5% completely untyped samples
of the total samples forwarded for further analysis. RNA was re-extracted from 30% fecal suspensions using the QIAamp Viral Mini RNA kit (Qiagen, Hilden, Germany) as per the manufacturer’s specifications for samples collected from 2007 to 2009 that were initially extracted using Trizol reagent (Invitrogen Life Technologies). Samples collected from 2010 to 2012 were initially subjected to RNA extraction using the Viral Mini RNA kit method; re-extraction was performed using the Trizol reagent. Polymerase chain reaction amplifying the VP6 region was performed to determine the presence or absence of rotavirus using primers described in Table 1 and random primed cDNA . For samples that were negative for the VP6 gene by PCR with GDC-0973 order http://www.selleckchem.com/products/SNS-032.html random primed cDNA, cDNA was synthesized using specific priming and amplified with the VP6 primers using the OneStep RT-PCR kit (Qiagen, Hilden, Germany). Samples that were negative by this method were recorded as negative on VP6 PCR with false positive ELISA. The samples positive for the VP6 gene were subjected to G and P typing using the standard primer sets as previously described . RNA from samples which were partially typed and VP6 PCR positive samples which remained untyped after re-extraction and application of the standard genotyping protocol were subjected to
specific priming for reverse transcription and amplification using the VP7F/R and Con2/Con3 primers and the One Step RT-PCR kit (Qiagen, Hilden, Germany),
followed by a second-round PCR with the standard primer set. Typing of samples that remained untyped was attempted using alternate primer sets targeting the consensus regions of the VP7 and VP4 genes (Table 1) . If present, the first-round product was sequenced for strains that were still G and P untyped (Fig. 1). Sequencing of the first-round amplicon was attempted for all VP6 positive, G- and P-untyped samples. Briefly, the amplicons were purified and sequenced in both directions with the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) using from the same primer pairs as in the first-round PCR. The sequences were resolved in the automated DNA sequencer, the ABI PRISM 310 Genetic Analyzer (Applied Biosystems), and the electropherograms were analyzed using sequencing analysis software (Finch TV, version 1.4.0). Consensus sequences were compared with available rotavirus sequences in GenBank for genotype confirmation using the Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). We explored an approach (Fig. 1) to further characterize partially and completely untyped samples for G and P typing of 57 partially typed and 308 untyped samples. Fifty-eight (58/308, 19%) of the untyped samples were negative for VP6 gene amplification after repeat extraction and VP6 PCR using both random and specific priming methods. These were considered ELISA false positives.