Selected RT-qPCR assays for detection of human noroviruses
Human noroviruses (hNoVs) and hepatitis A virus (HAV) are the leading cause of foodborne viral illness, and they exact a considerable economic and health toll worldwide. The detection of viral contamination in foods requires highly sensitive and accurate methods, due to the intrinsically low amounts of viruses and the complexity of the sample matrices. In recent years, a wide variety of nucleic acid-based molecular methods have been developed for the detection of hNoVs and HAV, displaying superior sensitivity, specificity, and speed. This chapter aims to provide a summary of the application of the molecular methods for the detection of the two important foodborne viruses.
- Human noroviruses
- hepatitis A virus
- foodborne viruses
- nucleic acid-based method
Enteric viruses have been increasingly recognized as the leading causative pathogens in foodborne disease outbreak, causing 66.6% of foodborne illnesses in the United States, compared with 14.2% and 9.7% for campylobacter and salmonella, respectively . A multitude of foodborne viral pathogens include (but are not limited to) human noroviruses (hNoVs), rotavirus, hepatitis A virus (HAV), hepatitis E virus, astrovirus, aichivirus, sapovirus, parvovirus, enterovirus, and adenovirus . Foodborne viruses are transmitted not only through contaminated food and water, but also in combination with close contact with infected individuals, aerosol contamination of projectile vomit, or through contamination of environmental surfaces. Potentially fecal-contaminated food, such as bivalve molluscan shellfish harvested in polluted water areas, fresh produce irrigated with contaminated water or harvested by an infected worker, and ready-to-eat foods prepared by an infected food handler are a means of infection [3—5]. Of all the foodborne viruses, hNoVs and HAV are the most important foodborne viral pathogens with regard to the severity of the associated illnesses and frequent occurrence worldwide . Both hNoVs and HAV display high environmental stability on contaminated objects, are abundantly excreted in human feces (e.g., exceeding 107 viral particles per gram of stool), and have a low infectious dose (1 to 100 infectious viral particles) , all of which contribute to the ease of transmission of the viruses within a community. It is commonly noted that one of the most efficient ways to prevent and control the foodborne viral infections is to implement a reliable surveillance system using rapid, sensitive, and precise diagnostics to identify the associated pathogens. Human NoVs do not replicate in cell culture. Wild-type HAV strains are not readily cultivated in vitro and the detection is impaired by their slow and inefficient growth in cell culture and lack of apparent cytopathic effect. Cell culture-based systems for determining virus infectivity are currently not available for hNoVs and wild-type HAV. Traditional diagnosis of these foodborne viral pathogens has been reliant on electron microscopy and immunological tests, but these methods lack sufficient sensitivity. While they may be useful for the detection of the viruses in clinical specimens that contain high amounts of viruses, for foods, which harbor potentially small quantities of viruses and may yet cause illness, it is not feasible to use these traditional laboratory methods to detect the viruses. This has led to the development of new, more sensitive and robust detection methods. In recent years, the majority of newly developed detection approaches are nucleic acid-oriented. Nucleic acid-based molecular methods have demonstrated a large improvement in speed, sensitivity, and accuracy of the detection of hNoV and HAV, bringing new insights into the etiology and diagnosis of foodborne viral disease. This chapter will touch upon a number of nucleic acid-based methods that have been developed and applied to detect the two epidemiologically important foodborne viruses.
2. Key notes from norovirus and hepatitis A virus
2.1. Norovirus genome and molecular diversity
Although “winter-vomiting disease” was described in 1929 , the responsible viral agent was not discovered until 1972 by Kapikian  from fecal materials derived from an outbreak of gastroenteritis among elementary school children in 1968 in Norwalk, Ohio. The virus was named Norwalk virus and was designated as the prototype strain for the group of viruses now called Noroviruses; in the literature, they were previously referred to as small round-structured viruses (SRSVs) by their surface morphology or Norwalk-like viruses. The Norwalk agent was the first enteric virus identified that specifically caused acute gastroenteritis in humans. Successful cloning and sequencing of the NoV genomes have led to progress in understanding viral genome organization and classification. Noroviruses are a member of the family Caliciviridae whose name is derived from the Greek word Calyx for cup . The viral genome is composed of a single strand of polyadenylated positive-sense, non-enveloped RNA size of approximately 7.6 kb. The linear RNA is organized into three open reading frames (ORFs), designated ORF1, ORF2, and ORF3. ORF1 encodes a 194-kDa protein that is cleaved by the viral cysteine protease into six non-structural proteins including p48, NTPase, p22, Vpg, protease, and RNA-dependent RNA polymerase. ORF2 codes for the major capsid protein VP1, which folds in two major domains: a shell (S) and a protruding (P) domain. The P domain is comprised of P1 and P2 subdomains. P2 is a highly variable region that is thought to be involved in the binding of the histo-blood group antigens (HBGAs), which are regarded as receptors and host-susceptibility factors for human infection [8, 9].
