Elimination of False-Positive Polymerase Chain Reaction Results Resulting from Hole Punch Carryover Contamination

Abstract

The collection of biological material (e.g., blood) directly onto filter paper for subsequent use in laboratory assays such as polymerase chain reaction (PCR), has become a common practice. Dried cells or fluid on the paper can be readily rehydrated and retrieved into a standard volume of an appropriate elution buffer but introduces a dilution factor to the sample. The use of a common cutting instrument for excising a standard-sized piece of paper that contains the material also introduces the potential for transferring biological material from one sample to subsequent samples, causing false-positive results by PCR. In the present study, filter-paper-collected blood that contained beak and feather disease virus was used to determine if viral DNA could be transferred between samples by a hole punch used to excise sequential filter papers. It was determined that false-positive results could be obtained at least 13 times after a positive sample. Subsequently, the efficacy of 4 methods of hole punch disinfection, flaming, VirkonS, bleach, and a bleach-ethanol combination, was assessed. The only effective and practical method to destroy DNA was a method where the hole punch was agitated in commercial bleach, rinsed in water, the water was displaced with 100% ethanol and air-dried. This method was simple, cheap, and relatively rapid, and allowed for the use of a single hole punch for a series of samples, without carryover contamination and consequent false-positive results.