Detection of Cow Milk in Water Buffalo Cheese by SYBR Green Real-Time PCR: Sensitivity Test on Governing Liquid Samples

Maria Feligini, Naima Alim, Ivan Bonizzi, Giuseppe Enne and Riccardo Aleandri

A real-time polymerase chain reaction (PCR) assay was developed to detect a bovine-specific mitochondrial DNA sequence in "buffalo" Mozzarella cheese by using primers targeting the cytochrome oxidase subunit 1 (COI) gene. Hot-start PCR, primer design, annealing and signal acquisition temperatures were exploited to obtain reliable analytical conditions which yielded a 134-bp amplicon from cow's DNA only. Water buffalo's DNA didn't originate any amplification product. DNA isolated from blood was used to test primers' specificity and to construct a calibration curve in order to quantify bovine DNA concentration in governing liquid. The method was capable to detect low amounts of cow's DNA in governing liquid samples. Due to low detection limit and fast, simple execution, the analytical protocol described in this work is suitable to become a common tool to detect the fraudulent addition of cow milk in water buffalo Mozzarella cheese.