INVESTIGATION OF SAMPLE STORAGE CONDITIONS FOR OPTIMIZED DETECTION OF FROG VIRUS 3 DNA IN CHELONIAN SWAB SAMPLES

The collection and storage of swab samples for molecular diagnostics is a routine component of wildlife health surveillance. The suitability of different sample storage conditions for maximizing the recovery of pathogen DNA in most species has not been assessed; therefore, the aim of this study was to identify a preferred storage method for swabs collected for the detection of frog virus 3 (FV3), a significant chelonian pathogen. Sterile swabs were inoculated in triplicate with a plasmid containing known quantities of FV3 DNA from 100 to 107 copies. Swabs were then stored under one of the following four conditions: 1) dry frozen at -20 °C; 2) immersed in phosphate-buffered saline (PBS) and frozen at -20 °C; 3) immersed in a nucleic acid preservative (RNAlater) and frozen at -20 °C; and 4) immersed in 100% ethanol at ambient room temperature. Swabs remained undisturbed under designated storage conditions for 14 d, at which point DNA extraction and conventional and quantitative PCR for FV3 detection were performed. Conventional PCR amplified down to the lowest expected FV3 target copy number (10,000 copies/swab) for the dry-frozen and PBS-frozen treatment groups. Conventional PCR amplification was inconsistent for the ethanol and RNAlater treatment groups. Quantitative PCR on dry-frozen samples successfully amplified as low as 100 FV3 target copies/swab with a mean recovery of 90%, with all other storage methods amplifying only down to 10,000 copies/swab. Findings suggest there is improved detection of pathogen DNA for samples stored from collection to extraction under the dry-frozen method. Swab sample storage recommendations for future applications should be observed within the context of study-specific objectives and target pathogens. Furthermore, failure to detect fewer than 100 copies/swab of FV3 from any storage method may have clinically significant ramifications and suggests that the differences in DNA recovery based on extraction method should also be examined.