Inhibition of PCR amplification by phytic acid, and treatment of bovine fecal specimens with phytase to reduce inhibition.
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.
Integrated Research Technology, LLC, c/o Quest Diagnostics Incorporated, 1901 Sulphur Spring Road, Baltimore, MD 21227, USA. IntegratedResTek@cs.com
Charles G
CG
Thornton
Selvin
S
Passen
D010832:6-Phytase / Q000737:chemistry; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005243:Feces / Q000737:chemistry / Q000382:microbiology*; D010833:Phytic Acid / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*
D004279:DNA, Viral; D004791:Enzyme Inhibitors; D010833:Phytic Acid; D010832:6-Phytase