Introduction

The genus Vibrio is a member of the family Vibrionaeceae and consists of 44 recognized species of which 12 species occur in human clinical specimens (Bergey’s Manual of Systematic Bacteriology 2005). V. cholerae O1 is the causative agent of pandemic cholera. V cholerae O139 (the Bengal strain) was reported as another cause of cholera (Ramamurthy et al. 1993). The epidemic causing strains of V. cholerae (O1 or O139 serogroups) produce cholera toxin (CT) which is the major contributing factor for profuse diarrohea (cholera gravis) characterized by severe diarrohea with rice water stools, devastating dehydration and electrolyte imbalance. V. cholerae non-O1 serogroups are widely distributed in the aquatic environment and are free living in nature. Strains of non-O1 V. cholerae greatly outnumber O1 strains in the environment and majority of these isolates lack the classical virulence factors such as cholera toxin and toxin co-regulated pilus.

Cholera Toxin (CT) encoded by ctxAB is responsible for the severe diarroheal symptoms elicited by V. cholerae (Kaper et al. 1995). CT is a potent A-B type exotoxin composed of 5 B subunits (ctxB) which binds holotoxin to the cell receptor and one A subunit (ctxA) which provides intracellularly toxigenic activity. V. cholerae O1 strains and O139 strains produce CT (Nair and Takeda 1993) but the expression of CT is rare in other serogroups of V. cholerae non-O1 (Said et al. 1994). However, there are atypical environmental strains that possess the ctx gene (Nair et al. 1988; Rivera et al. 2001). The toxigenic O139 serogroups having arisen from recombination with toxigenic O1 strains (Faruque et al. 2000b) and it has been hypothesized that V. cholerae non-O1/non-O139 strains can acquire genes for toxin production by transduction and might be the source of new epidemics.

PCR methods that target a single gene/sequence, multiple genes within V. cholerae, or two or more pathogens in a single PCR assay have been used by several researchers (Brasher et al. 1998; Bacteriological Analytical Manual 2001; Singh et al. 2002; Jing et al. 2003; Karunasagar et al. 2003; Panicker et al. 2004a; Fraga et al. 2007; Tarr et al. 2007; Khuntia et al. 2008). Tarr et al. (2007) developed a multiplex PCR for V. vulnificus, V. parahaemolyticus, V. mimicus and V. cholerae wherein the intra-specific variation in the conserved housekeeping gene viz., sodB was used as a source of marker for V. cholerae and as the house keeping genes are invariably present in all isolates this PCR method helps in the detection of all V. cholerae isolates irrespective of their toxigenic status. An effective PCR method for detecting enterotoxigenic Vibrio cholerae in food samples was described in Bacteriological Analytical Manual (2001). This PCR selectively amplifies a specific DNA fragment within the ctxAB operon of V. cholerae and detects only cholera toxin producing V. cholerae but does not provide information on non-cholera-toxigenic V. cholerae. A duplex-PCR that detects all V. cholerae isolates and simultaneously provides information on the choleratoxigenic potential is essential for risk analysis of food.

Cultured shrimp, mainly the black tiger shrimp (P. monodon) constitutes a major portion of Indian fishery export. During the year 2007–08, a total of 1,06,165 MT of shrimp was produced from a culture area of 1,22,078.80 ha (MPEDA 2008). Cultured shrimp processing waste is also a rich source for important by-products like chitin and carotenoids (Raghu et al. 2008). The importance of V. cholerae in the P. monodon aquaculture is mainly as a biological hazard that compromises post-harvest quality and leads to rejections from the importing countries. Rapid Alert System for Food and Feed (RASFF) of the European Union has issued alert notifications for the presence of either V. cholerae or V. cholerae non O1/non-O139 in frozen black tiger shrimp (P. monodon) imported by the European Union (EU) countries from shrimp exporting countries including India (http://ec.europa.eu/food/food/rapidalert/rasff_portal_database_en.htm).