According to the literature, NoVs have been genetically segregated into at least five genogroups (GI, GII, GIII, GIV, and GV) based on the complete amino acid sequences of the major capsid protein of 164 NoV strains . New genogroups consisting of canine NoVs have also been proposed [11-12]. GI, GII, and GIV are known to cause gastroenteritis in humans. GI and GII contain the majority of human strains, with GV viruses regarded as uncommon human pathogens. GIII viruses were identified in cow, while GV viruses infect mice. Due to their enormous genetic diversity, the viruses within genogroups can be further classified into genetic clusters or genotypes that are defined as containing 14—44% VP1 amino acid sequence difference, where strains have 0—14% difference . Accordingly, there are currently nine recognized genogroup I clusters and 22 genogroup II clusters. Despite their great molecular diversity, Genogroup II, genotype 4 (GII.4) variants have been responsible for the majority of outbreaks and cases in recent years, particularly those associated with person-to-person transmission [13—14]. The genotype GII.4 was first identified to predominate in outbreaks of gastroenteritis in the mid-1990s in countries on five continents , and new emerging variants have continued to evolve since then and have become the etiological agents for each of the four global gastroenteritis epidemics . Although the majority of reported NoV outbreaks and cases are derived from person-to-person transmission, it is estimated that approximately14% of them are attributed to food, and 37% of the foodborne outbreaks are caused by mixtures of GII.4 and other genotypes, 10% by all genotype GII.4, and 27% by all other single genotypes .
2.2. HAV genome and genotypes
HAV, first identified in 1973 by electron microscopy, is the most common cause of infectious hepatitis with annually causing about 1.4 million clinical cases and 200 million asymptomatic carriers worldwide . HAV is one of the most frequent causes of foodborne viral infection. In the United States, it is estimated that approximately 270,000 people become annually infected with hepatitis A, and most of the infection cases are not reported to health authorities . Epidemics associated with contaminated food or water can occur involving hundreds of thousands of people, such as the epidemic in Shanghai, China in 1988 affected almost 300,000 people due to the consumption of HAV-contaminated clams . Like other enteric viruses, HAV is resilient to environmental stressors. The virus is able to retain infectivity in acidic environments below pH3, and after refrigeration and freezing. HAV is a non-enveloped positive single-stranded RNA virus with a genome of approximately 7.5 kb in length. The virus is classified within the genus Hepatovirus of the family of the Picornaviridae . The viral genome consists of (i) a 5´-untranslated region (5´-UTR) of about 735 nucleotides; (ii) a single open reading frame (ORF) that is organized into three functional regions termed P1, P2, and P3; (iii) 3´-untranslated region (3´-UTR) with a polyadenylated A tail . The P region encodes the viral capsid polypeptides VP1—VP4, and the P2 and P3 regions encode the non-structural protein. The 5´-UTR is the most conserved region of the genome and therefore is favored for primer design in polymerase chain reaction (PCR) to detect most genotypes. HAV displays a high level of antigenic conservation throughout the viral genome. An immunological study identified the existence of a single human serotype of HAV , but ample genetic diversity still exists to classify HAV into six genotypes based upon differences of a 186-bp nucleotide sequence in the VP1—P2A junction region [24—26]. Genotype I, II, and III are associated with human infection, while genotype IV, V, and VI are found in simians . A genotype VII, designated SLF88, was proposed in an earlier study , but further analysis of the complete genome and capsid region of additional strains indicated that the genotype VII should be reclassified as genotype IIB [25, 26]. Genotype I and III can be each further divided into subgenotypes IA, IB, IIIA, and IIIB. Genotype I that comprises 80% human HAV strains studied is remarkably prevalent around the globe; subgenotype IA is more common than IB . Since genotype I predominates worldwide, genotyping alone is rarely used to determine the source of a chain of HAV transmissions or outbreaks. Genotype III includes most of the remaining human HAV strains. Genotype II contains two subgenotypes: IIA and IIB.
