The emergence of hepatitis E virus (HEV) as a zoonotic pathogen which may be transmitted by foods, especially through the pork supply chain, has resulted in a great deal of research being carried out by food and veterinary virologists across the globe to establish where links exist and how we may quantify the risks posed by the virus, and in developing methods to detect the virus in our foods [1]. In equal measure, there has been much concern from the food industry, especially the pork supply industry, as sporadic outbreaks of hepatitis E that are not travel related have been epidemiologically linked to food items. Ad hoc testing of retail pork-based products in various countries has resulted in a flurry of media activity, often negative in nature, with consequences that have had an impact on political and economic exportation policies [2].

There has been debate as to whether detection for HEV should be quantitative or qualitative and, as seen for the extraction procedures, a number of molecular methods have also been published for detection. Methods vary from detection of specific genotypes to pan-detection methods, and there is also variation in the controls used to validate the assays. Initially, most assays were designed to detect HEV in clinical samples and have been then taken forward for detection in food. This section focuses on PCR-based methods observed in the literature as this is the most likely approach.

While investigating the sero-prevalence of HEV in the south-west of France, Mansuy et al. (2004) [17] developed an assay, using the TaqMan format, with primers targeted against open reading frame (ORF)2 sequences. Although not stated directly, the assay was apparently universal for HEV. The assay was used to detect HEV in patients’ serum samples; subsequent sequencing was used to characterise the virus as gt3. Although no information on calibration was given, the limit of detection (LOD)was reported as an estimated 1 × 10³ copies mL⁻¹ serum. Similarly, in 2006, three publications described similar assays; a universal HEV assay using the same format as above and targeting ORF 2 sequences was developed by Enouf et al. [18] and Ahn et al. [19], and by Jothikumar et al. [20], who developed a universal RTPCR assay, targeting ORF3 sequences for clinical analysis, and quantitation was performed with DNA using a plasmid construct.

Quantitation using RNA calibration standards was performed by Ahn et al. [19] where HEV ORF2 sequences were cloned into a plasmid, which was then transcribed in vitro to produce cDNA with a detection limit of 168 genome equivalents (GE; this term is used to take into account the possibility that RNA fragments containing the primer sequences can be detected, and not always whole genomes).

The main difference here was that the assay by Jothikumar et al. [20] demonstrated specificity using a panel of non-HEV strains. None of these four assays included an AC.

These four assays were subsequently evaluated by Ward et al. [21], using a panel of pig faecal and serum samples. The assay of Jothikumar et al. [20] was found to be the most effective, detecting HEV in more samples than the other assays, and with greater sensitivity as judged by lower Ct values, suggesting the potential for this assay to be used in HEV detection in food.

For improved detection and to include a SPC, Ward et al. [21] merged the Jothikumar et al. (2006) assay with an in-house assay for feline calicivirus (FCV) to produce a multiplex RTPCR. They used feline calicivirus (FCV) as both a SPC and a heterologous IAC, adding virus particles to samples prior to NAA extraction. Plasmid standards were used to calibrate the multiplex assay. This assay was subsequently used to detect HEV in organs (including liver and muscle) and tissues of pigs at slaughter [22]. No HEV was detected in muscle samples, but the virus was detected in liver. Quantitative data was obtained.

To further determine the usefulness of PCR in HEV detection in food, in a survey of pork chops and pork livers sold at retail outlets in Canada, Wilhelm et al. [23] used the assay of Ward et al. [21] following the application of separate sample preparation procedures for each food type. For liver, 312 mg portions were added to a commercial lysis buffer and homogenised with ceramic beads. After centrifugation, 4 mL supernatant was used for NA extraction by commercial kit. The procedure for pork chops was similar except that after centrifugation, proteinase K digestion was performed, followed by an additional round of centrifugation prior to NA extraction. Approximately 57% of livers analysed had HEV nucleic acid detected, with no chops testing HEV-positive. Wilhelm et al. [24], revisiting the data from [23], considered that the likelihood of HEV detection was greater with liver than with pork chops, probably due to the latter being a more difficult matrix to process.

At a similar time, the assay of Kaba et al. [25], targeting ORF2 sequences, was developed to detect transmission in piglets and was used by Colson et al. [26] to detect HEV in figatellu, a sausage made from raw pig liver. In contrast to the study by Wilhelm et al. [23] the assay was not quantitative and no AC or SPC was included.

Detection of HEV in animal products became more prevalent in the literature with the main aim to detect HEV gt3, most commonly found in pork products. In addition, assays were improved to include relevant controls to validate the data.

HEV was detected in samples of pig liver sold at retail outlets in Germany by Wenzel et al. [27], using the RTqPCR assay described by Jothikumar et al. [20] using the human coxsackievirus B added to the sample prior to homogenisation as SPC, but did not include an AC control. An additional sample treatment was employed by Wenzel et al. [27] to include the testing of a capsid integrity-based RTqPCR approach to determine potential virus infectivity and as a comparison to straightforward NAA detection as previously described [28]. The detections of HEV in the two studies were broadly in accordance, but more evaluation of this approach is necessary before it can be used with confidence for suggesting infectivity; meanwhile, the caveat discussed above regarding infectivity assays remains pertinent.