The present study was taken up to screen samples from shrimp aquaculture system for potential cholera-toxigenic V. cholerae, develop a duplex-PCR method for the simultaneous detection of V. cholerae and differentiation of cholera toxin producing V. cholerae and determine the effect of fish and crustacean meats on the performance of the duplex-PCR.

Materials and methods

Samples from shrimp culture system

Water (n = 7) and post-larvae (n = 7) samples from seven Penaeus monodon (black tiger) shrimp hatcheries; pond water (n = 5), pond sediment (n = 5) and shrimp samples (n = 5) from five P. monodon aquaculture farms on the East Coast of India were screened.

Isolation and identification of V. cholerae

Thiosulfate Citrate Bile Sucrose Agar (TCBS Agar) was used for isolating vibrios. Serial ten fold dilutions of shrimp muscle, shrimp head, pond sediment and pond water were spread plated on TCBS agar, incubated at 37 °C for 24 h. The sucrose fermenting colonies on the TCBS plates were purified by streak dilution and identified to the species level by using the identification scheme of Noguerola and Blanch (2008). Isolates that were arginine dihydrolase negative, lysine decarboxylase positive, ornithine decarboxylase positive, indole positive, ONPG test positive, showed growth at 0%NaCl positive, growth at 8%NaCl negative, produced acid from sucrose were identified as V. cholerae. The V. cholerae isolates identified by the above mentioned schemes were confirmed by performing the tests described for V. cholerae species identification (Bergey’s Manual of Systematic Bacteriology 2005).

E.coli

Tergitol seven agar was used to determine E.coli counts and final counts were arrived after confirmation on Eosin Methylene Blue agar and by performing indole, methyl red, Voges-Proskauer and citrate (IMVC) tests.

Slide agglutination test for V. cholerae O1 and V. cholerae O139 and V. cholerae non-O1/non-O139

V. cholerae isolates were initially tested with polyvalent somatic O antiserum (Difco Vibrio cholerae antiserum Poly [Hikojima, Inaba, Ogawa], Becton, Dickinson and Company, Franklin Lanes, NJ, USA) and the isolates that gave positive agglutination reaction were classified as V. cholerae O1. Cultures that gave negative reaction with polyvalent somatic O antiserum were further tested for agglutination using V. cholerae O139 antiserum (V. cholerae antiserum O139 Bengal, Denka Seiken Co Ltd, Tokyo, Japan) and those V. cholerae cultures that gave positive agglutination reaction were classified as V. cholerae O139. V. cholerae cultures that gave negative reaction both with polyvalent somatic O antiserum and O139 antiserum were classified as V. cholerae non-O1/non-O139.

PCR

PCR for the detection of V. cholerae using species specific primers (Tarr et al. 2007) was performed using 1 μl of each sodB (Table 1) primer (10 μM stock) in a final PCR reaction volume of 20 μl. V. cholerae yield an amplicon of 248 bp. PCR for the detection of enterotoxigenic V. cholerae was performed (Bacteriological Analytical Manual 2001) using ctx primers (Table 1) at 0.5 μM concentration of each primer and 3% (v/v) APW lysate as template. ctxAB positive V. cholerae cultures yield an amplicon of 777 bp. PCR for V. cholerae O1 and V. cholerae O139 was performed as per Hoshino et al. (1998) using O1F2-1 and O1R2-2 primers (V. cholerae O1-rfb specific primers) and O139F2 and O139R2 primers (O139-rfb specific primers) (Table 1). V. cholerae O1 cultures yield an amplicon 192 bp whereas V. cholerae O139 cultures yield an amplicon 449 bp. V. cholerae non-O1/non-O139 cultures do not yield any of these amplicons. V. cholerae (MTCC 3906), V. vulnificus (MTCC 1145), V. alginolyticus (ATCC 17749) and V. parahaemolyticus (ATCC 17802) cultures were used for standardizing the PCR reactions.

