Introduction

Vibrio parahaemolyticus, a Gram-negative, facultative halophilic bacterium is commonly found in warm coastal waters worldwide (Twedt 1989). Shellfish and other bivalves accumulate this pathogen in their tissue during filter-feeding, and could cause gastroenteritis in humans who consumed raw or poorly cooked seafood (DePaola et al. 1990). Although the illnesses caused by this organism are usually self-limiting, in some severe cases dehydration or bloody diarrhea may persist, resulting in hospitalization of the patient and/or prolonged antibiotic treatment (Shevchuk et al.1986; Miyamoto et al.1969; Morris 2003). Sporadic cases of V. parahaemolyticus-related gastroenteritis due to the consumption of raw oysters in humans primarily along the coastal states in the United States and other countries such as Japan and South East Asia have been documented (Alam and Miyoshi 2003; DePaola et al.1990, 2003; Hara-Kudo et al.2003; Ijumba 1984; Robert-Pillot et al.2004). The first recorded outbreak in the United States was reported in 1971 in Maryland (CFSAN 2000) including 10 cases followed by a relatively larger outbreak in 1998 in the Pacific Northwest United States affecting 209 individuals and including 1 death (CDC 1999). This has led to the Interstate Shellfish Sanitation Conference (ISSC 2000), FDA and seafood industry to seriously consider the development of a nucleic acid-based rapid detection of this pathogen to ensure the safety of post-harvest processed (PHP) shellfish for human consumption at the highest level, thereby protecting consumer health. Real-time polymerase chain reaction (real-time PCR) methods of pathogen detection, using fluorogenic DNA-binding dyes or fluorescent-labeled oligonucleotide probes, have been shown to be effective, rapid and sensitive method for detection of vibrios (Fukushima et al.2003; Kim et al. 2008; Lo et al. 2008; Panicker et al.2004; Qin et al. 2008; Rosec et al. 2009; Wittwer et al. 2001). Using this approach, recently a nucleic acid-based real-time PCR with Taqman™ fluorescence probes targeting V. parahaemolyticus three hemolysin genes, the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh) (Tada et al.1992; Takeda 1982; Taniguchi et al.1985, 1986) and thermostable-related hemolysin (trh) genes have been described for rapid detection of total and pathogenic strains of this pathogen in shellfish (Membrillo-Hernandez 1997; Nordstrom et al.2007; Ward and Bej 2006). In another study, Taqman™ probe-based real-time PCR was developed for the detection of a newly emerged pandemic V. parahaemolyticus O3:K6 in Gulf of Mexico water targeting a segment of the ORF8 DNA (Okuda et al.1997; Myers et al.2003 Rizvi et al.2006). However, since the first appearance in 1996 in the U.S. causing an outbreak of gastroenteritis disease, the pandemic V. parahaemolyticus O3:K6 serogroup strain has not been further isolated. Therefore, the importance of this pathogen in the U.S. coastal waters and seafood harvested from these areas has become less significant. Although the Taqman™ probe-based real-time PCR has been shown to be effective for rapid and specific detection of this pathogen, this approach could be relatively costly. Therefore a cost-effective detection method for routine monitoring of PHP oysters that will maintain the highest level of product safety without compromising the market cost of the product is desirable. Recently, real-time PCR using DNA-binding fluorescent dye SYBR green has been reported for the detection of pathogenic V. parahaemolyticus in shellfish targeting a single gene tdh (Tyagi et al. 2009). In addition, conventional PCR detection of V. parahaemolyticus in Middle Black Sea Coast of Turkey by targeting the tlh, tdh and trh genes has been reported (Terzi et al. 2009). Here we describe a rapid, specific, sensitive and cost-effective multiplexed SYBR Green™ I-based real-time PCR detection of total and pathogenic V. parahaemolyticus in shellfish and gulf water targeting all three targeted genes tlh, tdh, and trh simultaneously in a single reaction in an effort to reduce shellfish-related V. parahaemolyticus-illnesses.

Materials and methods

Bacterial strains and microbiological media

All V. parahaemolyticus strains used in this study, along with other various Vibrio strains, are listed in Table 1. V. parahaemolyticus was cultured in T1N1 broth medium [10% (w/v) tryptone, 1% (w/v) NaCl] or on T1N3 agar plates (10% tryptone, 3% NaCl) (Atlas 1993) at 35°C overnight. All other strains were grown and maintained as follows: all V. vulnificus strains were grown on one-half strength marine agar or broth (Becton–Dickinson, Franklin Lakes, NJ); V. cholera was grown on Luria–Bertani (LB) agar or broth (Atlas and Bej 1990); V. alginolyticus, V. campbelli, V. furnisii, and V. mimicus were all grown on full strength marine agar or broth.

