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The second reaction mix fraction (F2) consisted of 4.4 l of 2X reaction mix, 200 nM of forward primer (F56_13 or gen2FSN), 1.5 l of 50 mM MgSO 4 , 0.1 l of ROX (25 M), and 200 nM of each probe (against either genotypes 1a, 1b, and 3a