Table 1 Details of the V. cholerae species specific and ctx specific primers
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Duplex PCR for the simultaneous detection of V. cholerae and differentiation of cholera toxin producing V. cholerae isolates (V. cholerae-duplex PCR)

A V. cholerae-duplex PCR method was developed by utilizing V. cholerae species specific sodB primers (Tarr et al. 2007) and ctxAB genes specific primers (Bacteriological Analytical Manual 2001). This method was designed to detect V. cholerae isolates and differentiate ctx toxin producing strains.

V. cholerae-duplex PCR reaction conditions

One ml of V. cholerae culture (24 h at 37 °C) grown in T1N1 (Tryptone 1%, NaCl 1%) was centrifuged at 10,000 rpm for 10 min. The cell pellet was resuspended in 100 μl of Tris-EDTA (TE) buffer, placed in a dry bath for 5 min at 95 °C and the crude lysate was used as template. The PCR amplification reactions were performed in a final volume of 20 μl. Each reaction mixture contained 0.5 μM each of PCR species specific primers, 0.5 μM each of ctxAB primers, 18 μl of master reaction mix containing 10 mM Tris-HCl, pH 8.3; 50 mM KCl, 1.5 mM MgCl2, 200 μM each dATP, dCTP, dGTP and dTTP, 1 U of Taq polymerase and 1.2 μl of crude lysate. PCR reaction was carried out in Minicycler (Minicycler PTC-150, MJ Research, MA, USA) programmed to perform a denaturation step at 93 °C for 15 min, followed by 35 cycles consisting of 40 s at 92 °C, 1 min at 57 °C and 1.5 min at 72 °C. The last extension step was extended to 7 min longer. Agarose gel analysis of V. cholerae-duplex PCR products (Sambrook and Russell 2001) was carried out by loading 10 μl of the PCR product in a 2% agarose gel containing 1 μg/ml Ethidium bromide in TAE buffer and electrophoresed. After appropriate migration with constant voltage of 5–10 V/cm (Electrophoresis Powerpack, Bangalore Genei, India) the agarose gel was scanned using a gel documentation system (Alpha Imager, Alpha Innotech Corporation, USA). V. cholerae cultures yield 248 bp amplicon; ctxAB positive V. cholerae cultures yield two amplicons viz., 248 bp and 777 bp.

End point dilution of V. cholerae-duplex PCR

The sensitivity of V. cholerae-duplex PCR was determined by making serial 10 fold dilutions of V. cholerae culture (grown in T1N1, incubated for 24 h at 37 °C) in normal saline ranging from undiluted to 10−6 viz., 106 V. cholerae cells, 105 V. cholerae cells, 104 V. cholerae cells, 103 V. cholerae cells, 100 V. cholerae cells, 10 V. cholerae cells and 1 V. cholerae cell. From each tube, 1 ml was withdrawn for template preparation and subjected to V. cholerae-duplex PCR.

Suitability of V. cholerae-duplex PCR in detecting V. cholerae in fish and crustaceans samples

Freshwater fish (carp, Labeo rohita), marine fish (tuna, Euthynnus affinis), black tiger shrimp (Penaeus monodon) and three spot crab (Portunus sangulnolentus) meats were used. All the food matrices were initially tested negative for the presence of V. cholerae. Fish, shrimp or crab meat (25 g) was homogenized with sterile alkaline peptone water (225 ml) and spiked with known number of V. cholerae (ctx positive) cells (106 cells) and incubated at 37 °C. The samples were drawn after 18 h of incubation for PCR analysis.

Results and discussion

Vibrio loads in shrimp hatchery and farm samples

Vibrio loads were higher in P. monodon hatchery samples than in aquaculture farm samples (Table 2). Shrimp post-larvae had maximum loads of Vibrios (2.1 × 105 CFU/g). Shrimp head portion had relatively higher counts of Vibrios (3.5 × 104 CFU /g) than shrimp muscle portion (1.4 × 104 CFU /g). Hatchery waters had higher Vibrio loads (2400 CFU /ml) than farm waters (150 CFU /ml). Sucrose non-fermenting Vibrios were higher in shrimp head portion (59%) and hatchery waters (51%) whereas more than 90% of the Vibrios in post-larvae, pond water, pond sediment and shrimp muscle portions were sucrose fermenters. Gomez-Gil et al. (1998) reported that Vibrio spp. isolated from the digestive tract of a population of healthy juvenile Litopenaeus vannamei consisted of both sucrose and non-sucrose fermenters whereas the haemolymph contained only non-sucrose fermenters.

