Inhibition of PCR amplification by phytic acid, and treatment of bovine fecal specimens with phytase to reduce inhibition

Abstract

Development of effective polymerase chain reaction (PCR)-based diagnostic tests using ruminant fecal specimens has been thwarted by excessive inhibition. A PCR system based on amplification of 1000 copies of bacteriophage λ-DNA was used as a model to evaluate inhibition levels in bovine feces. Dilution experiments using a bovine fecal specimen suggested that as little as 40 μg of feces (in a 100-μl PCR) affected the efficiency of amplification. It was discovered that phytic acid (the hexaphosphoric ester of inositol) is a powerful inhibitor of PCR. Above 0.3 mM phytate, the PCR is completely inhibited. In a very narrow range around 0.2 mM target-specific amplification proceeds efficiently. At concentrations between 10 and 100 μM, phytate nonspecific amplification (e.g., primer–dimer formation) is dominant. Below 10 μM, phytate target-specific amplification proceeds efficiently. A simple processing procedure using 50 units/ml of Aspergillus niger 3-phytase [E.C. 3.1.3.8] was developed that reduced PCR inhibition levels in bovine fecal specimens by approximately 500-fold.