*3.2.1. Reverse transcription-PCR*

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 [23], 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 [27]. A genotype VII, designated SLF88, was proposed in an earlier study [24], 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 [24]. 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

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 report‐ ers, 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 [31]. Dot blot hybridization assays were also used for detection of Norwalk viruses in 55 stool specimens from human volunteers with 27 samples tested positive [32]. A potential advantage of the direct hybridization technique is the low cost of the assay and decreased risk of cross-contamination [33]. However, the disadvantage is that the detection sensitivity is often low (approximately 104 virus particles) [28], thus limiting its practical application in detecting low numbers of viruses in clinical specimens, food, and environmen‐

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

human HAV strains. Genotype II contains two subgenotypes: IIA and IIB.

**3. Nucleic acid-based detection**

40 Nucleic Acids - From Basic Aspects to Laboratory Tools

tal samples.

**3.2. Nucleic acid amplification**

**3.1. Direct nucleic acid probe hybridization**

Since the first demonstration of the PCR process in 1985[34], 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 amplifica‐ tion of viral RNA. RT-PCR can generally be carried out eitherin a one-step ortwo-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 result‐ ing 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 success‐ ful 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 differ‐ ent 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 [36]. It is difficult to select a single oligonucleotide primer set with sufficient sensitivity and specificity to detect all the NoV strains [33]. 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 sensitivi‐ ty (10 to 1,000 times more sensitive than single round RT-PCR) has been achieved by implementation of this strategy [44].

HAV was one of the first enteric viruses for which RT-PCR assays have been developed [46]. 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 [48].

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 en‐ hanced by combining with the results of single-strand confirmation polymorphism (SSCP) analysis [49]. PCR-SSCP has been considered as a rapid and cost-effective approach to examining genetic diversities among hNoV strains [50] as well as HAV [51—53]. Hybridiza‐ tion assays such as dot blot and Southern blot have been used to identify and confirm NoVand 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 [58]. 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 ampli‐ fied products provides detailed molecular information not only for confirming the specifici‐ ty of the amplicons but also for classifying or subtyping virus strains [59].

#### *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. RTqPCR 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 [65]. 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 [66].


**Table 1.** Selected RT-qPCR assays for detection of human noroviruses

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 en‐ hanced by combining with the results of single-strand confirmation polymorphism (SSCP) analysis [49]. PCR-SSCP has been considered as a rapid and cost-effective approach to examining genetic diversities among hNoV strains [50] as well as HAV [51—53]. Hybridiza‐ tion assays such as dot blot and Southern blot have been used to identify and confirm NoVand 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 [58]. 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 ampli‐ fied products provides detailed molecular information not only for confirming the specifici‐

ty of the amplicons but also for classifying or subtyping virus strains [59].

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. RTqPCR 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

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 [65]. 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

*3.2.2. Quantitative real time RT-PCR (RT-qPCR)*

42 Nucleic Acids - From Basic Aspects to Laboratory Tools

detection of NoV and HAV is outlined in Tables 1 and 2.

Imperial Beach pier in San Diego [66].

There are two major groups of sequence-specific oligonucleotide probes: hydrolysis (e.g., TaqMan) probes and hybridization probes (e.g., molecular beacons and fluorescence reso‐ nance 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.


**Table 2.** Selected RT-qPCR assays of detection of hepatitis A virus

Kageyama and colleagues were the first to describe the junction-targeting RT-qPCR assay [67]. 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 [67]. 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 [74]. 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 [75]. 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 [76]. Six subtype-specific RT-qPCR assays using hydrolysis probes were developed for HAV detection and subtyping [77], 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 combi‐ nation with immunomagnetic separation [80]. Molecular beacon probes have also been used in another target amplification method: nucleic acid sequence-based amplification described below.
