title: 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.
authors.affiliation: School of Veterinary and Biomedical Sciences, South Street, Murdoch, Western Australia 6150. nicolai@bonne.no
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bonne
authors.forename: Phillip
authors.initials: P
authors.lastname: Clark
authors.forename: Patrick
authors.initials: P
authors.lastname: Shearer
authors.forename: Shane
authors.initials: S
authors.lastname: Raidal
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000821:virology*; D001717:Birds; D018173:Circoviridae Infections / Q000662:veterinary* / Q000821:virology; D017925:Circovirus / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D005189:False Positive Reactions; D005506:Food Contamination; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013048:Specimen Handling / Q000379:methods / Q000662:veterinary*
chemical_list: D004279:DNA, Viral