*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) [81]. 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 [84]. Several multiplex nucleic acid diagnostic platforms for simultaneous detection of a range of pathogenic enteric pathogens including hNoVs are now commercially available [85]. 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*

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

IB 5'-UTR 0.2 PFU, 63PFU <sup>D</sup> Cell culture, green

**Approach Genotype Target**

44 Nucleic Acids - From Basic Aspects to Laboratory Tools

IA, IB, IIA, IIB,

IIIA

Molecular beacons

TaqMan probe IA, IB, IIA, IIIA

**Region**

5'-UTR, VP1-2A, VP3, VP1, VP2-VP3

**Lmit of Detection**

50 copies for IIB, 500 for IA, IB, IIA and IIIB

IB 5'-UTR 491 copies Q

IB 5'-UTR 20 PFU D

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

SYBR Green Not reported VP3-VP1 2-4 log copies/l D Ocean water [66]

Not reported 5'-UTR 5 TCID50/g Q Oyster [65]

IA, IB 5'-UTR 10 copies or 0.05 Q Shellfish, stool [75]

**Detection/**

5'-UTR 40 copies or 0.5 D Cell culture [74]

**Quantitation Sample tested References**

Q Stool, serum [77]

Shellfish,

Seeded

springwater [83]

onion [76]

groundwater [80]

region sequencing and identified as IA by the subtype-specific RT-qPCR assays.

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 [86]. 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 RTqPCR 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.
