Optimization of DNA extraction and development of a Multiplex PCR for detection of sub-clinical mastitis caused by Staphylococcus aureus and Streptococcus agalactiae
Authors
R. M. P. C. D. Rajapaksha
Department of Zoology and Environment Sciences, University of Colombo, Sri Lanka.
D. K.Weerakoon
Department of Zoology and Environment Sciences, University of Colombo, Sri Lanka.
C. M. Nanayakkara
Department of Plant Sciences, University of Colombo, Sri Lanka.
D. Senevirathna
Genetech Research Institute, No. 54, Kithulwatta Road, Colombo 08, Sri Lanka.
I. C. Perera
Department of Zoology and Environment Sciences, University of Colombo, Sri Lanka.
The current study was carried out to establish the most suitable DNA extraction protocol to economically obtain pure genomic DNA from cow’s milk and to develop a multiplex PCR to detect sub-clinical mastitis caused by Streptococcus agalactiae and Staphylococcus aureus. Sodium iodide method possessed the least processing time (80 minutes), low cost (0.16$), highest yield (405.0±27.6 ng/ml) and highest purity (1.93±0.30) with up to 10 CFU/ml of minimal detection level. Multiplex PCR was able to detect both bacteria simultaneously up to 105 CFU/ml and individually 10 CFU/ml for S. agalactiae and 103 CFU/ml for S. aureus with high specificity of primers indicated by absence of any cross reactivity with each other and with frequent mastitis causing bacteria. Multiplex PCR is more efficient than conventional PCRs and cultures, integrated with the selected DNA extraction method has a high potential to develop as a commercially available kit for routine diagnosis of subclinical mastitis.
Keywords: DNA extraction, Mastitis, Multiplex PCR, S. agalactiae, S. aureus
