TaqMan[R] MCB real-time PCR approach to quantification of Perkinsus marinus and Perkinsus spp. in oysters.

ABSTRACT Several molecular diagnostic assays have been developed in an attempt to replace the traditional Ray's Fluid Thioglycollate Medium (RFTM) assay for detection and quantification of Perkinsus marinus in oysters. Real-time PCR technology is a state-of-the-art method currently used to diagnose disease intensity in vertebrates. We developed a simple (two-reagent) real-time PCR assay to quantify P. marinus (PMAR) and Perkinsus spp. (PERK) in oysters, using TaqMan[R] assays designed with Minor Groove Binder (MGB) probes on an Applied Biosystems 7500 Real-Time PCR System. Both PERK and PMAR assays demonstrate strong correlations ([R.sup.2] [greater than or equal to] 0.99) between parasite cell density and real-time PCR threshold cycle (CT) with amplification efficiencies [greater than or equal to] 99%. The PERK assay results in similar amplification plots for the three species tested (P. marinus, P. olseni and P. chesapeaki), whereas the PMAR assay detects only P. marinus. A strong correlation ([R.sup.2] > 0.90) was found between infection level determined by the traditional RFTM method and quantification by real-time PCR, based on internal standards prepared from P. marinus spiked oyster tissue. The PCR assays also detected Perkinsus in oysters diagnosed as negatives using the traditional method, suggesting that the described assay may be more sensitive. These assays provide a nonsubjective, specific and accurate quantification of P. marinus in oyster tissues and thus could potentially replace the traditional method in some applications.

KEY WORDS: TaqMan[R] real-time PCR, Perkinsus, oysters

INTRODUCTION

The long and continuing decline of eastern oyster (Crassostrea virginica) fisheries has been partly attributed to the persistent and oftentimes catastrophic effects of the diseases MSX (caused by Haplosporidium nelsoni) and Perkinsosis (caused by Perkinsus marinus). The ability to develop and execute effective management strategies has necessitated the development of efficient tools by which the prevalence and intensity of infections can be evaluated in natural populations. Traditionally, P. marinus infections have been assessed using the Fluid Thioglycollate Medium (RFTM) assay (Ray 1966). This method involves the culture of the parasite prior to staining and visualization, and thus it necessitates that the parasite be viable at the time of processing. Several molecular assays have been developed that not only streamline the diagnostic process but also can be used to assess prevalence of Perknisus spp. in archived or frozen tissues, which could expand our understanding of the patterns in disease occurrence and the environmental factors that may influence them (see review by Villalba et al. 2004). Primers that target either the nontranscribed spacer (NTS) or internal transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene complex have been used in P. marinus specific PCR assays (Marsh et al. 1995, Robledo et al. 1998, Audemard et al. 2004). General primers have also been designed to detect virtually all Perkinsus species (Casas et al. 2002).

Real-time PCR technology is a state-of-the-art method used to quantify initial target levels of RNA and DNA and has gained wide use in clinical laboratory diagnostics (Kaltenboeck & Wang 2005). Whereas standard PCR methods analyze product formation during the plateau phase (or endpoint), real-time PCR quantification occurs during logarithmic amplification of the target, which can be more accurately related to initial target concentration. This difference is critical for the development of assays comparable to the RFTM method, which provides not only presence/absence of the parasite but also infection intensity. The utility of real-time PCR approaches in quantifying Perkinsus spp. was demonstrated by Audemard et al. (2004), who reported real-time PCR methods for the detection and quantification of Perkinsus in environmental samples.

Here, we report the development of a simple, efficient real-time PCR assay to quantify P. marinus and Perkinsus spp. cell density in oyster tissues using TaqMan[R] Assays, designed with Minor Groove Binder (MGB) probes, detected on an Applied Biosystems 7500 Real-Time PCR System and analyzed with SDS software. This real-time PCR method overcomes a common problem in real-time PCR protocols, specifically the need for efficient amplification in order for the assay to be accurate in the quantification of the target. The TaqMan[R] Assays pair specific forward and reverse PCR primers to amplify a relatively short amplicon (<150 bp). The targeting of a small amplicon serves to one, increase amplification efficiency and two, reduce false negatives that may result from DNA rearrangement events that can occur within larger target regions, which is particularly important when targeting the NTS or ITS regions. The specificity of the real-time PCR measurement is increased with the TaqMan[R] internal probe prepared with a 5' fluorescent reporter dye, a 3'quencher and an MGB tail to allow increased stringency (Kutyavin et al. 2000). The fluorescence of unbound probe is quenched until the probe binds to the amplicon and the 5' exonuclease activity of Taq polymerase permanently releases the 5' fluorescent signal. If the probe is released without having been cleaved, as in the case of mismatches encountered during the exonuclease activity, the two ends will resume their close association and no signal will be delivered.

