A nested quantitative PCR assay for detection of the hard clam pathogen Mucochytrium quahogii (=QPX) in environmental samples

Abstract

Progress in understanding and managing QPX disease outbreaks in hard clams (Mercenaria mercenaria) has been limited by lack of insight into basic aspects of the biology and ecology of the opportunistic pathogen Mucochytrium quahogii (formerly QPX or Quahog Parasite Unknown). One barrier is that while several methods have been able to detect M. quahogii in seawater and sediment, its abundance was typically too low for reliable quantification by those methods. Here we describe the development and validation of a sensitive, M. quahogii-specific, nested quantitative PCR (nqPCR) assay following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. The assay reaches the theoretical limit of detection (LOD) of a PCR assay at 3 copies per reaction with excellent efficiency, linearity, and minimal sample PCR inhibition. The functionality of the assay was evaluated by quantifying M. quahogii in sediment and seawater samples, which revealed that M. quahogii was broadly distributed throughout the marine environment, detected in 75% of samples, with mean estimated abundance of 0.21 cells per mg sediment, 0.55 cells per ml bottom seawater, and 0.02 cells per ml surface seawater. M. quahogii was most prevalent and most abundant in sediment and bottom seawater samples, suggesting that the flocculent layer at the sediment-water interface is an important environmental reservoir where M. quahogii may interact with hard clams. This assay will serve as a valuable tool to better understand QPX disease dynamics and offers a model to guide development of similar assays for other important marine microbes typically present at similarly low abundance.

Mucochytrium quahogii (=QPX) Is a Commensal, Opportunistic Pathogen of the Hard Clam (Mercenaria mercenaria): Evidence and Implications for QPX Disease Management

“…In contrast, at site 20B in Great Kills Harbor, QPX disease is extremely low despite an even greater clam density (>250 clams/m 2 ). We also included a site in the Peconic Estuary in our survey since it was historically used as the receiving bay for the New York Shellfish Transplant Fishery [16].…”

“…For each sampling time point, we randomly selected up to 22 clams from the cohort of up to 30 clams collected; at some sites, clams were scarce and therefore number of clams assayed from each cohort varied from 7 to 22 individual clams (mean ± SD = 16 ± 2) for a total of 977 clams in 59 cohorts. Since pallial fluid was a new sample type, PCR inhibition testing was performed by spiking in linear plasmid to create a dilution series (1:10) to measure PCR efficiency and linearity [16,19]. A representative group of samples was assessed, revealing that inhibition was minimal with PCR efficiency and linearity within acceptable limits (10% compared to the standard curve) [20].…”

“…Environmental DNA was extracted with the MO BIO PowerSoil DNA isolation kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, eluted in 100 µL and stored at −20 • C until assayed. Samples were assayed in triplicate using the newly developed nested, quantitative PCR (nqPCR) assay for increased sensitivity, using 3 µL of template DNA [16,19]. Environmental samples were also assayed for total labyrinthulomycetes using a newly developed qPCR in triplicate using 1 µL of template DNA [16].…”

“…Samples were assayed in triplicate using the newly developed nested, quantitative PCR (nqPCR) assay for increased sensitivity, using 3 µL of template DNA [16,19]. Environmental samples were also assayed for total labyrinthulomycetes using a newly developed qPCR in triplicate using 1 µL of template DNA [16]. All quantitative data determined by qPCR (i.e., quantifiable results but not BLD) and nqPCR assays are expressed in terms of gene copies per mg tissue or sediment or mL pallial fluid or seawater.…”

“…Research focused on its pathobiology has revealed that QPX disease is likely facilitated by specific environmental factors, such as low temperature and high salinity, that give M. quahogii an advantage in the host-pathogen interaction [2,3,8]. However, there remain major gaps in our knowledge about the basic biology and ecology of M. quahogii.…”