Table 1 List of strains used in this study and the results from the specificity of the multiplexed real-time PCR targeting the tlh, tdh, and trh genes
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DNA purification for PCR optimization

Genomic DNA from an overnight (1.5 ml) culture of V. parahaemolyticus F113A was purified by following method described by Ausubel et al. (1987). Purified DNA was resuspended in Tris–EDTA (pH 8.0) buffer (Ausubel et al. 1987) and the concentration measured in a Lambda II spectrophotometer (Perkin-Elmer, Shelton, CT) at a wavelength of 260 nm.

Oligonucleotide primers

A segment of the thermolabile hemolysin encoding gene, tlh, was targeted for the detection of all V. parahaemolyticus; thermostable direct hemolysin encoding gene, tdh, and the tdh related gene, trh were also targeted for the pathogenic strains of V. parahaemolyticus. The description of the primers including the nucleotide sequence, position within the gene, length and the T m values are presented in Table 2. The T m values of all of the primers were determined using the equation [T m (°C) = 2(A + T) + 4(G + C)] (Bej et al. 1991; Rychlik and Rhoads 1989). All primers were custom synthesized by Integrated DNA Technology, Inc., Coralville, IA.

Table 2 Description of oligonucleotide primers, amplicon size and the melt temperature of the amplicons observed after real-time PCR amplification with SYBR Green™ I
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Optimization of PCR

Purified DNA from V. parahaemolyticus F113A was used for the optimization of the PCR (Bej et al. 1999). The PCR consisted of 1× PCR buffer [10× buffer consisted of 200 mM Tris–Cl (pH 8.4), 500 mM KCl], 1.5, 2.5 or 3.5 μl of PCR Enhancer mixture (Invitrogen, Inc., Carlsbad, CA), 2.5, 3 or 4 mM MgCl2; 0.2, 0.6 or 1 μM each of the tlh, tdh and trh primers, 200 μM of each dNTP (Sigma-Aldrich), 1×, 2× or 4× SYBR Green™ I nucleic acid fluorescent dye (Roche, Basel, Switzerland), three units of thermostable DNA polymerase (New England Biolabs, Beverly, MA) and autoclaved (121°C under 15 psi for 20 min) MilliQ water (Millipore, Bedford, MA) to bring the total reaction volume to 25 μl.

The PCR temperature cycling parameters used for the optimization of the amplification reaction are described below: initial denaturation of the template DNA at 95°C for 120 s followed by ample number of amplification cycles to surpass the threshold cycle by six cycles. These amplification cycles consisted of denaturation of the template DNA at 94°C for 15 or 20 s, primer annealing at 52°C, 56°C or 60°C for 15 or 30 s, and primer extension at 72°C for 25 or 45 s. All PCR amplification reactions were performed in a Cepheid™ Smart Cycler instrument (Cepheid, Sunnyvale, CA). Following PCR amplification, the sizes of the amplicons were confirmed by 2% (w/v) NuSeive 3:1 (FMC Bioproducts, Philadelphia, PA) agarose gel (Ausubel et al. 1987), and the results were documented digitally using a Kodak ds120 photo documentation system and 1D image analysis software (ver. 3) (Kodak, Rochester, NY).

Specificity of the PCR

The specificity of the primers to their respective targeted genes was confirmed by using the BLAST search program (www.ncbi.nlm.nih.gov) and by PCR amplification in multiplexed format on 44 V. parahaemolyticus strains and other Vibrio spp. using the PCR parameters described above (Table 1).

Sensitivity of the PCR

Purified genomic DNA from V. parahaemolyticus F113A was tenfold serially diluted (1.0 μg/μl to 0.1 pg/μl) in sterile distilled water. PCR amplifications were performed using optimized concentrations of reagents and temperature cycling parameters as described in the results section. Positive amplifications of the targeted gene segments were identified by the melt temperatures, which were generated by the melt curve analysis by the Cepheid™ Smart Cycler software (ver. 2d). The amplicon sizes were confirmed by gel electrophoresis using a 2% (w/v) NuSeive 3:1 agarose gel. This experiment was performed in triplicate to ensure a consistent level of detection.

