*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 [67] but provided less consistent signals [87]. A molecular beacon real-time NASBA method was developed to detect NoV GII from environmental samples, with 88% sensitivity compared to conventional RT-PCR [88]. 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 [89]. A multiplex NASBA and microtiter plate hybridization system was devel‐ oped to simultaneously detect HAV and rotavirus where 400 PFU/ml HAV were detected with reduced time and cost compared to monoplex system [90]. 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 [91].

### *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 [92]. 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 [93]. 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 [94].

#### **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 [101]. Recently, a sequenceindependent isothermal RNA amplification approach has been developed to amplify various enteric viral nucleic acids of hNoVs, HAV, and coxsackievirus for microarray analysis [102]. 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 [103]. Microarray detection of random-primed PCR products from a range of gastrointestinal viruses including Norwalk virus was reported recently [104].

### **3.4. Detection of infectious viruses**

*3.2.5. Nucleic Acid Sequence-Based Amplification (NASBA)*

46 Nucleic Acids - From Basic Aspects to Laboratory Tools

inoculum levels of 10(0) to 10(2) RT-PCR units [91].

102

and 103

*3.2.6. Reverse transcription loop-mediated isothermal amplification (RT-LAMP)*

**3.3. Nucleic acid amplification coupled with DNA microarray detection**

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 [67] but provided less consistent signals [87]. A molecular beacon real-time NASBA method was developed to detect NoV GII from environmental samples, with 88% sensitivity compared to conventional RT-PCR [88]. 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 [89]. A multiplex NASBA and microtiter plate hybridization system was devel‐ oped to simultaneously detect HAV and rotavirus where 400 PFU/ml HAV were detected with reduced time and cost compared to monoplex system [90]. 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

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

sensitivity and specificity of the RT-LAMP were 100% and 94% for GI, and 100% and 100% for GII, respectively [92]. 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 [93]. 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 [94].

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,

copies/tube for GI and GII. Compared to conventional RT-PCR, the clinical

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 [105]. 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 [106]. 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 [108]. 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 heatinactivated hNoVs [112].
