Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam ( Mercenaria mercenaria )

Abstract

We developed a real-time quantitative PCR (qPCR) assay targeting the rRNA internal transcribed spacer region of the hard clam pathogen QPX. The qPCR assay was more sensitive than was histology in detecting clams with light QPX infections. QPX was detected in 4 of 43 sediment samples but in none of 40 seawater samples.The thraustochytrid called QPX (for quahog parasite unknown) has caused high mortalities in hatchery-reared and wild hard clams (Mercenaria mercenaria, also known as quahogs) from Prince Edward Island (Canada) to Virginia (United States) since the late 1950s (17,22,25,29). In the summer of 2002, QPX infections appeared in the previously healthy Raritan Bay (off the coast of Staten Island in New York) M. mercenaria population, causing significant clam mortality and closure of the fishery (6). Management of hard clam populations affected by QPX disease is hampered by an incomplete understanding of factors controlling the occurrence and severity of QPX infections. Environmental factors, such as salinity and temperature, appear to be important (22), as do clam population density and the planting of seed from nonlocal sources (7). More quantitative information about the occurrence and progression of QPX disease in relation to these and other variables would support better prediction of, and response to, QPX outbreaks. QPX is thought to be an opportunistic pathogen (4, 7, 11), capable of growing outside its host. However, there is very little known about substrates that might support QPX organisms outside of hard clams (4). The abilities to detect and enumerate QPX cells in potential reservoirs would allow the dynamics of the QPX organism in the environment to be related to the occurrence of QPX disease, offering new insight into fundamental questions about the natural transmission mechanisms of the infection.The 18S ribosomal DNA (rDNA) primer pair QPX-F and QPX-R2 can be used in a standard PCR assay to detect the presence of QPX DNA in clam tissue samples (26). Unfortunately, the products are too long (ϳ650 bp), and often include too much primer dimer, for use in a SYBR green real-time quantitative PCR (qPCR) assay. The low sequence variability in rRNA genes made it difficult to design other primers specific for QPX 18S rDNA. Instead, we used our previously reported rRNA internal transcribed spacer (ITS) region (including ITS1, the 5.8S rRNA gene, and ITS2) sequences for QPX isolates from Massachusetts and New York (20) to develop a qPCR assay targeting the more variable ITS region (1).Development of QPX-specific real-time qPCR assay. The ITS regions of the thraustochytrids Schizochytrium aggregatum (ATCC 28209), Schizochytrium limacinum (ATCC MYA-1381), and Thraustochytrium aureum (ATCC 34304) (acquired from the American Type Culture Collection, Manassas, VA, and maintained in medium 790 Byϩ at 23°C) were PCR amplified with universal 18S and 28S rDNA primers (18S-RR and 28S46Rev) (Table 1), cloned and sequenced as described previously (20), and submitted to GenBank (http://www.ncbi.nlm .nih.gov) under ...

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

“…Increasing the amount of host tissue used in an assay can help re duce the frequency of false negatives due to sampling error (Iwanowicz et al 2015). As a preventative measure, the whole mantle could be used instead of, or in addition to, a sample of mantle tissue, as was done by Liu et al (2009) with Mercenaria mercenaria (Linnaeus, 1758) infected with QPX. Alternatively, DNA from a cross-section that includes samples of each mussel tissue (as is prepared for histopathological examinations) could be used in molecular assays.…”

“…Although most samples amplified using both 1:10 dilutions and undiluted DNA, some samples only amplified using one or the other. For future use of the end-point qPCR assay it is recommended to use both undiluted DNA and 1:10 dilutions, or to use a secondary purification along with both dilutions as was done by Liu et al (2009). In the qPCR assay all samples amplified from the 1:10 DNA dilutions, so the use of 1:10 dilutions is re com mended.…”

“…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.…”

A Re-Visit to the Evolution and Ecophysiology of the Labyrinthulomycetes

“…Clone libraries of SSU rRNA from water and environmental samples facilitate the investigation of natural communities and unknown lineages in various habitats (Massana et al, 2004a, b). Fluorescent in situ hybridisation probes (FISH) and quantitative PCR probes have also been developed for detection of thraustochytrids (Takao et al, 2007), and QPX from marine water simultaneously (Liu et al, 2009). …”

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location

“…Indeed, molecular diagnostic assays based on ITS are abundant in the plant pathology and human medical literature (see review by Iwen et al 2002), and have been developed for pathogens of aquatic species including bivalves (e.g. Audemard et al 2004, Maloy et al 2005, Liu et al 2009), crustaceans (e.g. Small et al 2007) and finfish (e.g.…”