For all specimens in food handling experiments the characterisation algorithm was followed as described in Figure 5.
Figure 5 A norovirus characterisation algorithm
Noroviruses genotypes were characterised by amplification and sequencing of region C (Sanger et al., 1977), and GII.4 viruses were further characterised by amplification and sequencing of the hypervariable P2 domain (Xerry et al., 2008; Allen et al., 2008). Amplification of 340-370bp of region C; the capsid gene within the S domain of the norovirus genome, was conducted using a semi-nested PCR. The thermal cycling conditions were a denaturation step of 94ᵒC for 1 minute, followed by 40 cycles at 94oC for 30 seconds, 55oC for 1 minute, 72oC for 1 minute and one cycle at 72oC for 5 minutes in the first round PCR reaction. Second round PCR reactions were conducted by a denaturation step of 94oC for 2 minutes, followed by 40 cycles at 94oC for 30 seconds, 55oC for 1 minute,
Negative Negative Positive Positive P2 domain PCR Negative Positive Genotyping PCR Region C typing (ORF 2) Detection PCR
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72oC for 1 minute, and a single cycle at 72oC for 5 minutes. These PCR total reaction volumes of 50µl comprised of 20µM of forward and reverse primer (Invitrogen), 1x PCR buffer (Invitrogen), 1.5mM MgCl2 (Invitrogen), 2.3mM dNTPs, 2U of Taq Polymerase (Invitrogen) and molecular grade water (Severn Biotech, UK) using oligonucleotide primers from published papers (Kojima et al., 2002, Gallimore et al., 2005). The sequence of the primers and probe used were as stated in Table 11.
Table 11 Primer sequences used in amplification of: first or second round PCR products in
the region C typing assay Round Primer
name Sequence (5’-3’) Amplicon size (bp) Original Reference
First GI FFN GGAGATCGAATCTCCTGCCCC 364 (Kojima et
al., 2002) (Gallimore et al., 2005) GI SKR CCACCCCACCATTRTACA (Kageyama et al., 2003) Second semi- nested GI FFN2 ATCTCCTGCCCGAWTWYGTAA 343 (Gallimore et al., 2005, Kojima et al., 2002)
First GII FBN TGGGAGGGCGATCGCAATCT 364 (Gallimore et
al., 2005)
GII SKR CCRCCNGCATRHCCRTTRTACAT (Kageyama et
al., 2003)
Second semi- nested
GII FBN2 GCGATCGCAATCTGGCTCCC 343 (Kojima et
al., 2002)
For further characterisation by amplification of the P2 domain, amplification of 790bp was conducted in the first round PCR and 512bp in the second round PCR of the P2 domain. This was set up in a total reaction volume of 50µl which comprised of 1x PCR Reaction Buffer (Invitrogen), 1mM each dNTP (Invitrogen), 1.5mM MgCl2 (Invitrogen), 20pmol of forward and reverse primer (Invitrogen), and 2U of Taq Polymerase (Invitrogen) and molecular grade water (Severn Biotech, UK). The amplicons were generated by a denaturation step of 94oC for 2 minutes, followed by 40 cycles at 94oC for 30 seconds, 55oC for 1 minute, 72oC
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for 1 minute for 40 cycles, followed by an extension step of 72oC for 5 minutes in the first round PCR reaction. Second round PCR reactions were conducted by a denaturation step of 94oC for 2 minutes, followed by 94oC for 30 seconds, 53oC for 1 minute, 72oC for 1 minute for 40 cycles and an extension step of 72oC for 5 minutes using nucleotide primers from published papers (Xerry et al., 2008). Amplification of the P2 domain for other genotypes used the same 50µl reaction mix; however, genotype specific oligonucleotide primers and annealing temperatures were adjusted as outlined in Table 12.