Table 2 Incidence of V. cholerae in black tiger shrimp culture system
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Incidence of V. cholerae in shrimp hatchery and farm samples

Two hundred ten Vibrio cultures isolated from water (n = 105) and post-larvae (n = 105) samples from shrimp hatcheries; 250 Vibrio cultures isolated and purified from pond water (n = 75), pond sediment (n = 75) and shrimp (n = 100) from aquaculture farms were screened for the presence of Vibrio species. Incidence of V. cholerae was not observed in shrimp hatchery samples as none of the 210 Vibrio cultures obtained from hatchery water or post-larvae were identified as V. cholerae. However, a total of 42 vibrio isolates out of the 250 vibrio cultures obtained from shrimp aquaculture samples were identified as V. cholerae by biochemical tests. The incidence of V. cholerae was higher in pond water (7.6%) than in pond sediment (5.2%). Shrimp head (3.6%) portion had relatively higher incidence of V. cholerae than shrimp muscle (1.6%). The incidence of V. cholerae in pond water, pond sediment, shrimp head portion and shrimp muscle as a percentage of the total V. cholerae isolates (n = 42) was 45.2%, 31%, 14.3% and 9.5%, respectively (Table 2). All the V. cholerae isolates obtained from shrimp aquaculture system were grouped as V. cholerae non-O1/non-O139 as they yielded negative agglutination results with polyvalent somatic O antiserum and O139 antiserum. All these V. cholerae isolates yielded the specific amplicon (248 bp) in the PCR employing sodB primers thereby confirming their identity as V. cholerae (Fig. 1). Three V. cholerae isolates were positive in the PCR employing ctxAB primers and yielded a single specific amplicon of 777 bp size. V. cholerae was absent but non-01 serotype V. cholerae was reported from Penaeus monodon ponds in India (Otta et al. 1999).

Fig. 1
figure 1

PCR for the detection of V. cholerae using species specific primers. Lane 1–3, V. cholerae isolates from shrimp pond water; Lane 4–5, V. cholerae isolates from shrimp pond sediment; Lane 6, V. cholerae isolates from shrimp muscle; Lane 7, V. cholerae (MTCC 3906); All V. cholerae yielded the species specific amplicon (248 bp) with sodB primers; Lane8, 100 bp DNA ladder (GeNei)

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V. cholerae-duplex PCR method for the simultaneous detection and differentiation of choleratoxigenic V. cholerae

In this study a V. cholerae-duplex PCR method was developed by utilizing V. cholerae species specific (Tarr et al. 2007) and ctxAB genes specific primers (Bacteriological Analytical Manual 2001). The reason for selecting the ctxAB and sodB primers for developing V. cholerae -duplex PCR was that the annealing temperatures for the ctxAB primers (55 °C) and sodB primers (57 °C) were nearer. This method was designed to detect V. cholerae isolates and simultaneously differentiate ctx toxin producing strains.

PCR amplification cycle conditions for the ctxAB primers was 34 cycles consisting of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C. The thermal cycling profile for the sodB primers was 35 cycles consisting of 40 s at 92 °C, 1 min at 57 °C and 1.5 min at 72 °C. The primer annealing condition of 1 min at 57 °C was used in this V. cholerae -duplex PCR. A simple template preparation procedure as mentioned in material and methods was used for the V. cholerae -duplex PCR. The crude lysate of V. cholerae culture was found sufficient for obtaining specific result in the V. cholerae -duplex PCR method. The V. cholerae -duplex PCR was initially standardized using V. cholerae (MTCC 3906), V. vulnificus (MTCC 1145), V. alginolyticus (ATCC 17749) and V. parahaemolyticus (ATCC 17802) cultures and ctx negative V. cholerae (laboratory culture collection) cultures. V. cholerae (ctx negative) cultures yielded a single amplicon (248 bp) whereas ctxAB positive V. cholerae cultures yielded two amplicons (248 bp and 777 bp). Other Vibrio species did not yield the V. cholerae specific amplicon. The results indicate that the V. cholerae -duplex PCR was specific to V. cholerae and the PCR cycle conditions were adequate for obtaining the desired result.