3. Nucleic acid-based detection
3.1. Direct nucleic acid probe hybridization
Direct nucleic acid probe hybridization was the first molecular technique developed for the detection of suite of enteric viral pathogens. This technique can be used in diagnostics in several major formats: solid-phase, solution or liquid-phase, and in situ hybridization. In hybridization assays, oligonucleotide probes (single-stranded RNA or cDNA) that are complementary to the target genomic sequence of interest were labeled with signal reporters, which include radioactive molecules, chemiluminescence, or fluorescent agents. After hybridization, the probe signal from the reporter can be visualized via radioactivity, fluorescence, or color development. Detection of the probe signal indicates the presences of nucleotide sequences of interest that have high sequence similarities to the probe. HAV has been detected using these techniques such as dot blot hybridization [28—30] and in situ hybridization . Dot blot hybridization assays were also used for detection of Norwalk viruses in 55 stool specimens from human volunteers with 27 samples tested positive . A potential advantage of the direct hybridization technique is the low cost of the assay and decreased risk of cross-contamination . However, the disadvantage is that the detection sensitivity is often low (approximately 104 virus particles) , thus limiting its practical application in detecting low numbers of viruses in clinical specimens, food, and environmental samples.
3.2. Nucleic acid amplification
Despite a number of reports describing the use of the direct probe hybridization technique, new molecular detection methods that incorporate the amplification of target nucleic acids are now being developed and predominantly used for the detection of foodborne viral pathogens in samples of different origins. Nucleic acid amplification offers an edge over direct probe hybridization by enhancing the detection sensitivity through amplifying target nucleic acids extracted from samples.
3.2.1. Reverse transcription-PCR
Since the first demonstration of the PCR process in 1985, this technology has been widely used for the detection of foodborne pathogens. As RNA cannot be directly used as a template for PCR amplification, reverse transcription-PCR (RT-PCR) is employed for the amplification of viral RNA. RT-PCR can generally be carried out either in a one-step or two-step format. One-step RT-PCR combines the first-strand cDNA synthesis reaction (reverse transcription) and PCR amplification in a single tube, minimizing reaction setup and risk of carryover contamination. Alternatively, the two-step RT-PCR starts with the reverse transcription of either total RNA or poly (A) RNA into cDNA using a combination of sequence-specific primers, oligo (dT) or random primers in the presence of reverse transcriptase. The resulting cDNA then serves as a template for the initiation of PCR amplification in a separate tube. Separation of reverse transcription and PCR processes allows greater flexibility when choosing primers and polymerase than the one-step RT-PCR system, which allows for the use of sequence-specific primers only.
The first RT-PCR assays for detecting NoVs were described within two years of the successful cloning and sequencing of the Norwalk virus genome in the early 1990s [32, 35]. Application of this technology has allowed the detection of NoVs from samples of different origins and has generated a great deal of sequence information on various NoV strains. The sensitivity and specificity of RT-PCR assays are strongly associated with primer design. RT-PCR tests to detect NoVs are challenged by the high molecular diversity of the viruses since new variant strains continue to evolve incessantly . It is difficult to select a single oligonucleotide primer set with sufficient sensitivity and specificity to detect all the NoV strains . Different primer sets targeting multiple regions of the viral genome have been designed and evaluated in RT-PCR assays. A highly conserved RNA-dependent RNA polymerase region has been favored for primer design and amplification ([37—39]. Other regions such as capsid region, 2C helicase and ORF3 regions have also been targeted for amplification [40—43]. In addition, RT-PCR assays using different primer combinations in nested (two primers) or semi-nested (one primer) format have been performed to increase the likelihood of NoV detection [44, 45]. It has been reported that higher detection sensitivity (10 to 1,000 times more sensitive than single round RT-PCR) has been achieved by implementation of this strategy .
HAV was one of the first enteric viruses for which RT-PCR assays have been developed . In contrast to NoVs, many human HAV strains across different genotypes can be amplified using a single pair of primers targeting genes coded for structural proteins, e.g., VP1—2A and VP3—VP1 junction regions [47, 48]. 5´-untranslated region (5’UTR) primers were also used in RT-PCR assays to detect HAV from clinical and environmental samples .