The increased specificity provided by the inclusion of TaqMan[R] probes allows discrimination of comparatively minor differences between sequences. The combination of these factors (short target amplicons, specific probes) results in a sensitive, specific and accurate determination of P. marinus and Perkinsus spp. cell density in oyster tissue that not only increases the efficiency of diagnosing Perkinsus spp. in oysters but also provides a new tool that may extend our understanding of this important oyster pathogen.

MATERIALS AND METHODS

In vitro Cultures

P. marinus (P-1) cultures were obtained from J. La Peyre (Louisiana State University) and maintained as previously described (Gauthier & Vasta 1995). Cultured P. olseni and P. chesapeaki were obtained from the American Type Tissue Culture Collection (ATCC 50984 and 50807, respectively) and were used to test assay specificity.

TaqMan[R] Assay Design

The assays were designed and synthesized as Custom TaqMan[R] Gene Expression Assays (Applied Biosystems, Foster City, CA) and the target sequences for the designs were submitted after having been formatted with their File Builder software.

Two target sequences were chosen for assay design (Fig. 1), both within the ITS region previously targeted by Casas et al. (2002), and referenced by Audemard et al. (2004). After aligning these regions with similar regions from other Perkinsus species (obtained from GenBank), one 231 bp region was identified that appears to be conserved across all species within this genus, and the Perkinsus spp. specific (PERK) assay was designed to anneal within this region. The second target sequence was a region that appears to be conserved in all P. marinus records but contains a higher frequency of substitutions relative to other species. The first P. marinus specific (PMAR) assay was designed to anneal within this region. P. olseni was identified as having the most similar sequence in the PMAR region and was chosen for testing the specificity of the PMAR assay despite the low probability of its occurrence in local oysters. Other species, such as P. chesapeaki, are considered more likely to coinfect oyster populations in North Carolina but exhibited greater differentiation relative to P. marinus.

[FIGURE 1 OMITTED]

Although the PERK assay efficiently reported all Perkinsus species tested (see results) and the first PMAR assay efficiently reported P. marinus, the initial PMAR assay exhibited cross-reactivity with P. olseni samples. This could be attributed to the fact that there was only a single-base mismatch in the probe binding site in P. olseni and only a single-base mismatch in each of the two primers. To enhance the specificity, two of the oligonucleotides in the PMAR assay were redesigned. The first was a slight shift of the reverse primer to incorporate one additional base mismatch, whereas retaining its melting temperature ([T.sub.M]) of about 60[degrees]C. The other modification was to reduce the [T.sub.M] of the probe. Ideally the MGB probes are designed to have a [T.sub.M] of 70[degrees]C (Applied Biosystems, Foster City, CA). However, to provide greater discrimination between a perfect match to P. marinus and the slight mismatch to P. olseni, the probe was designed with a [T.sub.M] of about 66[degrees]C. The changes in the PMAR primers and probes did not increase the potential for cross reactivity with the other Perkinsus species. For example, when compared with the P. chesapeaki sequence, the new PMAR forward primer contained 4 mismatched base pairs, the reverse primer contained five mismatched base pairs, and the probe contained six mismatched base pairs--more than sufficient to avoid the generation of a false positive.

Preparation of P. marinus Standards for Real-time PCR

Cell density of P. marinus stock culture was determined by triturating cells through a 25-gauge needle and 5-mL syringe to break-up clusters prior to performing replicate hemocytometer counts. Replicate preweighed uninfected oyster mantle tissue samples (courtesy of Horn Point Laboratory, University of Maryland) were inoculated ("spiked") with 5, 50, 500, 5,000 or 50,000 P. marinus cells in a 10-[micro]L volume of culture medium for the preparation of parasite cell density standards (parasites/g wet weight oyster tissue). These spiked tissue samples were immediately lysed and DNA extracted as described below.

DNA Extractions

Oyster tissue samples were resuspended in 400 [micro]L PureGene Cell Lysis Solution (D-5002) along with 2 [micro]L of a 20-[micro]g/mL stock solution of proteinase K and incubated at 55[degrees]C overnight or until all tissue was dissolved. Solutions were allowed to reach room temperature prior to the addition of 140-[micro]L PureGene Protein Precipitation Solution (D-5003), vortexed vigorously for 20 sec and placed on ice for 5 min. Tubes were centrifuged at top speed on a microcentrifuge for 5 min and supernatant transferred to a clean labeled tube. The DNA was precipitated with the addition of 400-[micro]L 100% isopropanol then gently inverted 50 times prior to centrifugation for 5 min. The DNA pellet was washed with 400-[micro]L 70% ethanol and allowed to air dry prior to the addition of 35-[micro]L double deionized water (dd[H.sub.2]0). DNA concentration ([micro]g/ [micro]L = A260 nm x 0. 05 x dilution factor) was determined on a Genesys 8 spectrophotometer and all samples were adjusted to 100 ng/[micro]L. The 260-nm/280 nm absorbance ratio was used to determine the purity of DNA.

