Optimisation of a triplex real time RT-PCR for detection of hepatitis E virus RNA and validation on biological samples

https://doi.org/10.1016/j.jviromet.2011.12.007Get rights and content

Abstract

The aim of this study was to optimise a two-tube reverse transcription triplex quantitative real time PCR (qRT-PCR) combining amplification of two loci with an internal amplification control (IAC) for detection and quantitation of hepatitis E virus (HEV) RNA and to validate its performance on a pool of biological samples. Optimisation was performed on serially diluted “home-made” RNA standards. The limit of detection was determined experimentally as 10 copies/μl of the RNA standard for both amplification targets. The qRT-PCR was validated on a cohort of samples originating from 48 wild boars (Sus scrofa), 17 fallow deer (Dama dama) and 28 mouflons (Ovis musimon), with nested RT-PCR used as a reference method. qRT-PCR was found to be more specific for the detection of HEV RNA in examined samples. HEV RNA was detected in samples of five more animals (one wild boar and four mouflons) in comparison with nested RT-PCR.

Highlights

Reverse transcription triplex quantitative real time PCR assay for detection and quantitation of hepatitis E virus (HEV) RNA was optimised. ► The assay was able to detect at least 10 copies of the HEV genome/μl of isolated RNA and allows quantitation based on RNA standards. ► Application on biological samples revealed that amplification of two HEV loci increased the likelihood of HEV RNA detection. ► The use of RNA internal amplification control facilitated monitoring of false negative results.

Introduction

Hepatitis E virus (HEV) is an important public health problem in many countries (Vasickova et al., 2005, Vasickova et al., 2007). The virus causes epidemics or smaller outbreaks most commonly in developing countries where the disease is usually associated with poor hygienic conditions and insufficient sanitation of drinking water. In industrialized countries, acute hepatitis E occurs sporadically. Due to the recognition of hepatitis E as a zoonosis, elucidating modes of HEV transmission is now an important issue. Risk factors for HEV infection include also direct contact with infected animals (especially domestic pigs, wild boar and deer), consumption of contaminated raw or insufficiently heat-treated meat, offal and shellfish (Meng, 2010a).

Currently, HEV is classified in the genus Hepevirus, family Hepeviridae (Carstens, 2010). The virus is non-enveloped and has icosahedral symmetry. Its genome contains single-stranded, positive-sense RNA, which consists of three partially overlapping open reading frames (ORFs; from 5′ to 3′ end ORF1, ORF3 and ORF2). ORF1 encodes a non-structural polyprotein, which is responsible for replication of viral particles and the diversity of the structural protein. The sole structural glycoprotein is encoded by ORF2 while a small phosphoprotein, which takes part in virus replication and virion morphogenesis, is encoded by ORF3 (Kabrane-Lazizi et al., 1999). The genome organisation of both human and animal strains is identical. Geographically distinct HEV isolates from mammals show a high degree of sequence similarity and currently segregate into four major genotypes. The relative conservation of genotypes 1 and 2 corresponds with their limited host range, primary circulation within humans and relation to epidemics in developing countries. In contrast, the diversity of genotypes 3 and 4 is related to their zoonotic origin from a variety of animal species. Distinct genotypes predominate in different geographical areas (Lu et al., 2006).

Due to the lack of an efficient cell culture system, the most common method for HEV detection is RT-PCR. Considering the heterogeneity of HEV strains circulating in humans and other animal species, many conventional RT-PCR and real time RT-PCR (qRT-PCR) systems have been developed (Erker et al., 1999, Jothikumar et al., 2006, Gyarmati et al., 2007, Ward et al., 2009). Despite the development of several PCR-based assays for detection of HEV, major problems regarding RNA quantitation based on plasmid DNA and identification of false or truly negative results remain. Due to the usage of plasmid DNA for quantitation, reverse transcription reaction (RT) as one of the crucial steps of qRT-PCR is dismissed. The accuracy of absolute quantitation depends entirely on the accuracy of used standards (Bustin and Mueller, 2005). According to Curry et al. (2002) the efficiency of RT is approximately 20%, but can vary within qRT-PCR systems and even qRT-PCR experiments. Therefore, the results of RNA quantitation based on DNA plasmids should not be taken into consideration. Many factors present in analysed samples have been shown to inhibit RT and PCR and these inhibitors can represent serious obstacles to accurate and reproducible quantitation of RNA or even for distinguishing between false and truly negative results. For that reason, the inclusion of an internal amplification control (IAC) such as in vitro transcribed RNA, armoured RNA or inactivated RNA virus is necessary (Fleige and Pfaffl, 2006).

The heterogeneity of viral genomes, especially of RNA viruses, affects the sensitivity and specificity of viral nucleic acid detection. Analyses of full-length genomes of various human and animal HEV strains revealed that the HEV genome could vary even in conserved regions. Therefore, the employment of more than one set of primers targeting different loci of the HEV genome increases the likelihood of its detection (Vasickova et al., 2009).

Based on this knowledge, the aims of this study were (i) to introduce and optimise a two-tube triplex qRT-PCR, which employs primers and probes targeting two different loci in the HEV genome, (ii) to prepare an RNA-based IAC to monitor the qRT-PCR assay and prevent possible false negativity, (iii) to prepare RNA standards to determine the limit of detection (LOD) of the triplex qRT-PCR assay and to quantify HEV RNA, and (iv) to verify the performance of the assay on samples originating from game animals using nested RT-PCR as the reference method.

Section snippets

Primers and probes for triplex qRT-PCR

To increase the likelihood of detecting different HEV strains by a single qRT-PCR reaction and with reference to Czech swine HEV (CZswHEV) sequences (Vasickova et al., 2009), two sets of published primers and probes were chosen. The forward primer JVHEVF (5′-GGTGGTTTCTGGGGTGAC-3′), reverse primer JVHEVR (5′-AGGGGTTGGTTGGATGAA-3′) and probe JVHEVP (5′-FAM–TGATTCTCAGCCCTTCGC-BHQ–1-3′) targeted the highly conserved 70 nt long sequences within overlapping parts of ORF3 and ORF2 of the HEV genome

Optimisation of the triplex qRT-PCR

Ten pmol of each primer per reaction led to primer-dimer interactions during the qPCR run and thus negatively affected qPCR efficiency. Therefore, the amount of primers had to be decreased to 4 pmol of the JVHEVR, JVHEVF, IS900qPCRR and IS900qPCRF primers and 6 pmol of the TqRev and TqFwd primers per qPCR. This decrease improved the qPCR efficiency and the LOD of the assay was not influenced in comparison with its monoplex formats even when higher amounts of primers were used. Non-specific PCR

Discussion

Amplification of multiple targets in the HEV genome can increase the sensitivity of detection (Vasickova et al., 2009). Therefore, a combination of several published (Jothikumar et al., 2006, Enouf et al., 2006, Zhao et al., 2007, Gyarmati et al., 2007), as well as sets of oligonucleotides designed “in-house” were tested on a cohort of Czech swine HEV isolates (Vasickova et al., 2009). The results (data not shown) revealed that the most specific and sensitive combination of primer sets are

Acknowledgements

We would like to thank Prof. Jiri Lamka (Charles University in Prague, Faculty of Pharmacy in Hradec Kralove) for the sample collection and Neysan Donnelly for grammatical correction of this manuscript. This work was supported by the Ministry of Agriculture (No. MZE0002716202), the Ministry of Education, Youth and Sports of Czech Republic (No. OC08045 and AdmireVet CZ 1.05/2.1.00/01.0006-ED0006/01/01) and by EC Grant Cost Action 929 ENVIRONET.

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