**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 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‐ tal 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.
