OPTIMIZING CONDITIONS FOR THE MULTIPLEX PCR TO IDENTIFY SUBTYPES OF HIV-1 The multiplex PCR assays for detection of HIV-1 subtypes were performed as two steps the following: (1) The first PCR performed using BECO3 and BECO5 primer pairs with annealing temperature is 63, 90C in 60 second maintaining; (2) The second multiplex-PCR (PCR2.0) was performed using 5, 5'E, 5'G, 3'A, 3'E and 3'G primers.