Interference of Real-Time PCR Quantification of Vibrio vulnificus by a Novel DNase from the Eastern Oyster (Crassostrea virginica)

Abstract

A novel DNase from oyster tissue with an optimum temperature at 65°C was found to interfere with the real-time PCR (Rti-PCR) quantification of Vibrio vulnificus. The DNase activity of oyster extracts at 65°C was 7.5-fold higher than that at 35°C after 30 min of DNA digestion and was linear at 50°C, 55°C, 60°C and 65°C for at least 80 min. The oyster DNase activity was strongly inhibited by EDTA, suggesting that EDTA sequestered the enzyme cofactor Mg++. The Ct values from Rti-PCR amplification of DNA from V. vulnificus cells heated at 65°C or 70°C in the presence of oyster tissue were larger than the Ct values from DNA derived from cells heated at these temperatures in the absence of oyster tissue, which suggested that oyster DNase is a potential PCR inhibitor. These observations suggest that Rti-PCR inhibition was most probably due to the DNase entering the cells at 65°C and 70°C. Activated carbon coated with benonite was used to remove oyster DNase and other PCR inhibitors, resulting in a minimum detection level of 10 CFU of V. vulnificus per gram of oyster tissue, which was a 10-fold higher level of detection sensitivity than the 100 CFU/g obtained with the Wizard DNA purification system.