After amplifying a target of interest, post-amplification analysis is necessary to interpret the results. The simplest method is to run the amplified products on ethidium bromide- or SYBR Green dye-stained agarose gels. A band that is of the expected size to that of the positive control and/or molecular weight markers is considered a positive result. However, this method does not provide additional reassurance as to the specificity of the amplification. Analysis of the RT-PCR products by restriction fragment length polymorphism (RFLP) could discriminate genetic variants of HAV of different origins; this resolution has been enhanced by combining with the results of single-strand confirmation polymorphism (SSCP) analysis . PCR-SSCP has been considered as a rapid and cost-effective approach to examining genetic diversities among hNoV strains  as well as HAV [51—53]. Hybridization assays such as dot blot and Southern blot have been used to identify and confirm NoV- and HAV-specific amplicons [32, 54—57]. The reverse line blot hybridization method was utilized to detect NoV RT-PCR products and to genotype the virus strains . DNA microarray technology has been used to analyze amplified products for detecting and genotyping hNOVs and HAV discussed in the section below. Direct sequencing of amplified products provides detailed molecular information not only for confirming the specificity of the amplicons but also for classifying or subtyping virus strains .
3.2.2. Quantitative real time RT-PCR (RT-qPCR)
A RT-qPCR system combines amplification of target nucleic acids with amplicon detection in the same reaction tube, eliminating the necessity of further post-amplification analysis. RT-qPCR is currently the method of choice for the detection of hNoVs and HAV in many molecular diagnostic laboratories. This method has become the gold standard for quantification of viral load based on a reference standard curve. Two principle approaches are commonly utilized for the detection of enteric viruses in RT-qPCR assays: DNA-binding fluorogenic dyes and sequence-specific oligonucleotide probes. Selected application of these approaches for detection of NoV and HAV is outlined in Tables 1 and 2.
SYBR Green is commonly used as a dye for the quantification of double-stranded DNA in qPCR methodology. The binding of SYBR Green is non-specific; it binds indiscriminately to any double-stranded DNA including non-specific amplification and primer-dimers. To distinguish virus-specific amplified products from non-specific primer-dimers, a melting curve analysis is generally used as the virus-specific products have a higher dissociation temperature. A number of SYBR Green-based RT-qPCR assays have been developed for the detection of hNoVs by targeting different genomic regions such as the capsid region [60, 61] and RNA polymerase [61—64]. SYBR Green-based RT-qPCR employing 5´—UTR region primers was used to detect as low as 5 Tissue Culture Infectious Dose 50% (TCID50) per gram in seeded oyster samples . Using SYBR Green RT-qPCR with VP3—VP1 junction primers, HAV could be detected in all eight tested ocean water samples with viral loads varying from 90 to 3523 HAV copies/L near the mouth of Tijuana River, and 347 to 2656 copies/L near the Imperial Beach pier in San Diego .
|Approach||Genogroup||Target Region||Lmit of Detection||Detection/ Quantitation||Sample tested||References|
|SYBR Green||Not reported||RNA polymerase||Not reported||D||Stool|||
|GI||Capsid||1 RT-PCR unit||Q||Stool|||
|Not reported||RNA polymerase||Up to 5 logs||D||Stool|||
|GI/GII||Capsid in GI/|
RNA polymerase in GII
|25,000 RNA copies/gram||D||Stool|||
|Not reported||RNA polymerase||6 log GII cDNA copies/100ml||Q||Enviromental water|||
|TaqMan probe||GI/GII||ORF1-ORF2 junction||2.0x10∧4RNA||Q||Stool|||
|GI/GII/GIV||ORF1-ORF2 junction||<10GII, <100GI RNA copies||Q||Stool/water|||
|GI/GII||Capsid||50 GII copies, 500 GI copies||Q||Stool|||
|GI/GII/GIV||ORF1-ORF2 junction||16.9GI, 6.3GII, 43GIV copies||Q||Stool, vomitus, anal swab|||
|GI/GII||ORF1-ORF2 junction||23GI, 33GII copies||D||Shellfish & springwater|||
|GI/GII||Capsid||3~7 RT-PCR unit||Q||Strawberry|||
|Hybridization probe||GI/GII||ORF1-ORF2||50 copies||D||Stool|||
|GI/GII||ORF1-ORF2||1~10GI, 1~100GII RNA copies||D||Stool|||
|Approach||Genotype||Target Region||Lmit of Detection||Detection/ Quantitation||Sample tested||References|
|SYBR Green||Not reported||VP3-VP1||2-4 log copies/l||D||Ocean water|||
|Not reported||5’-UTR||5 TCID50/g||Q||Oyster|||
|TaqMan probe||IA, IB, IIA, IIIA||5’-UTR, VP1-2A, VP3, VP1, VP2-VP3||50 copies for IIB, 500 for IA, IB, IIA and IIIB||Q||Stool, serum|||
