Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction

A nonisotopic ligase chain reaction (LCR) assay was developed to detect the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovine leukocyte adhesion deficiency (BLAD). Two sets of diagonally opposed discriminating LCR primers that differentiate the normal and BLAD allele were designed so that the 3′ end of each primer overlapped the D128G mutation. These discriminating primers were synthesized with a 5′ biotin and could be captured using streptavidin-coated microtitre wells. A common set of primers that abut these discriminating primers were also synthesized and 3′-tailed with digoxigenin-ddUTP. Captured LCR products were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos TM 530 and the entire assay including DNA isolation can be completed within 8 h.