Author:
Alyethodi R. R.,Singh U.,Kumar S.,Alex R.,Sengar G. S.,Raja T. V.,Deb R.,Prakash B.
Abstract
Abstract
Background
DNA testing in the cattle industry undergoes multiple hurdles. Successful genotyping involves the transportation of samples from the field to the laboratory in a chilled environment followed by DNA extraction, and finally, a specific genotyping protocol is followed. Various researches are focused on overcoming these issues. Microcards offer blood transportation at ambient temperature. Direct PCR methods can save the time of DNA extraction but available only for simplex PCR. Tetra Primer-Amplification Refractory Mutation System based Polymerase Chain Reaction (T-ARMS PCR) can make DNA testing faster in a low-cost setting. The present study was aimed to design, optimize, and validate a T-ARMS PCR for faster DNA testing of SNP responsible for Complex Vertebral Malformation (CVM)-an important genetic disease of the cattle industry. Further, a direct T-ARMS PCR from whole blood was developed to avoid the DNA extraction steps. Lastly, using the optimized protocol, genotyping of blood spotted on Microcard eliminates the need for cold chain maintenance in the transportation of samples.
Results
The present study demonstrated a novel T-ARMS PCR-based genotyping of the SNP rs438228855, which is responsible for CVM. Here, wild genotypes were recognized by 389 bp and 199 bp bands in agarose gel, while the carrier genotype showed an additional 241 bp band. The developed protocol was validated using PCR-Primer Introduced Restriction Analysis (PCR-PIRA) and sequencing. The present study further established a direct T-ARMS PCR for this SNP from whole blood. Different conditions such as heparin and EDTA treated blood, the need for pre-treatment, and two different DNA Polymerases for the direct PCR were optimized. Finally, our optimized protocol successfully genotyped the whole blood samples dried on Insta™DNA cards.
Conclusions
The present study reported the usefulness of primer modified T-ARMS PCR for detecting CVM for the first time. To the best of our knowledge, direct PCR in T-ARMS PCR has never been reported. Lastly, the use of microcards in the developed protocol can make the assay useful in the DNA testing of field samples.
Publisher
Springer Science and Business Media LLC
Reference40 articles.
1. Kanae Y, Endoh D, Nagahata H, Hayashi M. A method for detecting complex vertebral malformation in Holstein calves using polymerase chain reaction--primer introduced restriction analysis. J Vet Diagnostic Investig. 2005;17(3):258–62. https://doi.org/10.1177/104063870501700309.
2. Thomsen B, Horn P, Panitz F, Bendixen E, Petersen AH, Holm L-E, et al. A missense mutation in the bovine SLC35A3 gene, encoding a UDP-N-acetylglucosamine transporter, causes complex vertebral malformation. Genome Res. 2006;16(1):97–105. https://doi.org/10.1101/gr.3690506.
3. Agerholm JS, Bendixen C, Andersen O, Arnbjerg J. Complex vertebral malformation in Holstein calves. J Vet Diagnostic Investig. 2001;13(4):283–9. https://doi.org/10.1177/104063870101300401.
4. Alyethodi RR, Kumar S, Deb R, Alex R, Singh U, Sharma S, et al. Using PCR-PIRA based genotyping for identifying complex vertebral malformation allele in Frieswal young bulls in India. Iran J Vet Res. 2018;19:44-7.
5. Gabor M, Miluchová M, Trakovická A, Riecká Z, Candrák J. Vavriš\’\inová K. detection of complex vertebral malformation carriers in Slovak Holstein cattle by high resolution melting analysis. Acta Vet Brno. 2012;62(2-3):239–48. https://doi.org/10.2298/AVB1203239G.
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