Detection of V. parahaemolyticus in pure culture

A stationary-phase V. parahaemolyticus F113A culture (4.6 × 107 cfu/ml) grown overnight culture was tenfold serially diluted to extinction. Then cells were collected by centrifugation at 9,300×g for 10 min. The supernatant was discarded, and the cells were resuspended in 100 μl of Instagene™ Matrix consisting of specially formulated Chelex™ resin (BioRad Laboratories, Hercules, CA). The samples were then incubated at 56°C for 10 min, vortexed intermittently for 1 min and then boiled for 6 min. For each 25 μl PCR, 3 μl of the Instagene™ treated boiled sample was used as a source of the template DNA. All experiments were conducted in triplicate in order to evaluate the reliability of the sensitivity of detection.

Detection of V. parahaemolyticus in gulf water

An overnight culture of V. parahaemolyticus F113A was inoculated in fresh T1N1 broth in 1:10 ratio, and grown at 37°C until the OD450nm reached 0.2. The culture was centrifuged and cell pellet was resuspended in autoclaved Gulf of Mexico water (gulf water from herein). The viable plate count on T1N3 agar plate was determined to be 4.6 × 107 cfu/ml. The culture was then subjected to tenfold serial dilution to extinction in 10 ml of autoclaved gulf water and enriched at 37°C for 5 h in a rotary shaker incubator set at 150 rpm. The gulf water was collected from Dauphin Island, AL and the salinity was determined to be 27 ± 3 ppt (n = 5) using a refractometer (Reichert Scientific Instruments, Buffalo, NY). The cells were then collected by centrifugation; supernatant was carefully discarded, and the cells were resuspended in 100 μl of Instagene™ Matrix (BioRad) and used for PCR amplification as described above. To determine the consistency of the level of detection, all experiments were conducted in triplicate.

Detection of V. parahaemolyticus in seeded oysters

All oyster samples were obtained from a local seafood store in Birmingham, AL. A pure culture of V. parahaemolyticus F113A was grown in T1N1 broth until the OD450nm reached to 0.2. The viable plate count of 107 cfu/ml was determined on T1N3 agar plates (Atlas 1993). The culture was then tenfold serially diluted in T1N1 broth to extinction and 10 ml of each dilution was added to an additional 50 ml of T1N1 broth and 1 ml of homogenized oyster shell stock (ISSC, 2000). After 5 h of enrichment in a rotary shaker incubator (Innova 4000) set at 150 rpm and 37°C, 1 ml aliquots were collected by centrifugation at 9,500×g for 10 min. The supernatant was carefully discarded using a micropipette, and the cell pellet was resuspended in 100 μl of Instagene™ Matrix (BioRad) and treated to release template DNA as described above. For each SYBR Green™ I PCR, 3 μl of the supernatant of the boiled samples were used as a source of template DNA. Unseeded oyster homogenate shell stock (1 ml) was enriched and treated in Instagene™ matrix as described above, and used as a control. All experiments were conducted in triplicate in order to evaluate the reliability of the sensitivity of detection.

Detection of V. parahaemolyticus in natural oysters

A total of 24 oysters were collected from the Gulf of Mexico water near Dauphin Island, Alabama during the month of June 2007. Collected oysters were immediately chilled and cleaned with 70% (v/v) alcohol with a scrubbing brush using standard method (Kaysner et al. 1992; NSSP 1997). The oysters were shucked, and the shell stock homogenized in an autoclaved Waring Blender (Fisher Scientific). An aliquot (30 ml) of the samples from each group was then enriched for 5 h in T1N1 broth as described above. Enriched samples were aliquoted and stored in −80°C until used for PCR. DNA from the enriched samples were purified using the Instagene™ Matrix as described above and 3 μl was used for the multiplexed PCR amplification. Purified genomic DNA from V. parahaemolyticus F113A strain consisting of all three hemolysin genes were spiked into 3 μl Instagene™ treated oyster homogenate and PCR amplified to determine that negative detection of the samples was not due to the inhibition of the reaction by the oyster tissue matrix.

