Development and Validation of a qPCR Assay & Water Filtration Procedure for Detecting New Zealand Mud Snail Environmental DNA

Thursday, September 12, 2013: 9:00 AM
Fulton (Statehouse Convention Center)
John Wood , Pisces Molecular, Boulder, CO
Patrick Power , Pisces Molecular, Boulder, CO
We have developed and validated a qPCR assay and water filtration procedure for detecting the presence of environmental DNA shed by the invasive and widespread aquatic nuisance species New Zealand Mud Snail (NZMS), Potamopyrgus  antipodarum.  Our qPCR assay targets the junction of two P. antipodarum mitochondrial genes, rather than using primers in a single, highly conserved mitochondrial gene such as Cytochrome Oxidase I (CO1),  as a way of decreasing the likelihood of getting false PCR positives due to cross-reactivity in taxonomically complex filtered water samples.  Additionally, our qPCR assay uses a serial dilution series of plasmid DNA containing the NZMS target primer and probe sequences as a positive control and to generate the standard curve.  This allows an absolute quantitation of the efficiency and sensitivity of the qPCR assay and the number of NZMS target DNA molecules in a sample; the demonstrated sensitivity is less than 10 molecules per reaction.  The specificity of the assay for NZMS has been confirmed by testing DNA samples from a wide variety Hydrobiidae snails and showing no cross-reactivity.  Finally, we have developed and tested a water filtration procedure for collecting and filtering water samples in the field using hand tools and requiring no external power source.