Following addition of 1 µL (1.9U) of Taq polymerase and 100 µL mineral oil, first-round PCR is carried out for 30 cycles (1 min 94oC, 2 min at 50oC, and 2 min at 72oC. A cooling cycle is used to bring the samples to 17OC at the completion of the experiment. The second-round typing reaction if performed using 0.5 to 5 µL of the first-round product (5 µL if no visible product; 0.5 µL if visible product) is mixed with 45 µL reaction mixture in a final volume of 50 µL. If less than 5 µL of first round product is added, the remaining volume, to complete 50 µL, is 10mM TrisHCl (pH 8.3)-2.5mM MgCl¬2. The reaction mixture is 19.5 µL water, 16 µL dNTPs, 5 µL 10X Buffer II, 3 µL 25 mM MgCl2, 1 µL of the primer cocktail (containing 20 µM each of Con3, 1T-1, 2T-1, 3T-1, and 4T-1), and 0.5 µL (2.5U) Taq. The samples are overlaid with mineral oil and taken through 25 rounds of PCR (same cycles as first round). The samples are then analyzed by gel electrophoresis and product size enables determination of VP4 genotype. Other primer sets and/or sequencing is used to distinguish genotype when/if the initial primers fail.