Results

Primer selection and optimization of real-time PCR

The primers for the tlh gene target amplified the expected 173 bp DNA fragment for each of the 44 V. parahaemolyticus strains tested, however tlh amplification was negative for the rest of the non-parahaemolyticus vibrios and non-vibrio species. This suggests that the R-TLH-731 and L-TLH-933 primers are specific for the detection of genus V. parahaemolyticus. Only 26 (~59%) of the V. parahaemolyticus strains tested positive for tdh and only 19 (~ 43%) for trh, showing that not all of the isolates tested were pathogenic. Amplification of tlh, tdh and trh was evident by the fluorescent detection displaying the expected amplicon melt temperature values (Table 2) and was confirmed by gel electrophoresis (not shown).

To achieve consistent and detectable level of fluorescence during the melt-temperature analysis for all amplicons, the tlh primer was sufficient at 0.2 μM concentration, whereas the tdh and trh primer needed to be 0.6 μM. The SYBR Green™ I dye was most favorable at a 2× concentration. The MgCl2 concentration of 3 mM along with 2.5 μl of enhancer mixture was found to be optimum for the assay.

The following PCR cycling parameters were used for efficient and consistent amplification of the targeted gene sequences: initial denaturation of the template DNA for 120 s at 95°C followed by 6 cycles of amplification cycles surpassing the threshold cycle (C t ). The amplification cycles consisted of a denaturation of the template DNA at 94°C for 15 s, annealing at 56°C for 15 s, and extension for 25 s at 72°C for consistent results.

Sensitivity of detection

A minimum of 10 pg of purified genomic DNA from V. parahaemolyticus F113A displayed a detectable level of positive amplification for all three gene targets with the C t value of 27.36 ± 1.53 (Table 3). The amplification was evident with the DNA melt temperatures (Fig. 1) and confirmed by gel electrophoresis displaying the DNA bands of expected size of 173 bp, 217 bp, and 270 bp for tlh, trh and tdh, respectively (data not shown). Increasing C t values with the expected melt temperatures were observed as the amount of initial template DNA increased (Table 3). This level of detection of genomic DNA is comparable to approximately 103 cfu V. parahaemolyticus (Atlas and Bej 1990). A good linear correlation of the samples with positive PCR amplification was found between the C t values and the concentration of purified DNA (n = 3; r 2 = 0.99) (Fig. 2).

Table 3 Sensitivity of PCR detection on purified Vibrio parahemolyticus DNA
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Fig. 1
figure 1

Optics graphs generated by the Cepheid™ Smart Cycler displaying the amplicon melt curves and temperatures of the hemolysin genes following real time PCR of the purified genomic DNA from V. parahaemolyticus with SYBR Green™ I dye. The decline of the fluorescent signals with increasing temperatures indicates the dissociation of SYBR Green™ I dye from amplicons. The melt temperature values were generated by the Smart Cycler software when one-half of the double-stranded amplicons were denatured. a The melt temperature value of the tlh gene, b the melt temperature value of the tdh gene, c the melt temperature value of the trh gene, d the melt temperatures following multiplexed PCR targeting tlh, tdh and trh genes, e the melt temperatures following multiplexed PCR without any DNA added in the reaction (negative control)

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Fig. 2
figure 2

Standard curve for the amount of purified DNA versus C t values. The error bars indicate the standard deviation from three independent experiments

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Detection of V. parahaemolyticus in pure cultures

The level of detection of an unenriched V. parahaemolyticus in pure culture was 107 cfu/ml with a C t value of 24.56 ± 1.53 and amplicon melt temperatures of 84.45 ± 0.13°C (tlh), 81.83 ± 0.24°C (tdh), and 80.08 ± 0.06°C (trh) (Table 4). This level of detection was consistent with three individual reactions.

Table 4 Sensitivity of PCR detection of Vibrio parahemolyticus in pure cultures without enrichment
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Detection of V. parahaemolyticus in seeded gulf water

The minimum level of detection of all three hemolysin genes following 5 h enrichment of gulf waters samples of a serial dilution consisted of an inoculum of 10 cfu/ml prior to enrichment. The observed C t value for this sample was 25.35 ± 0.06 and the amplicon melt temperatures were approximately 84.46 ± 0.04, 81.72 ± 0.14, and 81.43 ± 0.2°C for targeted genes tlh, tdh, and trh, respectively (Table 5). This level of detection was found to be consistent in all replicates.

