Detection and quantification of the free-living stage of the parasitic dinoflagellate Hematodinium sp. in laboratory and environmental samples

Overwintering of the parasitic dinoflagellate Hematodinium perezi in dredged blue crabs (Callinectes sapidus) from Wachapreague Creek, Virginia

“…Included in all PCR assays was a negative control that consisted of no DNA, and a positive control consisting of a sample of H. perezi DNA that had routinely amplified in past PCR studies. We did not use a quantitative PCR method of Li et al (2010) because it was optimized for environmental samples, not hemolymph samples.…”

“…Hemolymph smears using vital stains, prepared histological slides, PCR assays and in some cases altered body coloration indicative of advanced infections have been used for many years (see Stentiford & Shields, 2005). Molecular studies have progressed from diagnosis using PCR assays with genus-specific probes (Hudson & Adlard, 1994;Gruebl et al, 2002;Hamilton et al, 2007;Small et al, 2007Small et al, , 2012, to more refined tools for detection in environmental samples (Frisher et al, 2006;Hamilton et al, 2011), and for real-time PCR in environmental samples (Li et al, 2010;Hanif et al, 2013), as well as the development of more advanced or novel tools, such as the application of high performance liquid chromatography to detect pathogens (Troedssson et al, 2008), use of microsatellite markers for population genetics (Pagenkopp Lohan et al, 2014), and development of a novel dinoflagellate/viral nucleo-protein technique for visualizing the parasite in tissues (Gornik et al, 2013). However, none of these studies have compared methodologies using the epidemiological reference points of sensitivity and specificity.…”

Disease ecology of Hematodinium perezi in a high salinity estuary: investigating seasonal trends in environmental detection

“…Raw qPCR numbers were then used to calculate cells l −1 under the assumption that 300 copies of rDNA exist per cell (Li et al 2010). A cell count was performed to test this assumption, using a neutral red stain (as described by Li et al 2010) to identify parasite cells from a heavily infected blue crab.…”

“…A cell count was performed to test this assumption, using a neutral red stain (as described by Li et al 2010) to identify parasite cells from a heavily infected blue crab. A comparison was made between the estimated cell count obtained with a hemocytometer and by calculating cells ml −1 from the raw copy number generated by qPCR.…”

“…Also included in each run were the negative and positive controls from each batch of purifications. The purification negative control was used to determine back-171 Dis Aquat Org 124: [169][170][171][172][173][174][175][176][177][178][179] 2017 ground amplification and set the limit for what was con sidered negative within that batch of DNA purifications.Raw qPCR numbers were then used to calculate cells l −1 under the assumption that 300 copies of rDNA exist per cell (Li et al 2010). A cell count was performed to test this assumption, using a neutral red stain (as described by Li et al 2010) to identify parasite cells from a heavily infected blue crab.…”