Thornton Charles G, Passen Selvin
Integrated Research Technology, LLC, c/o Quest Diagnostics Incorporated, 1901 Sulphur Spring Road, Baltimore, MD 21227, USA.
J Microbiol Methods. 2004 Oct;59(1):43-52. doi: 10.1016/j.mimet.2004.06.001.
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 lambda-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 microg of feces (in a 100-microl 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 microM, phytate nonspecific amplification (e.g., primer-dimer formation) is dominant. Below 10 microM, 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.