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Table 12 Primers and annealing temperatures for generation of both first and second round PCR products of the P2 domain domain GII-4 specific PCR assay,
for specific norovirus GI and GII genotypes used in validation experiments of the intra-genotyping assay
Norovirus
genotype Round Primer names Primers (5’-3’) Amplicon size (bp) Annealing temp ℃ References GI-3 First round
Second round semi nested
P2 GI-3 F P2 GI-3 R P2 GI-3 FN TCWAAYTCAAGRGTCCCTTCT GCTTCMCCTCTAGTGGGGGCCT TTCWYTMATYAAWKCWATGAT 682 662 50 40 (Xerry et al., 2010, Xerry et al., 2008)
GI-6 First round P2 GI-6 F
P2 GI-6 R TCAAATTCTCGTGTCCCTGTG GTTCATTRCAGAAGTGGGTAAT 646 45 (Xerry et al., 2010)
GI-7 First round P2 GI-7 F
P2 GI-7 R GCTAACTCCAGAGTGCCCGCA GCGGCTTCACCTCGGATTGGTG 674 50 (Xerry et al., 2010)
GII-4 First round GII 4P2F GANGATGTCTTCACAGTCTCTT 790 53 (Xerry et al.,
2008) GII 4P2R CATTCCTGGGGGAGTAGACA
Second round nested N-P2F TGGCARTGYACGACTGATGG 512 50 (Allen et al., 2008)
N-P2R2 CTRAAGAAAAGAAGYTGCTCA
GII-5 First round P2 GII-5 F
P2 GII-5 R GACGCAGTTTTCACCGTCTCAT CACTCCTGAGGCACCAGACA 794 48 (Xerry et al., 2008) GII-6 First round
Second round semi nested
P2 GII-6 F P2 GII-6 R P2 GII-6 FN GAGGACGTGTTCACTGTTTCTT CATTCCTGGGGTATGAGACA CACCAACTGTTGAATCAAAAA 827 753 45 45 (Xerry et al., 2008)
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All PCR products were resolved on a 1% (w/v) agarose gel by electrophoresis. Gels were made using Agarose MP (Roche Diagnostics Ltd) and 1x Tris-Borate EDTA (TBE) (Invitrogen). Electrophoresis was performed at 110V/cm2 on a 13.2cm x 14.2cm gel, for 100 minutes. Products were visualised after immersion of the gel in an ethidium bromide tank containing 5mg/l ethidium bromide (Sigma Aldrich) in 1x TBE buffer (Invitrogen) for 15 minutes. The gel was washed three times with water and visualised under a UV light using the GelDoc system (BioRad Laboratories Ltd, Hertfordshire). The amplicons were measured against a 100bp ladder (Invitrogen) run in parallel on the agarose gel. Amplicons of the correct size were purified from solution using the AMPure Agencourt® system. Amplicons were purified using 65µl of Agencourt® Ampure®magnetic beads (Beckman Coulter, UK) added to 45µl of the PCR product in solution in a 96 well plate. Agencourt®® Ampure®magnetic beads and PCR products were mixed ten times by pipette until the colour of the mixture appeared homogenous. The homogenous sample was then incubated at ambient temperature for three minutes. The 96 well plate was placed on the Agencourt® SPRIplate 96R (Beckman Coulter) and allowed to incubate for five-ten minutes. The colourless solution was aspirated and discarded and 200µl of 70% ethanol was added to each reaction well and incubated at ambient temperature for 30 seconds. The ethanol was aspirated, discarded and this was repeated two more times. Once the ethanol was removed the 96 well reaction plate was air-dried for 15 minutes at ambient temperature before 40µl of RNase free water was added to elute the DNA. Excess purified PCR product was stored at -20oC.
DNA was set up to be sequenced in an ABI MicroAmp optical 96 well plates (Applied Biosystems) by the addition of 2pmols/µl of primer and 30ng of purified DNA template to a total reaction volume of 6µl for each sequencing reaction (Sanger et al., 1977). All
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sequencing was performed by PHE in-house genomics and sequencing service using BigDye Chemistry version 3.1 (Applied Biosystems) and analysed by the Genetic Analyser 3730XL (Applied Biosystems). Nucleotide sequence contigs were generated from trace sequence data using the Assembler tool in seqMan Pro (DNASTAR version 12.2), ClustalW multiple alignment and phylogenetic analysis was also performed using algorithms in MegAlign (DNASTAR version 12.2). For region C typing, contig sequences were trimmed to position 5331 at the 5’ and position 5653 at the 3’ end, and the trimmed sequences were aligned by ClustalW multiple alignment in MegAlign. The P2 region defined as ranging from amino acid 279 to 405 (Prasad et al., 1999), was then used to confirm sequence homology of the P2 domain. Contig sequences were trimmed to these amino acid positions, and the trimmed sequences were aligned by ClustalW multiple alignment in MegAlign.