Table 5 Sensitivity of PCR detection of Vibrio parahemolyticus in gulf water after 5 h enrichment
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Detection of V. parahaemolyticus in seeded oysters

The sensitivity of detection of V. parahaemolyticus in 1.0 ml of oyster shell stock homogenate with initial inoculum of 101 cfu V. parahaemolyticus exhibited positive results of C t value of 27.42 ± 0.18 and T m values of 84.19 ± 0.52 (tlh), 81.32 ± 0.67 (tdh) and 80.70 ± 0.23 (trh) following 5 h of enrichment (Table 6). This detection level was reproducible for three individual samples tested. The positive results were confirmed by the post-amplification melt temperature analysis and gel electrophoresis (not shown). Oyster tissue samples without any added V. parahaemolyticus exhibited negative amplification results confirming that the positive amplifications and sensitivity of detection were a direct result of the seeded culture and not pre-existing V. parahaemolyticus in the oyster samples.

Table 6 Sensitivity of PCR detection of Vibrio parahemolyticus in oyster homogenate after 5 h enrichment
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Detection of V. parahaemolyticus in natural oysters

Multiplexed PCR amplification on enriched 24 oyster homogenates exhibited positive amplification of tlh on 14 samples (~58%); three of these 14 tlh-positive samples (~21%) were positive for tdh. Also, one of the 14 tlh-positive samples (0.7%) amplified for the trh, but was negative for the tdh gene. The PCR amplified DNA were confirmed for the tlh (174 bp); tdh (270 bp); and trh (217 bp) by agarose gel (data not shown). Oyster samples spiked with V. parahaemolyticus F113A exhibited amplification of all three targeted hemolysin genes suggesting that negative amplification was not due to the inhibition by oyster tissue matrix.