|IA, IB||5’-UTR||10 copies or 0.05||Q||Shellfish, stool|||
|IA, IB, IIA, IIB, IIIA||5’-UTR||40 copies or 0.5||D||Cell culture|||
|IB||5’-UTR||491 copies||Q||Shellfish, springwater|||
|IB||5’-UTR||0.2 PFU, 63PFU||D||Cell culture, green onion|||
|Molecular beacons||IB||5’-UTR||20 PFU||D||Seeded groundwater|||
There are two major groups of sequence-specific oligonucleotide probes: hydrolysis (e.g., TaqMan) probes and hybridization probes (e.g., molecular beacons and fluorescence resonance energy transfer probes); both groups are homologous to the internal region of amplified products. TaqMan probes have been frequently used in RT-qPCR for detecting hNOVs [67—73]. While RNA polymerase and capsid genes are the primary targets for amplification, within the NoV genomes, a junction of ORF1—ORF2—polymerase—capsid has been demonstrated to be the most highly conserved region that can serve as an effective target for amplification. Kageyama and colleagues were the first to describe the junction-targeting RT-qPCR assay . Their studies showed a better detection rate in 80 of 81 (99%) stool samples positive by electron microscopy, compared to conventional RT-PCR assays that detected 77% when targeting the polymerase and 83% when targeting the capsid N/S regions, respectively, in the same panel of stool specimens . In an effort to design assays capable of detecting all genogroups of these highly diverse viruses, this ORF1—ORF2 junction region has become the most widely used for primer and probe design in RT-qPCR tests. A TaqMan RT-qPCR assay using a probe for the 5´-UTR of HAV genome was able to detect 40 copies of RNA transcripts and 0.5 infectious units (IU) in cell culture strains and clinical fecal specimens, respectively . Constafreda et al. (2006) also developed a TaqMan RT-qPCR method targeting 5´-UTR for quantitative detection of HAV from clinical specimens (stool and serum) and shellfish samples, and the detection limit was 0.05 IU or 10 copies of single-stranded RNA transcripts . Nested RT-PCR assays combining conventional PCR, nested PCR and qPCR have been used to detect as low as 0.02 plaque forming units (PFU) of HAV from cell culture and 63 PFU from green onions . Six subtype-specific RT-qPCR assays using hydrolysis probes were developed for HAV detection and subtyping , with limit of detection at 50 genome copies/assay for subtype IIB, 500 genome copies/assay for IA, IB, IIA, and IIIB, and 5,000 genome copies/assay for IIIA. Thirty-five clinical stool and serum specimens were tested with this method. Only a single discrepant result was observed for a serum specimen provided as IB subtype by VP1/2A region sequencing and identified as IA by the subtype-specific RT-qPCR assays.
Hybridization probes including molecular beacons and fluorescence resonance energy transfer (FRET) probes have been used in RT-qPCR assays for detecting NoVs [78, 79]. Abd El Galil et al. showed that a molecular beacon RT-qPCR assay targeting the HAV highly conserved 5´-UTR region could detect as lower as 20 PFU HAV in seeded groundwater samples in combination with immunomagnetic separation . Molecular beacon probes have also been used in another target amplification method: nucleic acid sequence-based amplification described below.
3.2.3. Multiplex PCR
Multiplex PCR-based methods are designed to simultaneously amplify more than one target nucleic acids using different sets of primers in a single reaction. Primers of different targets should not be complementary to each target and can work efficiently at the same annealing temperature during PCR.
A multiplex RT-PCR method was developed for simultaneous detection of HAV, norovirus, and poliovirus type (PV1) using three different sets of primers with detection limits of ≤ 1 infectious unit (HAV and PV1) and 1 RT-PCR unit (NoV) . Noroviruses (GI and GII) and HAV from different food matrices such as lettuce, strawberry, green onions, and bivalve mollusks were detected using a multiplex RT-qPCR assay [82, 83]. A multiplex RT-qPCR for simultaneous detection and quantification of GI, GII, and GIV noroviruses was recently reported . Several multiplex nucleic acid diagnostic platforms for simultaneous detection of a range of pathogenic enteric pathogens including hNoVs are now commercially available . Although multiplex PCR allows rapid and cost-effective detection of several targets (viruses) in a single reaction, different targets may compete with each other for resources such that a highly abundant target would get more chances to be amplified, thus preventing less abundant ones from getting detected. In addition, multiplex PCR tend to have decreased sensitivity as compared to standard single PCR.