All the 42 V. cholerae isolates obtained from shrimp aquaculture system were subjected to V. cholerae -duplex PCR. Thirty nine V. cholerae cultures yielded a single amplicon of 248 bp indicating that they were V. cholerae but non-choleratoxigenic. Three V. cholerae cultures yielded two amplicons viz., species specific 248 bp and cholera toxin specific 777 bp, thereby indicating that they were cholera toxin producing strains of V. cholerae (Fig. 2). The specificity of the V. cholerae -duplex PCR was confirmed by screening the remaining 208 Vibrios cultures isolated from aquaculture ponds. None of the samples yielded the specific amplicons in V. cholerae -duplex PCR indicating the specificity of V. cholerae -duplex PCR. The V. cholerae -duplex PCR and the dichotomous scheme of Noguerola and Blanch (2008) yielded the same result; in both the cases the same cultures were identified as V. cholerae.

Fig. 2
figure 2

Duplex PCR for the simultaneous detection of V. cholerae and differentiation of cholera toxin producing V. cholerae isolates. Lane 1, Negative control; Lane 2–3, Cholera toxin negative V. cholerae isolates from shrimp pond water; Lane 4–6, Cholera toxin positive V. cholerae isolates from shrimp pond water; Lane 7, V. cholerae (MTCC 3906); Lane 8, 100 bp DNA ladder (Gene Ruler™, Fermentas)

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V. cholerae was not detected in shrimp hatchery water and post-larvae. The results indicate that, even though V. cholerae was detected in shrimp pond sediment, shrimp head and shrimp muscle samples majority of the isolates were found to be non-choleratoxigenic as the isolates did not yield the ctxAB specific amplicon in V. cholerae -duplex PCR. Only 7% of the V. cholerae isolates (three isolates) were potentially cholera-toxigenic (Table 2). These three V. cholerae isolates were obtained from pond water and they agglutinated neither with polyclonal somatic O antiserum nor with O139 antiserum; thereby grouping them as V. cholerae non-O1/non-O139 serogroup. These three isolates failed to yield O1 specific or O139 specific amplicon when tested in PCR (Fig. 3) reconfirming that the isolates were indeed V. cholerae non-O1/non-O139. The faecal indicator, E. coli was detected in aquaculture farms but not in shrimp hatchery samples and the mean E. coli levels were high in pond water (123 ± 87 CFU/g) than in shrimp head (16 ± 16.7 CFU/g) and shrimp muscle (12 ± 11 CFU/g). This may suggest that the ctx positive V. cholerae in pond water might have entered through faecal pollution either through the source water or through feed. Farm made feeds were reported positive for V. cholerae (Raghavan 2003). However, pond sediment samples had higher mean E. coli levels (204 ± 133 CFU/g) than pond water (123 ± 87 CFU/g) but ctx positive V. cholerae could not be isolated from these sources which suggests that the relationship between faecal bacteria and presence of ctx positive V. cholerae is non-existent. A negative correlation was observed between total vibrio counts and E. coli (r = −0.54) in the shrimp culture system. The incidence of V. cholerae observed in aquaculture ponds might also have been due to natural inhabitation. The autochthonous existence of V. cholerae in environment has been previously reported by several researchers (Colwell et al. 1981; Lee et al. 1982). Moreover, the presence of V. cholerae could not be correlated with faecal indicators (Hood and Ness 1982; Filetici et al. 1997).