Discussion

In this study, we have developed and optimized a multiplexed real-time PCR assay using SYBR Green™ I DNA-binding fluorescent dye targeting hemolysin genes for the detection of V. parahaemolyticus in gulf water and shellfish. For the detection of total V. parahemolyticus, a species-specific gene tlh was targeted; and for virulent strains two other hemolysin genes, tdh and trh were used (Bej et al. 1999; Terzi et al. 2009; Tyagi et al. 2009; Ward and Bej 2006). These 2 hemolysin genes specifically detect the pathogenic strains of V. parahaemolyticus that has recently been validated by Espiñeira et al. (2010). Real-time PCR with SYBR Green™ I DNA-binding fluorescent dye targeting tdh or the trh gene of V. parahaemolyticus has been reported (Fukushima et al. 2003). However, a comprehensive detection of all three hemolysin genes using SYBR Green™ I have not been described. In fact, to the best of our knowledge, this is the first study in which a SYBR Green™ I-based real-time multiplexed PCR amplification targeting three genes segments simultaneously has been optimized and applied in shellfish (shell stock) and in gulf water. The strategy for the primer selection for the multiplexed PCR assay was to choose segments of the targeted genes (tlh, tdh and trh) that will generate distinguishable melt temperature values enabling us to identify the unlike fragments upon completion of the PCR. In our experience, at least 1°C difference between two targeted genes was necessary for the Cepheid™ Smart Cycler system to distinguish unlike fragments and reliably display distinct melt temperature values. Additionally, the differences in the amplicon lengths were kept within 100 bp so that the amplification of all three genes remains equally efficient. The use of the enhancer reagent in the reaction helps keep the formation of primer artifacts to a minimum level. Although in previous studies tlh, tdh and trh genes were shown to be specific for V. parahaemolyticus (Bej et al. 1999; Terzi et al. 2009; Tyagi et al. 2009; Ward and Bej 2006), however, since we have selected new primer sets in this study, it was necessary to test the specificity of the assay. The selected primers amplified only V. parahaemolyticus. It helped avoid false negative detection and generated realistic C t values for DNA and cfu. Multiplexed PCR with SYBR Green™ I enabled us to establish reliable and specific detection of this pathogen in shellfish and gulf water. Multiplexed PCR amplification of the V. parahaemolyicus culture without enrichment exhibited detection level of 106 cfu. A recent study by Terzi et al. (2009) also showed similar level of detection in pure cultures V. parahaemolyicus for all three targeted genes in single-tube reaction. However, 5 h enrichment of the oyster homogenate shell stock significantly improved the sensitivity of detection to 10 cfu in 10 ml of gulf water and 1 g of oyster tissue homogenate. Detection of 1 cfu of V. parahaemolyticus in oyster tissue homogenate following either 8 h (Rizvi et al. 2006) or an overnight (Ward and Bej 2006) enrichment by real-time PCR and Taqman™ probes has been reported. Haldar et al. (2010) has reported similar level (10–100 cells) of detection of several pathogenic Vibrio including V. parahaemlytics. In another study by Nordstrom et al. (2007), V. parahaemolyticus was detected at <10 cfu following MPN enrichment. In this study, we were able to achieve detection of initial inoculum (prior to enrichment) of 10 cfu of V. parahaemolyticus in 10 ml Gulf of Mexico water or 1 ml homogenized shell stock after 5 h of enrichment, which is well within the minimum level of detection recommended by the ISSC for safe consumption of oysters. Also <10 cfu V. parahaemolyticus in 1 g of oyster tissue homogenate is considered as “non-detectable” level and therefore safe for human consumption (ISSC 2000). A biochemical test by the identification of a β-type hemolysis on Wagatsuma blood agar referred as the Kanagawa phenomenon was considered to be closely associated with the pathogenicity of V. parahaemolyticus (Miyamoto et al. 1969; Wagatsuma 1968). However, this method of detection is laborious and lengthy. Current real-time PCR assay with SYBR Green™ I is rapid (~8 h to complete), specific to the targeted genes and reliable for the detection of V. parahaemolyticus in shellfish and gulf water. The standard curve from the multiplexed PCR amplification on purified V. parahaemolyticus DNA exhibited a good linear correlation among serially diluted samples. This was used to provide us with a reliable means to compare C t values and correlate the amount of V. parahaemolyticus DNA present in samples to that with cfu. Also, the levels of inhibition of PCR by the shellfish or gulf water samples matrix could be estimated. In this study, the C t values for enriched oyster tissue homogenate was approximately 1.5 cycles higher than gulf water samples indicating that oyster tissue components may have had an inhibitory effect on the PCR amplification. The C t values for gulf water samples were <1 cycle higher than V. parahaemolyticus in pure cultures, suggesting that gulf water had a negligible inhibitory effect on the real-time PCR. However, for SYBR Green I-based PCR detection requires an accurate T m value to confirm the amplification of a targeted gene segment. Therefore, the C t values may not be essential for confirming detection, but are indicative of the presence of the targeted amplicons. All positive PCR amplifications exhibited expected T m values suggesting that the multiplexed PCR assay developed in this study is specific, reliable and reproducible. Enrichment of V. parahaemolyticus in oyster tissue homogenate and in gulf water allowed us to ensure detection of viable cells. In addition, an enrichment period enabled us to achieve the sensitivity of detection at a level that is in compliance with the current ISSC guidelines. In this study, the oyster shell stock was enriched in the ISSC-recommended T1N1 growth medium whereas no recommendation was specified for testing the presence of this pathogen in gulf water. It has been documented that the gulf water is a nutrient-rich environment mostly due ot the agricultural run-offs (U.S. Geological Survey and U.S. Government Department of Interior, http://water.usgs.gov/nawqa/sparrow/gulf_findings/primary_sources.html). The high level of nutrient and warm summer temperature often leads to eutrophication followed by hypoxic conditions in the coastal gulf water spreading miles offshore (Boesch et al. 2009). Due to the high nutrient content, the gulf water was used in this study for enrichment of V. parahaemolytius without addition of any nutrient medium. Moreover, Julie et al. (2010) correlated the importance of ecological parameters such as nutrient, salinity, turbidity, chlorophyll A and temperature and the level of total and pathogenic V. parahaemolyticus in sediment, water, and mussels prior to and day of collection from the French Atlantic coast. They have used the PCR method targeting the hemolysin genes to assess the level of this pathogen. The relevance of the study by Julie et al. (2010) to this report is that the level of the V. parahaemolyticus in environmental water modulates with the changing environmental parameters therefore enrichment step is crucial for samples with low counts on the targeted pathogen. The results from this study suggests that multiplexed real-time PCR with SYBR Green™ I is cost-effective, specific and sensitive and capable of simultaneously detecting species-specific and pathogenic strains of V. parahaemolyticus in shellfish and gulf water. This means of rapid detection will help the shellfish industry implement routine monitoring of oysters and keep track of global transmission of this pathogen, which often occurs through cargo ballast water. The current study offers a workable rapid detection assay for total species and pathogenic strains of V. parahaemolyticus, which will help seafood industry maintain a steady supply of safe oysters, thus prevent disease outbreaks and protect consumer health.