3.2.4. Digital PCR
Digital PCR has recently been described as a novel approach to nucleic acid detection and quantification. It is a different method of absolute quantification relative to conventional quantitative PCR, because it directly counts the number of target nucleic acid molecules rather than relying on reference standards. This improvement is achieved by partitioning sample amplification reactions into tens to thousands of picoliter-scale compartments on microfluidic chips or microdroplets so that each mini-reaction contains zero or one copy of the target nucleic acid molecule. A comparative study of digital RT-PCR (RT-dPCR) and RT-qPCR for the detection and quantification of HAV and NoVs in lettuce and water samples was recently reported . This RT-dPCR assay showed that the sensitivity was either comparable or slightly (around 1 log10) decreased to that of RT-qPCR for detecting viral RNA and cDNA of HAV and NoV, but the viral recoveries were found to be significantly higher than that of RT-qPCR for NoV GI and HAV in water, and for NoV GII and HAV in lettuce. In addition, this RT-dPCR was more tolerant to inhibitory substances present in lettuce.
3.2.5. Nucleic Acid Sequence-Based Amplification (NASBA)
Nucleic acid sequence-based amplification is an isothermal target amplification process for amplifying RNA. A NASBA reaction consists of reverse transcriptase, T7 RNA polymerase, and RNase-H with two target sequence-specific oligonucleotide primers. A NoV NASBA assay showed equivalent analytical sensitivities with RT-PCR using the NoV GII primer sets described by Kageyama et al  but provided less consistent signals . A molecular beacon real-time NASBA method was developed to detect NoV GII from environmental samples, with 88% sensitivity compared to conventional RT-PCR . This NASBA technology has been applied to detect as little as 0.4 ng of HAV RNA/ml using primers targeting the VP1 and VP2 capsid genes . A multiplex NASBA and microtiter plate hybridization system was developed to simultaneously detect HAV and rotavirus where 400 PFU/ml HAV were detected with reduced time and cost compared to monoplex system . Using established primer pairs, multiplex NASBA assays were developed for simultaneous detection of HAV and NoV GI and GII in spiked ready-to-eat foods. All three viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) RT-PCR units .
3.2.6. Reverse transcription loop-mediated isothermal amplification (RT-LAMP)
RT-LAMP is a one-step non-PCR nucleic acid amplification that is performed at a constant temperature between 60 and 65°C. Unlike NASBA, it requires only two enzymes instead of three, namely, reverse transcriptase and DNA polymerase. A genogroup-specific RT-LAMP assay has been developed using 9 and 13 specially designed primers containing mixed bases for genogroup I (GI) and II (GII), respectively, and showed the limits of detection between 102 and 103 copies/tube for GI and GII. Compared to conventional RT-PCR, the clinical sensitivity and specificity of the RT-LAMP were 100% and 94% for GI, and 100% and 100% for GII, respectively . Commercial loopamp NoV GI and GII detection kits were evaluated using 510 clinical fecal specimens; the sensitivity of GI (83.3%) was less than that of GII (97.4%) with regard to genogroup-specific RT-qPCR . A single tube, real-time HAV RT-LAMP assay using seven primer sets was applied to identify three different subgenotypes of HVA (IA, IB, and IIIB) with detection limits of 0.4—0.8 focus forming units per reaction .
