Samples showing an OD value of >0.150 were reported as positive.
An internal control was included in all runs, and the run was repeated if the internal control did not fall in the expected range. Genotyping was performed on the antigen positive samples. RNA Alisertib was extracted using the QIAamp Viral RNA Mini Kit. Complementary DNA was synthesized using random primers (Pd(N)6 hexamers; Pharmacia Biotech) and 400 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies) and was used as template for VP7 and VP4 (G and P) typing in PCRs using published oligonucleotide primers and protocols to detect VP7 genotypes G1, G2, G3, G4, G8, G9, G10, and G12 and VP4 genotypes P, P, P, P, P, and P . Samples which failed to type the first time were confirmed to be rotavirus positive by PCR to detect the VP6 gene. If the VP6 PCR was positive, alternate primer
sets were used to attempt genotyping. Samples which were VP6 negative were re-extracted by Trizol method and subjected to a repeat VP6 PCR to confirm or rule out the presence of rotavirus . A total of 1191 children were recruited from the 3 sites over the study period and rotavirus was detected in 458 children using the antigen detection ELISA, accounting for 39% of the cases of diarrhea. The detection rates of rotavirus varied from 26% in Vellore to 40% in Delhi and 50% in Trichy. The proportion SP600125 solubility dmso positive each year did not vary by site, with higher whatever rates in Trichy and lower rates in Vellore in each year of surveillance. Of the children recruited, 60% were male, with mean age of 10.1 months (+SD 7.4) versus 40% female with an average age of 11.6 months (+SD
7.6). The median age of rotavirus positive and negative cases was 10 months. Of the children who tested positive for rotavirus, 63% were less than 1 year of age, 26% 1–2 years of age and 11% between ages of 2 and 5 years. Rotavirus was detected throughout the year from the sites in south India compared to the site in the north India where the rates of detection where much higher during March–April, as compared to the other months (Fig. 1). Of the 458 samples which tested positive by ELISA, genotyping was attempted for 453 strains (98%). Fifty-eight (13%) of the ELISA positive samples were negative on genotyping, and when tested for VP6 gene they were all negative even after re-extraction of samples by another method (Fig. 2a). Of the 395 samples, 96% were G-typed and 91% were P-typed. Both G and P type was obtained for 315 (80%) strains. The most prevalent G and P type combinations were G1P (133/395, 33%), G2P (69/395, 17%) and G9 P (43/395, 11%) (Fig. 2b, Table 1). We detected G12 strains, in combination with P and P, from both the north and south Indian sites, with more G12 P strain from north India.