TaqMan[R] Reagents

The PERK specific primer/probe mix containing 18 [micro]M each of forward and reverse primers and 5-[micro]M TaqMan[R] probe, which was obtained as a 20x stock solution (ABI 4331348, Applied Biosystems, Foster City, CA). The PMAR specific primer/probe mix was prepared manually by diluting each 100-[micro]M primer (ABI 185312336-1,2) and 100-[micro]M probe (ABI 185312336) to 18 [micro]M and 5 [micro]M, respectively for a 20x stock solution. The TaqMan[R] Universal PCR MasterMix (ABI 4324018) containing AmpliTaq Gold DNA Polymerase, AmpErase without UNG, dNTPs with dUTP, and buffer components optimized as a 2x solution.

Real-time PCR

Reagents were added in the following proportions to each well of 96-well optical reaction plates (ABI 4306737): 12.5-[micro]L MasterMix, 1.25-[micro]L prime/probe, 9.25-[micro]L dd[H.sub.2]O, and 2-[micro]L of template (100 [micro]g/[micro]L) for a 25-[micro]L reaction volume. Plates were sealed with ThermalSeal RT (Excel Scientific, Inc). Real-time PCR was conducted on an ABI 7500 Real-Time PCR System using the ABI 7500 System SDS Software using the default temperature program (95[degrees]C/10 min; 40 cycles of 95[degrees]C/15 sec followed by 60[degrees]C/1 min) for a total run time of 1.5 h.

Specificity and Sensitivity of PERK and PMAR Assays

The specificity of PERK and PMAR primer/probe reagents was determined by conducting real-time PCR as described above on separate aliquots of 10-ng DNA from P. marinus, P. olseni and P. chesapeaki and observing each of the amplification plots relative to nontemplate controls. The sensitivity of the two assays was determined separately on 10-fold serial dilutions of P. marinus target DNA (10 pg-0.001 fg) in the presence of 100 ng oyster DNA.

Quantification of Perkinsus spp. in Naturally Infected Oysters

A comparison was made between P. marinus infection intensity determined by real-time PCR and the traditional RFTM assay using mantle tissue. Thirteen intertidal oysters were collected from Masonboro Sound at the UNCW Center for Marine Science (30 ppt, 24[degrees]C ambient conditions). Two ~5[mm.sup.2] pieces of mantle tissue were removed and placed in either 10-mL RFTM medium with antibiotics for the traditional method (Ray 1966, Mackin 1962, Craig et al. 1989) or microcentrifuge tube for real-time PCR analysis. Determination of parasite cell density in unknown samples was accomplished by including three or more standards (described above) for each real-time PCR run. The ABI 7500 software automatically plots the relationship between cell density and threshold cycle ([C.sub.T]) and converts [C.sub.T] values of unknowns to parasite cell density. The [C.sub.T] value represents the number of cycles required for target amplification to reach the exponential phase and is therefore inversely related to initial target concentrations.

Statistical Analysis

Linear regression analysis was conducted to determine the relationship between parasite cell density and [C.sub.T] value and between real-time PCR determination of infection intensity and semiquantitative estimates using the RFTM method.

RESULTS

Specificity and Sensitivity of PERK and PMAR Assays

The PERK assay resulted in similar amplification plots for P. marinus, P. olseni and P. chesapeaki DNA (Fig. 2A), whereas the PMAR assay resulted in amplification of P. marinus but not P. chesapeaki or P. olseni (Fig. 2B). The sensitivity of the PERK and PMAR assays were similar, detecting down to 10 fg P. marinus DNA in the presence of 100-ng oyster DNA (Fig. 3).

[FIGURES 2-3 OMITTED]

Standard Curves for PERK and PMAR Assays

A strong correlation ([R.sup.2] [greater than or equal to] 0.99; P < 0.0001) was observed between P. marinus cell density in oyster tissue and [C.sub.T] determined by PERK and PMAR real-time PCR assays (Fig. 4). Amplification efficiency ([[10.sup.(-1/slope)] -1]) for both assays was determined to be [greater than or equal to] 99%. These parasite cell density standards were included in all PCR runs to determine infection intensity in field collected oyster tissue described later.

[FIGURE 4 OMITTED]

Quantification of P. marinus in Naturally Infected Oysters

A strong correlation ([R.sup.2] [greater than or equal to] 0.90; P < 0.0001) was found between infection intensity determined by the traditional RFTM mantle tissue assay and both PERK and PMAR real-time PCR assays based on internal standards prepared from P. marinus spiked oyster tissue (Fig. 5). Both PCR assays detected parasites in oysters (2 out of 13) diagnosed as uninfected by the traditional RFTM method. All 13 oysters tested positive by PCR, but only 11 tested positive by the RFTM method.

