title: Development of a fast and economical genotyping protocol for bovine leukocyte adhesion deficiency (BLAD) in cattle.
abstract: Fast and economical means of assaying SNP's are important in diagnostic assays, especially when a large number of animals have to be screened for a genetic disease. This study was aimed at the development of a fast and economical screening assay for bovine leukocyte adhesion deficiency (BLAD) which is an important genetic disease of cattle industry. Four primers were designed where the outer primers amplify a 354 bp amplicon of CD18 gene carrying the polymorphism responsible for BLAD. The specifically designed inner primers in conjunction with the modified reaction mixture and cyclic conditions ensured amplification of either of wild or mutated alleles. Together with outer primers, the inner primers generated typical banding pattern in agarose gel which discriminated the normal animal against the carrier. We successfully used this protocol in 200 bulls for genotyping the BLAD allele which confirmed by sequencing, showing a cent percentage concordance. With the developed assay the need for restriction digestion or use of costly equipment viz. real time PCR was eliminated. This genotyping assay ensured fast and economical genotyping and could be adopted in every laboratory with a minimum equipment requirement of thermocycler and gel documentation system.
authors.affiliation: ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
authors.forename: Rafeeque R
authors.initials: RR
authors.lastname: Alyethodi
authors.affiliation: ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
authors.forename: Umesh
authors.initials: U
authors.lastname: Singh
authors.affiliation: ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
authors.forename: Sushil
authors.initials: S
authors.lastname: Kumar
authors.affiliation: ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
authors.forename: Rajib
authors.initials: R
authors.lastname: Deb
authors.affiliation: ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
authors.forename: Rani
authors.initials: R
authors.lastname: Alex
authors.affiliation: ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
authors.forename: Sheetal
authors.initials: S
authors.lastname: Sharma
authors.affiliation: ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
authors.forename: Gyanendra S
authors.initials: GS
authors.lastname: Sengar
authors.affiliation: ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
authors.forename: B
authors.initials: B
authors.lastname: Prakash
keywords: BLAD; Frieswal; PCR–RFLP; SNP genotyping; T-ARMS PCR