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

Abstract

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 bleachethanol 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.

Laser Cutting Eliminates Nucleic Acid Cross-Contamination in Dried-Blood-Spot Processing

“…For instance, false-positive results for beak and feather disease virus (BFDV) were propagated into 13 samples processed after a BFDV-positive sample (1). BVDF contamination was removed by bleach, water, and ethanol washes, followed by air drying, but such steps are laborious and poorly suited for highthroughput labs.…”

Assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease

“…Dried blood spots were excised from the filter paper using a stationary hole puncher (OfficeWorks) according to the methods previously described by Bonne et al (2008), deposited into 1.5 ml Eppendorf tubes and DNA extracted using a QIAamp DNA Blood Mini kit (Qiagen). PCR for detection of BFDV DNA was performed as described by Ypelaar et al (1999).…”

Where are the weapons of mass destruction — the Mycobacterium paratuberculosis in Crohn's disease?

“…How was the microtome knife, tissue receiving platform and water bath (where the sections are spread out before being mounted on slides) decontaminated between cuts and between patients and the various controls? 60,61 Were the tissues of more than one patient, or control, cut on the same day, in other words in series at the microtome? Were stain baths free of floaters or other contaminants?…”