[FIGURE 5 OMITTED]

DISCUSSION

The results presented suggest that the TaqMan[R] MGB approach provides an efficient and accurate method for not only quantifying the prevalence and intensity of P. marinus infections but also a tool for investigating patterns of coinfection involving other species of Perkinsus. The combination of the genus-specific (PERK) that targets an 86 bp region and the species-specific (PMAR) assays that targets a 72 bp region can be used to evaluate the extent to which positive Perkinsus diagnosis could be attributed to species other than P. marinus. Although not all reported Perkinsus species were tested, the PERK assay recognized both P. marinus and P. chesapeaki, which share the lowest sequence identity within the target region, based on the data available in GenBank. The specificity of the PMAR assay is demonstrated by the lack of recognition for P. olseni, the species with the greatest sequence identity to P. marinus within the target region.

Our results demonstrate that the PCR assays are an accurate alternative to the traditional mantle smear RFTM method for the assessment of disease intensity. Parasite cell density standards, prepared by spiking oyster tissue samples of known wet weight with known numbers of cultured P. marinus, were used to determine parasite density in naturally infected oysters and these estimates correlate well with those obtained using the RFTM method on the same oysters. Further, the linear relationship between the two methods allows us to convert the PCR data to familiar terms (Mackin Scale) if needed. Such agreement between methods allows for integration of data sets that rely on the different methods.

The reliance of the assay on the relatively easy to sample mantle tissue is also advantageous. Research has shown that although mantle tissue can be used fairly accurately to assess disease prevalence and intensity in oyster populations (Bushek et al. 1994, Oliver et al. 1998), the traditional RFTM method is known to result in false negative diagnoses at low infection levels (Choi et al. 1989). A major advantage of the PCR approach is the increased sensitivity with which mantle tissue samples can be analyzed, thus minimizing the likelihood of a false negative diagnosis. In this study, PERK and PMAR assays detected infections in oysters that were diagnosed as negative by the RFTM method.

Although the traditional RFTM method is approximately 3-fold less expensive, there are several advantages and particular applications of the real-time PCR assays: First, they can be used to distinguish between P. marinus and other Perkinsus spp. The ability to determine the extent to which infected oysters harbor more than a single species is of considerable interest. Nonspecific quantification could result in overestimation of P. marinus levels in areas where different species co-occur. Whereas the small numbers of natural oysters evaluated during this study exhibited similar infection intensities when tested with the generic and specific assays (suggesting an absence of multiple infections), a more substantial study would provide a more definitive answer to this question. Further, the ability to specifically attribute infections to P. marinus may facilitate the evaluation of other species to act as potential reservoirs.

A second advantage is that the PCR assays eliminate the subjectivity associated with estimation of infection intensity using a semiquantitative rating system. The potential for subjective error poses a problem when comparing year-to-year samples, particularly when there are multiple investigators involved. Third, the assays are extremely sensitive, detecting down to 10 fg of P. marinus DNA in the presence of 100 ng oyster DNA. This sensitivity is likely to result in a more accurate assessment of disease prevalence and intensity. Fourth, the assays provide an accurate, quantitative measure of parasite cell density in oyster tissue samples. The linear relationship between known parasite cell density and [C.sub.T] is used to quantity infection level in unknown tissue samples. Fifth, these assays can be accomplished in 1-2 days if needed as opposed to 5-7 days required for the RFTM assay. The assays are also relatively simple molecular protocols with only two reagents involved in setting up the PCR reaction. Lastly, the assays can make use of frozen samples or archived DNA, whereas the RFTM method requires that parasites be kept alive until processing. This attribute is allowing us to assess oyster disease patterns in North Carolina over the past several years from archived samples. Oyster tissue can also be sent through the mail in cell lysis solution without refrigeration for processing elsewhere.

The advantages associated with the adoption of real-time PCR strategies for the quantification of Perkinsosis in oysters may be offset in part by the greater expense associated with such techniques. Further refinement and automation of the assay will undoubtedly reduce the costs, and the advantages of the real-time assays will, in some cases, more than justify the additional costs. The linear relationship between the PCR assay determination of parasite cell density and the traditional semiquantitative rating scale allows us to relate results from the two methodologies.

ACKNOWLEDGMENTS

The authors thank J. La Peyre for providing initial P. marinus cultures and S. Alexander for hatchery reared uninfected oysters; T. Alphin and S. Colosimo for their advice and help with oyster field samples and the DNA core facility for use of its ABI 7500 Real-Time PCR System. The comments of two anonymous reviewers improved the manuscript. This research was funded in part by the Marine Biotechnology program at the UNCW Center for Marine Science.

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