Int. J. Environ. Res. Public Health 2012, 9 2.3. Laboratory Investigation 2.3.1. Nucleic Acid Extraction, PCR and RT-PCRs 898 The viral nucleic acids were extracted from the faecal specimens using guanidium isothiocyanate (Invitrogen, Carlsbad, CA; Imperial, OH, USA) or spin columns (Qiagen, Hilden, Germany) as per manufacturer’s instructions. All specimens were tested by conventional RT-PCR targeting 220 nucleotides of the RVA VP6 gene as described earlier [20]. Genotyping of RVA strains was carried out by multiplex PCR of VP7 and VP4 genes according to the protocols described previously [17,21,22]. Newly designed primers NSP2-AF: GCCATCAGACAGAGAATGTGTTGCA (primer positions: 112–136), NSP2-BR: CCAATCAGTCACAAGAGTCCATAGT (primer positions: 340–316) and NSP2-CR: TTGTCTGCCGAAGCTAAAACATCC (primer positions: 432–409) based on CAL-1 strain (AY238383) targeting against 321 bp (primers NSP2-AF and NSP2-CR) and 229 bp (primers NSP2-AF and NSP2-BR) of NSP2 gene were employed to detect the presence of RVB in the faecal specimens by semi-nested RT-PCR assay. PCR conditions involved an initial reverse transcription of 30 min at 50 °C, followed by PCR activation at 95 °C for 15 min and 35 cycles of amplification (1 min at 94 °C, 30 sec at 55 °C and 1 min at 72 °C) with final extension at 72 °C for 7 min. The presence of NoV GI and GII, AdV, HAstV, EV and AiV RNA in the faecal specimens was detected by amplification of RdRp region A (126 bp), hexon (300 bp), ORF 1a (289 bp), 5′ NCR (404 bp), 3C-3D junction (265 bp) regions, respectively as described earlier [23-28]. This was followed by genotyping of NoV, HAstV and EV strains with primers specific for VP1 [region C, 330 (GI)/344 bp (GII)], ORF2 (232 bp), VP1/2A (450 bp) regions, respectively as described previously [18,29,30]. 2.3.2. Nucleotide Sequencing and Phylogentic Analyses All of the PCR products were electrophoresed in 2% agarose gel containing ethidium bromide (0.5 µg/mL) and visualized under UV transilluminator. PCR amplicons were excised from the gel for purification (QIAquick, Qiagen, Hilden, Germany) and cycle sequencing was carried out using BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). The sequences were collected from automated DNA sequencer ABI 3130XL (Applied Biosystems). Sequence identity was determined through BLAST and multiple sequence alignment was carried out with CLUSTAL W program. The phylogenetic analysis of aligned sequences was carried out using MEGA 4 [31]. The phylogenetic tree was generated with neighbor-joining algorithm and Kimura 2- parameter distance model. The reliability of phylogenetic tree was tested by applying bootstrap test with 1000 bootstrap replications. 2.3.3. Nucleotide Sequence Accession Numbers The nucleotide sequences of the strains examined in the study have been deposited in GenBank under the accession numbers, HQ268736–HQ268770 for RVA, HQ268794–HQ268804 for RVB,