15 sec/60 C for 30 sec/72 C for 2 min followed by a final elongation step at 72 C for 7 min. Second round PCR was performed on 4 μL of primary PCR product in a final volume of 50 μL containing 200 nM of primers JVHEV-F and 3159N.…”

Section: Methodsmentioning

confidence: 99%

High Prevalence of Hepatitis E in Humans and Pigs and Evidence of Genotype-3 Virus in Swine, Madagascar

Temmam¹,

Besnard²,

Andriamandimby³

et al. 2013

The American Society of Tropical Medicine and Hygiene

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Abstract. Hepatitis E virus (HEV) causes an orofecal disease transmitted through poor hygiene environments, contaminated food (mainly pork products), or by contacts with infected animals. Very little data are currently available regarding the disease in the Southwestern Indian Ocean Islands. We report the first sero-and viro-survey for HEV in human and swine in Madagascar. A seroprevalence rate of 14.1% (60 of 427) was measured in slaughterhouse workers. Seroprevalence to HEV in pigs was estimated to 71.2% (178 of 250), strongly suggesting the existence of a zoonotic cycle. Three out of 250 pig livers (1.2%) tested HEV RNA-positive by quantitative polymerase chain reaction. Phylogenetic analyses based on 1-kb sequences of the ORF 2-3 identified these viruses as HEV genotype 3. Sequences clustered in a distinct Malagasy sub-clade, possibly representative of a new sub-genotype, for which the date of emergence was estimated around 1989. Further studies are needed to confirm other transmission routes of HEV to humans, especially through non-zoonotic cycles.

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“…These assays include conventional reverse transcriptase PCR (RT-PCR) as well as realtime RT-PCR and reverse transcription-loop-mediated isothermal amplification (10) for the detection of HEV RNA in serum and plasma or in fecal samples. While consensus assays have been developed for the detection of HEV genotypes 1 to 4 (5,8), no studies have been performed so far to compare the abilities of laboratories to detect HEV RNA.…”

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confidence: 99%

Standardization of Hepatitis E Virus (HEV) Nucleic Acid Amplification Technique-Based Assays: an Initial Study To Evaluate a Panel of HEV Strains and Investigate Laboratory Performance

et al. 2011

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The performance of hepatitis E virus (HEV) RNA nucleic acid amplification (NAT)-based assays has been investigated using a panel of HEV-containing plasma samples. The panel comprised 22 HEV-positive plasma samples representing 10-fold serial dilutions of HEV genotypes 3a, 3b, 3f, and 4c obtained from blood donors. Two negative-control plasma samples were included. All samples were blinded. The plasma samples were prepared as liquid/frozen materials and distributed to participants on dry ice. Laboratories were requested to test the panel using their routine HEV assays and to score samples as either positive or negative and could optionally return data in copies/ml for HEV RNA. Twenty laboratories from 10 different countries participated in the study. Data were returned by all participating laboratories; 10 laboratories returned quantitative data. All assays except one were developed in-house using conventional or real-time reverse transcriptase PCR (RT-PCR) methodologies. There was a 100-to 1,000-fold difference in sensitivity between the majority of assays, independent of the virus strain. Although the quantitative data were limited, for the samples in the range of ϳ6 to 4 log 10 copies/ml, the standard deviations of the geometric means of the samples ranged between 0.38 and 1.09. Except for one equivocal result, HEV RNA was not detected in the negative samples. The variability of assay sensitivity highlights the need for the standardization of HEV RNA NAT assays.

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Universal detection of hepatitis E virus by two real-time PCR assays: TaqMan® and Primer-Probe Energy Transfer

Cited by 89 publications

References 26 publications

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

High Prevalence of Hepatitis E in Humans and Pigs and Evidence of Genotype-3 Virus in Swine, Madagascar

Standardization of Hepatitis E Virus (HEV) Nucleic Acid Amplification Technique-Based Assays: an Initial Study To Evaluate a Panel of HEV Strains and Investigate Laboratory Performance

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