High-resolution typing for HLB-DRB1*15 and-DRB1*16 by fluorescence-marked sequence-specific priming (TaqMan assay)

Sequence-specific primed polymerase chain reaction (PCR-SSP) is widely used in HLA laboratories The TaqMan method, which is described here for high-re solution typing of HLA-DRB1*15 and -DRB1*16, does not require elaborate and time-consuming post-PCR detection steps. In this one-tube assay, conventio nal PCR-SSP and fluorescence detection of the amplicon with a doubly labele d fluorescent probe are combined: a fluorogenic hybridization probe (FHP) l abeled with a spectral resolvable fluorescent reporter dye (FAM or TET) at its 5' terminus and a common quencher dye (TAMRA) at its 3' terminus is cle aved by the 5' nuclease activity of Tao DNA polymerase only if the target s equence is amplified. An increase of fluorescence intensity indicates a suc cessful amplification. For high-resolution typing of HLA-DRB1*15 and -DRB1* 16 alleles we designed two FHPs and 14 specific primer mixes (7 for DR15 an d 7 for DR16). Amplification of the specific sequence was detected by a FAM -labeled FIB, whereas amplification of the internal control was detected by a TET-labeled FHP. We were able to type all heterozygous DRB1*15/DRB1*16 s ubtype combinations. For evaluation, 60 HLA-DRB1*15-positive and 40 HLA-DRB 1*16-positive individuals were typed and the results were compared with con ventional PCR-SSP DR15/16 subtyping. There were no discrepancies between th e two methods. The TaqMan method is an alternative to conventional PCR-SSP typing which is suitable for routine use in HLA laboratories.