Inclusion of the remaining 39 untyped samples and 57 partially typed samples for reverse transcription and amplification with the One Step RT-PCR, using specific priming for VP7 and VP4, resulted in resolution of both G and P genotypes for an additional 45 samples. We subjected the remaining partially typed and untyped samples (n = 51) to specific priming for VP7 and VP4 RT using alternate primer sets ( Table 1). This

led to determination of both G and P types for 8 strains and partial typing for 35 strains (12 G untyped and 23 P untyped). Seven samples remained completely untyped ( C59 wnt molecular weight Fig. 2). Of the original 57 partially typed samples, 22 remained partially typed. Only one sample which failed to type in

the second-round PCR for either VP7 or VP4 had a first round product for both genes and these were sequenced and the strain identified as G11P[25]. The most common G and P types isolated were G1 (n = 100/307, 32%) and P[8] (n = 157/307, 51%), respectively ( Table 2). Use of a standard protocol for genotyping had resulted in 308/2226 (13.5%) samples being untyped for G and P types and 57/2226 (2.5%) being partially typed for either G or P type. The approach we used, as shown in Fig. 1, is to sequence the first-round G and P amplification product, if available. If not present, the presence of rotavirus is confirmed by performing VP6 PCR using both random and specific I-BET151 cost priming approaches after re-extraction. If VP6 is positive,

specific priming with standard G and P primers or alternate primer sets was carried out to attempt genotyping of these samples. Application of the VP6 PCR for confirmation resulted in the identification of 58/2226 (2.6%) false positive ELISA results. A recent publication has indicated the sensitivity unless and specificity of the Premier Rotaclone kit to be 76% and 100%, respectively [12]. It is possible that the ELISA false positives identified in this study could be due to degradation of the nucleic acid in the samples, but it could also be due to variation in test performance characteristics depending on the laboratory and the types of samples included for evaluation. In the remaining 307 untyped and partially typed samples, alternate extraction methods with the standard primer sets resulted in typing of both G and P types in 256 (83%) and partially typing in 43 (14%) samples. Hence, use of the standard primer sets resulted in G or P or both types in 97% of the samples obtained from India. The lack of initial typing may be because of the inefficiency of the extraction followed by random priming or because PCR inhibitors may be carried over from extraction.