Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus

Abstract

Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10 000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 µl of serum, yielding a theoretical detection limit of 5 cells ml -1 hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen.

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

“…Similarly, we have found that the addition of BSA can counteract the inhibitory effects of remaining environmental inhibitors during diagnosis of Hematodinium using RT PCR. Thus, the use of BSA in the RT PCR protocol for diagnosis and quantification of Hematodinium in environmental samples is essential when inhibition is likely, as has been reported by others (Nagle et al, 2009).…”

“…(H.J. Small unpublished results), and based on an alignment of available SSU rRNA gene sequences, the primers developed by Nagle et al (2009) would also amplify the DNA of all Hematodinium and Hematodinium-like spp., as well as that of many other Syndiniales. As such, the above assays are genus-specific at best, but may also cross react with organisms in closely related genera.…”

“…Frischer et al (2006) and Nagle et al (2009) developed real-time PCR assays for the detection and enumeration of the Hematodinium sp. infecting C. sapidus targeting regions of the SSU rRNA gene.…”

Development of quantitative PCR assay for detection of the trematode parasite Proctoeces maculatus in the blue mussel Mytilus edulis

“…The Qiagen DNeasy Blood and Tissue Kit, for example, has been shown to be very efficient (Bletz et al 2015) and could be used for future DNA extractions, but this would increase the cost and processing time of the assay considerably. Additionally, DNA extraction methods are unable to completely separate DNA from PCR inhibitors (Liu et al 2009, Nagle et al 2009). For this reason, dilutions were used in the PCR assays in order to decrease PCR inhibition.…”

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

“…Based on the previous work of Nagel et al (2009), qPCR detection was used as an indicator of infection status in both hemolymph and muscle tissue. The ITS2 primer set and the protocol described in Hanif et al (2013) were used for all samples in this study.…”