Extracted viral RNA is then taken through nested PCR utilizing primers which are complimentary to the 3' ends of both viral RNA strands within gene segment 9, which encodes viral protein 7 (VP7). These primers are used for first strand synthesis. Primer RVG9 and six serotype-specific primers (aBT1, aCT2, aET3, aDT4, aAT8, and aFT9) which are located in six variable regions on the gene 9 and correspond to G-serotypes 1, 2, 3, 4, 8 and 9, are used in second amplification. Briefly, 5 µl of RNA with 25 pmol of each primer Beg9 and End9 is denatured for 5 min at 97°C and cooled on ice. Thereafter, 8 µl of RT reaction mixture containing 1.5 µl of 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 8.3), 2 µl of 25 mM MgCl2, 2 µl of 2.5 mM of each dATP, dCTP, dGTP and dTTP, 10 U AMV reverse transcriptase (Promega) and 20 U Rnasin ribonuclease inhibitor (Promega), is added to the sample-primer mixture and incubated at 42°C for 60 min. PCR buffer mixture (35 µl), containing 3.5 µl of 10X PCR buffer, 2 µl of 2.5 mM peach dATP, dCTP, dGTP and dTTP, 2.5 U Ampli Taq DNA polymerase (Perkin Elmer) and 27 µl of water are added to RT reaction. The reaction mixture is denatured at 94°C for 3 min, followed by 30 cycles (1 min at 94°C, 2 min at 42°C, 3 min at 72°C) and a final extension of 5 min at 72°C.