Volume 6, Issue 5, October Issue - 2018, Pages:808-815
Authors: K. M. Chandrashekar, Shrikrishna Isloor, D. Rathnamma, B.M. Veeregowda, Raveendra Hegde, R. Sharada, G. S. Naveen Kumar, Nagendra R. Hegde
Abstract: The present study explores the application of conventional PCR and Mole v/s Avogadro’s number using single copy genes of Staphylococcus aureus and Escherichia coli for assessing bacterial load in milk samples from mastitis cases. The nuc and uidA genes produced the lowest detection limit for Staph. aureus and E. coli respectively. The present findings estimated that, as few as 60 and 89 genome copies or organisms of Staph. aureus and E. coli were detected, respectively. Based on the LOD of molecules, standard graphs for these genes were generated and this knowledge was applied to milk samples from the field. Spiking known amount of genomic DNA (which in turn indicates organisms based on Avogadro’s number) in LB broth and pasteurized milk was carried out to compare the input with output and simulating field conditions. A total of 90 samples from subclinical cases of mastitis were collected from four organized farms located at various villages. Out of 90 samples, 29 (32.22 %) were showed culture and duplex PCR positive. Of these, 12 (13.33 %) were found to be positive for E. coli alone, nine (10 %) were found to be positive for Staph. aureus alone, and eight (8.88 %) carried a mixed infection for both. In conclusion, this study suggests that quantification of bacteria directly from milk by the conventional PCR can be an alternative to time consuming conventional culture method and expensive real-time PCR, but requires extensive standardization.