Diez-Valcarce et al. [29] proposed the use of murine norovirus (MNV) as a suitable SPC for analysis of foods for enteric viruses, and it was incorporated in the fully controlled method for HEV detection in pork liver and muscle samples; this method was used in the studies of Di Bartolo [30] and Berto et al. [31] both of which used the sample treatment procedure of Bouwknegt et al. [32]. In each study, the assay of Jothikumar et al. [20] was utilized, and was modified by the use of an RNA IAC, as designed by Diez-Valcarce et al. [33]. No quantitation was performed in either study. Subsequent studies by Di Bartolo et al. [34] and De Sabato et al. [35] used the same method, but the assay was calibrated using plasmid-transcribed RNA sequences identical to those of the HEV target sequences to produce fully quantitative results. Garcia et al. [36] modified the sample treatment procedure by incorporation. of a commercial phenol:chlororm based reagent prior to chloroform extraction; they calculated the extraction efficiency of this procedure as being approximately 50%.

A triplex qRTPCR assay, incorporating the primer sets of Gyarmati et al. [37] and Jothikumar et al. [20], and a heterologous IAC, was used as the basis of a method to detect HEV in wild boar and mouflon [38]. Sample treatment was similar to that of Bouwknegt et al. [32]. RNA standards were used for quantification, and the LOD of the assay was determined to be 50 GE. Son et al. [39] also used the assay of Jothikumar et al. [20] for HEV detection in pig liver and quantified the data using RNA standards. No SPC or AC was reported. No LOD was reported.

A survey of foods containing raw pork liver was carried out in France by Pavio et al. [40]. The foods included figatellu, dried liver, dried and fresh sausages, and liver paste (quenelle), and HEV was detected in samples of each type. No SPC was used and, again, the assay of Jothikumar et al. [20] was employed, as modified by Barnaud et al. [41] to include an EAC consisting of HEV RNA. Quantitative data were reported, and as the assay was calibrated using HEV RNA, these can be regarded as accurate. The assay of Barnaud et al. [41] was subsequently used in a survey of pig liver and meat obtained at French slaughterhouses [42], and to detect HEV in the muscle juice of experimentally infected pigs [43].

Comparing nine in-house sample treatment procedures for detection of HEV in pig liver sausages, Martin-Latil et al. [44] selected as optimal a process based on the use of polyethylene glycol (PEG) as a virus particle flocculant. Artificially contaminated food samples were used as test materials. MNV was used as SPC. qRTPCR was performed using the duplex HEV/MNV assay of Martin-Latil et al. [45] using HEV RNA calibration standards, MNV acting as both SPC and a heterologous IAC. The sample treatment procedure of Martin-Latil et al. [44] was employed to facilitate quantitative detection of HEV in liver samples using a subsequent digital RTPCR assay (Martin-Latil et al. [46].

An evaluation of virus extraction procedures was performed by Hennechart-Collette et al. [47]. Six procedures involving permutations of sample size, homogenisation liquid (distilled water; dH₂O) volume, and homogenisation techniques were examined, using pork liver, sausage and figatellu that had previously [44] been found to be HEV-contaminated. The qRTPCR of Jothikumar et al. [20], quantified using RNA standards, was used to evaluate the effectiveness of each procedure by comparing the GE copy numbers obtained.

Szabo et al. [48] reported that a sample treatment based on the use of a commercial phenol:chloroform-based reagent gave a better recovery (4.90%) of HEV in artificially contaminated pork products than the procedures of Colson et al. [26], Di Bartolo et al. [30] and Martin-Latil et al. [44]. The RTPCR assay of Jothikumar et al. [20], with quantitation using RNA standards, was employed to detect viral RNA. LODs of the final method were reported as 2.9 × 10³ GE/5 g raw sausage and 5.3 × 10⁴ GE/2 g liver sausage. The method was applied to the analysis of liver sausages and raw pork sausages purchased at retail. MS2 bacteriophage was added to the food samples before processing. MS2 was detected in a separate RTPCR to HEV, thus functioning as both SPC and heterologous EAC. The method of Szabo et al. [48] was subsequently used in Swiss surveys that detected HEV in local salami-type sausages made from raw cured pig or game liver and meat [49,50]; the LOD was determined as being 1.56 × 10³ and 1.56 × 10² per g of liver sausages and raw meat sausages, respectively [49]. The LOQ of the Szabo et al. [48] method was determined as 3.15 log GE/g [51], although this was performed using plasmid DNA and is therefore not likely to be accurate. The Szabo et al. [48] method was evaluated in a ring trial involving nine German and Swiss laboratories using artificially contaminated liver sausage as test material, and shown to be highly repeatable and reproducible. It was reported [52] that significantly higher recoveries of internal control swine mitochondrial sequences could be obtained with the method of Szabo et al. [48], by increasing the intensity and time of the bead-beating step.

A relatively simple sample treatment procedure was used by Boxman et al. [53] in a method to detect HEV in porcine blood products used as food ingredients. Again, the assay of Jothikumar et al. [20] was used, with a standardised HEV RNA oligonucleotide [34] as EAC. Quantitation was performed with DNA standards. The method was subsequently used to detect HEV in pork liver, meat, and pate, and in wild boar meat samples [54].

Using a sample treatment based on vacuum filtration, Mykytczuk et al. [55] analysed pork products including pate, sausages, and liver for HEV. Following an in-house conventional PCR to screen for HEV-positive samples, digital droplet RTPCR (ddRTPCR) was performed using the primer/probe set of Jothikumar et al. [20] to quantify the viral load. The efficiency of detection was calculated using recovery of the SPC, and was found to be at least 1% in the majority of samples tested.