3.3. Nucleic acid amplification coupled with DNA microarray detection
DNA microarray technology consists of numerous individual target-probe hybridization reactions that are performed in a single assay. The intrinsic ability of this technology to simultaneously analyze thousands of specific DNA sequences presents a significant advantage in parallel identification of a broad spectrum of microbial pathogens. Additionally, a DNA microarray composed of well-designed probes has the potential to discover novel viruses or pathogens not well-represented in the current sequence database. Not surprisingly, this technology, coupled with virus-specific monoplex or multiplex RT- PCR amplification, has enabled sensitive detection and identification of a number of enteric viruses including hNoVs and HAV from clinical specimens, environmental samples, and virus-infected cell cultures [95—100]. However, unbiased amplification with virus-specific PCR is often complicated by the existence of enormous genetic diversities in foodborne viruses. It would be advantageous to develop amplification approaches that do not rely on specific pathogen sequence information, yet can produce sufficient target nucleic acids from minute amounts of starting materials of viral, bacterial, plant, and animal origins for microarray analysis . Recently, a sequence-independent isothermal RNA amplification approach has been developed to amplify various enteric viral nucleic acids of hNoVs, HAV, and coxsackievirus for microarray analysis . Utility of this microarray platform and amplification strategy allows not only discerning genotypic information on hNoVs but also detection of mixed viral agents (hNoVs and HAV) present in the same fecal specimen . Microarray detection of random-primed PCR products from a range of gastrointestinal viruses including Norwalk virus was reported recently .
3.4. Detection of infectious viruses
As mentioned above, there are no efficient cell culture systems available for hNoV propagation, and wild-type HAV strains are difficult to grow in vitro. In absence of effective culture-based infectivity assays, development of rapid and sensitive molecular methods for reliably detecting infectious vial particles to determine virus infectivity is a key issue for the application of food risk management. Integrity of the virus capsid and its genome are essential for virus infectivity; both have been targeted for the development of methods for predicting virus infectivity. Nuanualsuwan and Cliver (2002) described a method in their effort to correlate RT-PCR data with virus infectivity . In their study, HAV, vaccine poliovirus, and feline calicivirus were inactivated by ultraviolet light, hypochlorite, or heating at 72°C. They observed that the inactivated viruses, which were treated with proteinase K and ribonuclease before RT-PCR, did not yield positive amplicons . Integrated RT-qPCR approaches have been used to discriminate the infectivity status of NoVs based on the assumption that infectious virus particles would more efficiently bind to the appropriate receptors than non-infectious viruses [106, 107]. Long-range RT-qPCR has been used to test the integrity of the NoV genome following 72°C heat treatment . Recent studies on the use of nucleic acid intercalating dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA) in conjunction with RT-PCR or RT-qPCR to distinguish between infectious and non-infectious enteric viruses including hNoVs and HAV have been reported [109—111]. However, PMA RT-PCR could not differentiate infectious Norwalk virus from non-infectious Norwalk virus, although it was able to differentiate selectively between infectious and noninfectious murine NoV, coming to the conclusion that PMA RT-PCR can be used to detect intact, potentially infectious viruses only under specified conditions [108, 110]. A real-time NASBA combined with enzymatic treatment of proteinase K and RNase has been developed to discriminate the infectious from the heat-inactivated hNoVs .
4. Concluding remarks
Over the past few years, nucleic acid-based molecular methods have been developed, refined, and used for the detection of hNoVs and HAV that are most commonly associated with the transmission of foodborne viral disease. In particular, RT-qPCR has emerged as a preferred method for the detection and quantification of the viruses due to its high sensitivity, reproducibility, speed, and minimization of risk of carry-over contamination. Recent advances in high-throughput next-generation sequencing (NGS) technologies have opened new avenues for genomic research and diagnostic applications. It is expected that utilization of NGS in viral metagenomics and whole genome sequencing will highly improve the opportunities for identifying viruses of different origins including those that are too divergent to be detected by PCR or other molecular approaches. However, the identification of hNoVs and HAV in vast ranges of food matrices is still a demanding task that is largely attributed to some factors such as intrinsically low quantities of contaminated viruses and broad chemical composition of food, which may inhibit the activity of enzymes used in the molecular detection. This requires a meticulous investigation into sample preparation procedures to obtain acceptable recovery of viral RNA for downstream analysis. Nucleic acid-based molecular methods have a disadvantage in that the majority of the reported assays for detection of foodborne virus contamination do not have standardized protocols, and can vary from laboratory to laboratory. This could be due to several reasons, such as different approaches required for preparation of viral nucleic acids from different test matrices, and a lack of multi-laboratory validation of promising procedures. There is a need for harmonized standards and quality control of the reagents used. Moreover, in many cases, molecular methodology has focused on merely detecting the presence of viral nucleic acids that is not necessarily associated with the detection of infectious particles, although some studies stated above have shown promising differentiating results. Research on developing new methods to accurately determine the virus infectivity is eventually needed to gauge health risk.
The author is grateful to Marianne Solomotis and Michael Kulka for reading this manuscript.