Real Time-PCR coupled with melt curve analysis for detecting the authenticity of camel milk

Abstract

The study evaluated the use of Real Time-Polymerase Chain Reaction (RT- PCR) to detect the adulteration of camel milk with goat, cow milk. DNA was isolated from camel milk, camel milk powder, camel milk soap, cow milk, and goat milk using DNA extraction kit. RT- PCR amplified a single piece of DNA into millions of copies. The camel specific primers were designed using the primer- 3 online software and quantification of the isolated DNA was carried out by RT- PCR system through DNA standard curves and cycle threshold (Ct) values. The detection limit of DNA template was in the range of 0.001–0.002%. The reaction mixture (20μL) contained 10 μL SYBR Green master mix, 0.3 μL of 10 μM of each primer and 5 μL DNA. Thermal cycling consisted of an initial denaturation at 95 °C for 1 min, followed by 40 cycles for 15 s at 95 °C and 60 °C for 30 s. The primer pairs used were confirmed for their PCR efficiency, and specific products were evaluated by melt curve analysis. Results indicated positive amplification for the camel milk, camel milk powder, and camel milk soap but negative amplification for cow and goat milk. In conclusion, the RT- PCR based identification is a low cost and appropriate method for camel milk and its products. Although, the yield of DNA from camel milk soap after isolation is low but the isolated DNA segment was easily identified.