Title: Designing multiplex PCR tests for simultaneous screening of bovine leukocyte adhesion deficiency, bovine citrullinemia and factor XI deficiency genetic diseases in cattle
Authors: Anshuman Kumar, Ravi Kumar D, Vineeth MR, Govind Mohan, S Jayakumar, Saket K Niranjan and ID Gupta
Source: Ruminant Science (2017)-6(2):215-220.
Cite this reference as: Kumar Anshuman, Kumar D Ravi, MR Vineeth, Mohan Govind, Jayakumar S, Niranjan SK and Gupta ID (2017). Designing multiplex PCR tests for simultaneous screening of bovine leukocyte adhesion deficiency, bovine citrullinemia and factor XI deficiency genetic diseases in cattle. Ruminant Science 6(2):215-220.
Bovine Leukocyte Adhesion Deficiency (BLAD), Bovine Citrullinemia (BC) and Factor XI deficiency (FXID) are most prevalent lethal genetic disorders in cattle, causing death of homozygote animal at young stage and severe adverse effects on viability in heterozygous condition also. These disorders propagate unabated in absence of screening of carrier bulls in herds, worldwide. After identifying the loci for these disorders, the breeding animals; especially bulls are being screened separately through DNA based tests in various countries including India. Under this study, an attempt was made to detect the BLAD, BC and FXID mutations simultaneously after multiplexing of these conventional PCR based DNA tests. Two multiplexed PCR protocols – one for BLAD and FXID and another for BC and FXID loci screening were developed. Along with modifying the primers, the PCR components and conditions were also standardized for developing both multiplexed PCR protocols. In both of the protocols, loci were amplified and further analysed by restriction fragment length polymorphism (RFLP) using TaqI and AvaII restriction enzymes, respectively. After PCR-RFLP, the products were resolved in gel electrophoresis, which were easily identifiable to ascertain their genotypes. These protocols produced the results without compromising accuracy, reducing overall time and cost incurred in separate PCR tests and may be used for rapid screening of these genetic disorders in a herd.
Agerholm JS, Bendixen C, Andersen O and Arnbjerg J (2001). Complex vertebral malformation in Holstein calves. Journal of Veterinary Diagnostic Investigation 13(4):283-289.
Alyethodi RR, Singh U, Kumar S, Deb R, Alex R, Sharma S, Sengar GS and Prakash B (2016). Development of a fast and economical genotyping protocol for bovine leukocyte adhesion deficiency (BLAD) in cattle. Springer Plus 5(1):1442.
Dennis JA, Healy PJ, Beaudet AL and O’ Brien WE (1989). Molecular definition of bovine argininosuccinate synthetase deficiency. Proceedings of the National Academy of Sciences 86(20):7947-7951.
Gaur U, Sathe TG, Roy A, Sunkara PSS, Patel RK and Sri Venkatesh P (2012). Polymorphism in Arginosuccinate synthase gene in Indian Holstein. International Journal of Veterinary Science 1(3):115-117.
Grupe S, Dietl G and Schwerin M (1996). Population survey of citrullinemia on German Holsteins. Livestock Production Science 45(1):35-38.
Henegariu O, Heerema NA, Dlouhy SR, Vance GH and Vogt PH (1997). Multiplex PCR: Critical parameters and step-by-step protocol. Biotechniques 23(3):504-511.
Kalendar R, Lee D and Schulman AH (2011). Java web tools for PCR, in silico PCR, and oligonucleoide assembly and analysis. Genomics 98:137-144.
Kotikalapudi R, Patel RK, Kushwah RS, and Sunkara PSS (2014). Identification of citrullinaemia carrier and detection of new silent mutation at 240bp position in ASS1 gene in normal Holstein cattle. Genetika (Serbia) 46(2):515-520.
Mahdi M, Ashwani S, Dubey PP, Singh A, Tantia MS, Bina M, Kumar V and Joshi BK (2010). PCR based identification of bovine leukocyte adhesion deficiency syndrome (BLAD) carriers in Karan Fries bulls. Indian Journal of Animal Sciences 80(5):433-435.
Markoulatos P, Siafakas N and Moncany M (2002). Multiplex polymerase chain reaction: A practical approach. Journal of Clinical Laboratory Analysis 16(1):47-51.
Marron BM, Robinson JL, Gentry PA, and Beever JE (2004). Identification of a mutation associated with factor XI deficiency in Holstein cattle. Animal Genetics 35:454-456.
Mondal K, Chakravarti S, Ghosh AK, Kumar S, Nayak B, Nandi S, Sarkar U, Deb R, De A and Biswas J (2016). Novel identification of Factor XI deficiency in Indian Sahiwal (Bos indicus) cattle. Molecular biology reports 43(4):213-219.
Natonek M (2000). Identification of BLAD mutation in cattle with the PCR-RFLP method. Biuletyn Informacyjny-Instytut Zootechniki 38(4):29-33.
Padeeri M, Khoda VK, Grupe S, Mukhopadhya PN, Manfred S and Mehta HH (1999). Incidence of hereditary citrullinaemia and bovine leukocyte adhesion deficiency syndrome in Indain dairy cattle (Bos taurus, Bos indicus) and buffalo (Bubalus bubalis) population. ArchivTierzucht Dummerstorf 42:347-352.
Patel RK, Soni KJ, Chauhan JB, Singh KM, and Sambasiva Rao K (2007). Factor XI deficiency in Indian Bos taurus, Bos indicus and Bubalus bubalis. Genetics and Molecular Biology 30(3):580-583.
Sambrook J and Russell DW (2001). Molecular cloning: A laboratory manual III. Cold Spring Laboratory Press; NY, Cold Spring Harbour.
Schwerin M, Parkanyi V, Roschlau K, Kanitz W and Brockmann G (1994). Simultaneous genetic typing at different loci in bovine embryos by multiplex polymerase chain reaction. Animal Biotechnology 5:1, 47-63.
Shuster DE, Kehrli ME, Ackermann MR and Gilbert RO (1992). Identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in Holstein cattle. Proceedings of the National Academy of Sciences 89:9225-9229.
Zhang Y, Fan X, Sun D, Wang Y, Yu Y, Xie Y and Zhang Y (2012). A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle. Journal of Animal Science and Biotechnology 3(1):24.
Zsolnai A and Fésüs L (1996). Simultaneous analysis of bovine κ-casein and BLAD alleles by multiplex PCR followed by parallel digestion with two restriction enzymes. Animal Genetics 27:207-209.