Materials and Methods Supplementary

Materials and methods – supplementary

Total nucleic acid was extracted from the stool specimens essentially as outlined previously 21. Briefly, an approximate 10% (w/v) stool suspension was prepared in PBS buffer and vigorously mixed. Five hundred microlitres of this suspension was then subjected to an extraction step with 100ul of analytical grade chloroform. The aqueous phase was separated out by centrifugation at 1000×g for 1min. Two hundred microlitres of the above aqueous phase was then extracted for nucleic acid with the QIAsymphony SP automated instrument (Qiagen) using the DSP virus/Pathogen Mini Kit version 1 ( and eluted in 110ul according to the manufacturer's instructions. The eluted RNA was then reverse transcribed and analysed for the presence of norovirus genogroup I and II by real-time (RT)-PCR as previously described 21,22.

Quantitative PCR was performed by including a dilution (10-fold) series of a recombinant pUC57 plasmid containing the norovirus group II real-time PCR target sequence in each real-time PCR run. Six standards, ranging from 1.6 x 107 to 1.6 x 102 genome equivalents were employed to calculate the viral load present in each specimen. Sequential samples from each patient were quantified together on the same run in order to minimise variation. Isolation of viral RNA from archived biopsy specimens was achieved using RecoverAll Total Nucleic Acid Isolation Kit (Applied Biosystems/ Ambion).

Viral genome sequencing was carried out to determine the phylogenetic relationships of the nucleotide sequences derived from the individual patients’ stools and the archived duodenal biopsies from the index patient. In the case of the stools, a region of the viral genome (383bp) extending from the conserved target site (ORF1/ORF2 overlap) used by the real time assay into the capsid VP1 (ORF2) gene was selected. Real-time PCR amplification of the 383bp amplicon was achieved by pairing the Cog2F forward primer 5’-CARGARBCNATGTTYAGRTGGATGAG-3’22 and used in our real-time assay with a new generic group II reverse primer, ORF2R, 5’-CCACCIGCATANCCRTTRTACAT-3’ and including the norovirus group II specific TaqMan® probe, ring2, 5’-Joe-TGGGAGGGCGATCGCAATCT-BHQ1-3’. For the Norovirus positive biopsy specimens from case #1, a second real-time PCR assay amplifying a shorter region (199bp) within the above target site was designed in order to provide informative sequence data to permit a phylogenetic analysis. A forward primer, 5’-CTATTGCGGCACCTGTAGCGG-3’, perfectly matched to the index case stool sequence data was paired with the ORF2R reverse primer and a new TaqMan® probe, 5’-6FAM-CAGGTGAAATACTATGGAG-MGBNFQ-3’, again matched to the index case sequence. RT-PCR was carried out on a Rotor-Gene™ 6000 apparatus under the following conditions: incubation at 50 °C for 30 min followed by denaturation at 95 °C for 2 min and then 50cycles of amplification with denaturation at 95 °C for 15 s and annealing/extension at 54 °C for 1 min, acquiring on either the FAM or JOE channels. Real-time PCR amplicons were cleaned up with QIAquick PCR purification kits (Qiagen) and subjected to DNA sequencing (BigDye ® Terminator v3.1 cycle sequencing kit part no.4336917; Applied Biosystems, Warrington UK) on the ABI 3130 genetic analyser using the original primers and an additional internal forward primer PolF1, 5’-AGCACRTGGGAGGGCGATC-3’, for the stool specimens and NORF1, 5’-ACAAGCCCCTGGTGGAGAGTT-3’ for the biopsy specimens. Nucleotide sequences were merged using the SeqMan Pro module of the Lasergene (version 8.1.5) sequence analysis software suite ( and their phylogenetic relationships were established using the MegAlign (Clustal V) module.