Fig. 3
figure 3

PCR for testing the O1 and O139 status of ctx bearing V. cholerae isolates from shrimp pond water. Lanes 1–3, ctx positive V. cholerae isolates from shrimp aquaculture system failed to yield O1 specific or O139 specific amplicon indicating that the isolates were V. cholerae non-O1/non-O139; lane 4, Known V. cholerae O1 isolate from laboratory culture collection; lane 5, V. cholerae (MTCC 3906) – V. cholerae O139; lane 6, Known V. cholerae non-O1/non-O139 from laboratory culture collection; lane 7, 100 bp DNA ladder (GeNei)

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The structural genes for the ctx element reside on a filamentous phage ctxФ (Waldor and Mekalanos 1996). CTXФ is found in all epidemic V. cholerae isolates but is rarely recovered from the non-O1/non-O139 V. cholerae environmental isolates. A DNA probe study showed that a small percentage of environmental strains of V. cholerae non-O1 have the ctx gene (Nair et al. 1988). Virulence genes including ctxAB were found among environmental strains from Calcutta, India (Chakraborty et al. 2000). Occurrence of ctxA was found among 10% on non-O1/non-O139 environmental isolates from Brazil (Rivera et al. 2001). Clinical toxigenic V. cholerae isolates are closely related to non-toxigenic environmental strains (Jiang et al. 2000) and CT genes are highly mobile among environmental isolates. The spread of CT genes in the environment can be facilitated by the exposure of CTXФ positive strains to sunlight (Faruque et al. 2000a). Genetic and phenotypic evidence strongly suggests that the O139 strain arose from a V. cholerae O1 strain by horizontal gene transfer (Bik et al. 1995; Comstock et al. 1996; Waldor and Mekalanos 1994; Faruque et al. 2000b). Similarly, V. cholerae non-O1/non-O139 strains can also acquire toxigenic genes for toxin production by transduction and therefore might be the source of new epidemics. Hypothesis of the existence of a TCP-independent mechanism for infection by CTXФ was proposed by Jiang et al. (2003). The choleratoxigenic V. cholerae (non-O1/non-O139) isolated from shrimp aquaculture system showed greater genetic similarity with ctx negative V. cholerae than amongst ctx positive V. cholerae (Rao and Surendran 2010).

Keeping these in view, the screening of environmental and food samples for the presence of ctx positive V. cholerae, especially within the non-O1/ non-O139 serogroup, is of paramount importance. The V. cholerae duplex-PCR method developed in this study would be a useful tool to screen for ctx positive V. cholerae. Even though the existence of ctx carrying non-O1/non-O139 V. cholerae isolates was very low in shrimp culture system, the ecological significance of ctx genes among these V. cholerae non-O1 and non- O139 isolates in the shrimp aquaculture environment needs to be further investigated. Study on the expression of ctx gene by the ctx positive non-O1/non-O139 V. cholerae was not performed. Additional study on the expression of ctx genes would unravel the ecological significance of ctx genes in non-O1/non-O139 V. cholerae

The autochthonous existence of non-O1/non-O139 V. cholerae in aquatic environment has been reported from several areas world over. Non-O1/non-O139 V. cholerae appears to constitute part of the microflora of prawns (Nair et al. 1991) and oysters (McIntyre et al. 1979; Salamaso et al. 1980). While, non-O1/O139 V. cholerae may be occasionally found in shrimp, there is no known risk of cholera associated with these serotypes in shrimp. Risk assessments by FAO/WHO presented a very small risk of acquiring cholera through consumption of imported warmwater shrimp (FAO/WHO 2005). However, RASFF alert notifications (numbers: 2005.778, 2008.AXE, 2008.BDH) were issued for the presence of non-O1/O139 V. cholerae in black tiger shrimps exported from India. (http://ec.europa.eu/food/food/rapidalert/rasff_portal_database_en.htm). The biohazard status of non-O1/non-O139 V. cholerae which appears to be autochthonous bacterial flora in the black tiger shrimp culture system needs to be reassessed. It is proposed that the mere presence of V. cholerae non-O1/non-O139 in cultured black tiger shrimp need not be considered as the criterion for biohazard leading to rejection of shrimp exports but ctx carrying V. cholerae non-O1/non-O139 may be considered potential public health risk. However, further studies are needed to establish V. cholerae non-O1/non-O139 as native flora of black tiger shrimp culture system. Other virulence factors associated with V.cholerae are tcpA, hlyA, zot, ace, rtxA, etc. Fraga et al. (2007) reported that V. cholerae isolates from the environment lacked ctxA and tcpA but contained hlyA (98·7%), rtxA (99·0%), toxR (98·7%) and stn-sto (1·9%) The present study did not search for other virulence genes and further studies on the prevalence of these genes in ctx positive non-O1/non-O139 would aid in risk assessment.

The sensitivity of V. cholerae-duplex PCR was determined using templates prepared from different V. cholerae cell concentrations. The ctxAB specific primers yielded an amplicon only when the concentration of V. cholerae cells was 1000 V. cholerae cells/ml and above whereas the species specific sodB primers yielded an amplicon at a concentration as low as 100 V. cholerae cells/ml. The intensity and thickness of both the amplicon bands decreased with decrease in V. cholerae cell numbers. The sensitivity attained was similar that of reported sensitivities for multiplex PCR. The sensitivity of the multiplex PCR for the detection of V. cholerae targeting ctxA, ace, zot, tcpA and toxR was 102 CFU/ml (Jing et al. 2003). The sensitivity of multiplex PCR for the detection of toxigenic and pathogenic V. cholerae from direct water sources using specific primers targeting ompW, ctxB, rfbG, zot and tcpB was 5 × 104 V. cholerae cells per reaction (Goel et al. 2007). A duplex PCR targeting the genes gyrB and tl for specific identification of V. parahaemolyticus could detect as few as 250 CFU/ml in pure cultures (Vongxay et al. 2006). In the multiplex PCR (vvh fragment and viuB) for V. vulnificus, the sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 103 CFU/ml of pure culture (Panicker et al. 2004b)

The suitability of V. cholerae-duplex PCR in detecting V. cholerae in fish (rohu, tuna) and crustaceans (black tiger shrimp, three spot crab) samples was studied to know whether inhibitory substances present in these foods might adversely affect the performance of PCR. All the above mentioned food matrices did not show any inhibitory effect on V. cholera duplex-PCR as all the spiked samples yielded the V. cholerae species specific amplicon and ctx specific amplicon (Fig. 4). The specificity of the V. cholerae duplex-PCR is further strengthened by the fact that the primers yielded the specific amplicon even in the presence of initial resident Vibrio populations ranging between 60 CFU /g and 4.6 × 104 CFU/g in these food matrices (Table 3). The V. cholerae duplex-PCR can be of use in the post-harvest quality analysis of fish and crustaceans.

Fig. 4
figure 4

Performance of V. cholerae duplex-PCR in different food matrices. Lane 1, Tiger shrimp, (Penaeus monodon); Lane 2, 3 spot crab (Portunus sangulnolentus); Lane 3, Freshwater fish (carp, Labeo rohita); Lane 4, marine fish (tuna, Euthynnus affinis) meats spiked with V. cholerae (ctx positive) cells yielded positive PCR result for the presence of Vibrio cholerae species specific amplicon (248 bp) and cholera toxin specific amplicon (777 bp amplicon); Lane 8, 100 bp DNA ladder (GeNei)

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Table 3 Effect of different fish/shrimp matrices on the performance of V.cholerae-duplex PCR
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In this study, we studied the incidence of V. cholerae in black tiger shrimp aquaculture system and detected non-O1/non-O139 V. cholerae in aquaculture samples but not in hatchery samples. Seven percent of the non-O1/non-O139 V. cholerae isolates were potentially cholera-toxigenic. This work reports a new method viz., V. cholerae-duplex PCR method by utilizing V. cholerae species specific sodB primers and ctxAB genes specific primers. V. cholerae duplex-PCR may be particularly usefulness in the risk assessment of fish and shrimp as V. cholerae non-O1/non-O139 strains can acquire toxigenic genes for toxin production and therefore might be the source of new epidemics.