Vibrio cholerae is a Gram-negative, comma shaped facultative pathogen responsible for causing cholera. The global incidence of cholera was about 2.8 million cases per year, with 91,000 deaths (1). V. cholerae O1 has been the etiological agent for several cholera epidemics. The serogroup O139 was responsible for cholera outbreaks in India and other countries in Asia during 1992 (2) and was also isolated during the outbreak in November 2000 in India (3) and March–April 2002 in Bangladesh (4).

Vibrio cholerae O1 and O139 serogroups express toxin coregulated pilus which confers the bacteria the ability to colonize the intestine while the cholera toxin is associated with secretory diarrhea (5). Depending on severity, the infectious dose for V. cholerae varies from 10⁶ to 10¹¹ cells (6).

Toxigenic and non-toxigenic non O1, non-O139 have been documented as incriminating in several outbreaks in developing countries (7–10). In non-CT-producing vibrios, virulence factors such as type 3 secretion systems, hemolysin (HlyA), repeat in toxin (RTX), and heat-stable enterotoxin have major roles in causing infections (11). Hasan et al. (10) reported 98% V. cholerae strains carried hemagglutinin protease hap (98%) irrespective of their source, i.e., clinical or environmental. Other virulence factors present are T6SS (94–99%), rtxA (96%), toxR (87%), and hlyA (83%), and all these virulence factors might be responsible for diarrhea caused by non-toxigenic non-O1/non-O139 variants.

Vibrio cholerae in the viable but non-culturable (VBNC) state can express virulence factors required to produce infection (12). The VBNC cells have the capacity to revert to the culturable state and colonize the intestine (13) the mechanism of which is largely unknown (14). These organisms may go undetected if conventional culture based methods are used (15). Conventional identification of V. cholera, which may be done by biochemical tests, is time consuming and laborious. Available commercial biochemical identification systems, such as dipstick test used for the detection of O1 and O139 strains, are not always accurate (16). V. cholerae has been shown to possess similar biochemical properties with other species in the Genus Vibrio and Aeromonas, hence complicating an accurate identification (17).

Compared to conventional PCR, real-time PCR is less labor intensive, more safe, and rapid due to the elimination of gel electrophoresis. It has greater sensitivity and can detect minute amounts of target amplicons that might be missed by the conventional PCR. Real-time PCR can directly target genomic DNA and thus eliminate extensive incubation periods (18). Furthermore, VBNC cells can be detected which might be missed by culture-based methods. The ompW sequence is highly conserved among V. cholerae species belonging to different biotypes and/or serogroups (17). Hence, the ompW gene could be used as a target for species-specific detection, identification, and quantification.

A number of assays exist for the detection of V. cholerae (19–24) but many of these assays lack empirical data for reproducibility and repeatability. Some of these assays have not been validated in terms of detecting non-specific products that might accompany the amplification reaction. Furthermore, a number of assays are based on virulence factors that might not be present in certain strains and might yield false negative results.

The aim of this study was to develop an assay that detects and quantifies both O1/O139 and non O1/O139 disease causing strains of Vibrio spp. In addition, the assay would be able to quantify VBNC cells that cannot be detected or quantified by conventional methods.

We have developed a real-time assay with designed primers for the detection and quantification of V. cholerae. The assay was based on SYBR Green PCR Mastermix and targeted the ompW gene, which is present in all species of V. cholerae. Initially, the physical properties of primers were assessed, followed by validation of sensitivity, precision, specificity, and melt curve analysis.

The LC describes nucleotide arrangement and composition of a sequence and the likelihood of PCR success of each primer. LC values of 80 and higher serve as excellent candidate primers. The primers developed had LC values of 89 and 95 for reverse and forward primers, respectively. Low-complexity regions such as Simple Sequence Repeats, imperfect direct or inverted repeats, triple-stranded DNA structures, and G/C quadruplexes (32) were unlikely to be formed if primers with high LC values are used. The parameter “Primer Quality” determines the possibility of primer dimer formation since dimers reduces the PQ value. The designed primers had PQ values of 87 and 93 for reverse and forward primers, respectively. Thus, these high values suggest that self-complementarity was not apparent. Two terminal C/G bases, recommended for increased PCR efficiency (33) were present in the designed primers.

The efficiency of a PCR assay is the amount of DNA that is amplified in each cycle. An efficiency of 100% indicates the target DNA has been doubled. The efficiencies obtained for the replicates 1 and 2 were 89.16 and 97.37%, respectively. Generally, efficiencies ranging from 90 to 100% are considered to be satisfactory. Inadequate primer design, production of non-specific amplicons and primer dimers may be responsible for reduced efficiencies (34). This is, however, only an estimate of the PCR efficiency and a real test sample, such as food, may contain inhibitory substances that decrease the PCR efficiency (35).

The precision of the assay was assessed by calculating both repeatability (intra-assay precision) and reproducibility (inter-assay precision). The coefficient of variation (CV%) for the repeatability ranged from 0.24 to 1.32 for both replicates. The CV% for the reproducibility varied from 1.08 to 3.79. The reproducibility is an important parameter since changed conditions such as different equipment and operators might affect the outcome. Pipetting and other human errors might account for poor precision. The precision usually increases with decreasing gene copy concentration (34) but this pattern was not observed for the developed assay. Retesting is required if the % CV of the PCR replicates exceeded 30% (36). All the CV% values for the assay were acceptable.

Specificity is ability to detecting chosen gene in the presence of non-specific DNA (34). The specificity is an important parameter since, in clinical and food samples, DNA from a wide range of organisms might be present. The developed assay was able to correctly detect the 7 V. cholerae and gave C_T values that ranged from 18.778 to 23.706. Though the assay did not give any C_T values for the 22 non-V. cholerae strains (Table 5), two E. coli strains—E. coli EIEC 2V, E. coli EHEC BH29 showed C_T values of 38.439 and 35.073 respectively. Since the cut point C_T value for ABI StepOne real-time machine is between >8 and <35, these C_T values of E. coli strains can be considered as negative results.

The LOD is the lowest gene copy number that the assay is able to consistently detect (37). A satisfactory LOD is 10 gene copies per reaction, and the assay was able to meet this requirement by consistently detecting 5.46 copies of the gene. The LOD sheds light on how sensitive the assay is.

The assay was evaluated for its ability to detect V. cholerae O1/O139 and non-O1/non-O139 in food and environmental samples over different dilutions. It was observed that drinking water, pond water, shrimp, and boiled rice spiked with these strains registered C_T values that ranged from 16.33 to 26.78 (Table 6).

To assess if the assay is affected by interference from non-target DNA, unspiked drinking water, pond water, shrimp, and boiled rice were examined by qPCR. Before this assessment, absence of V. cholerae was confirmed. Results showed that no C_T values were obtained for these unspiked food and water samples. Thus, this assay is suitable for detecting both V. cholerae O1/O139 and non-O1/non-O139 in food and environmental samples since non-specific amplification was not seen in negative controls.

Melt curve analysis was done to assess whether secondary products such as primer dimers or non-specific products were formed. The melt curve gave a single peak with a Tm value of about 78.46°C. Agarose gel electrophoresis of SYBR Green PCR products gave a single band at 191 bp. These results suggest that the amplification was specific and only one type of amplicon was produced. Non-specific products hamper the efficiency of the assay and affect precision. Non-specific products were absent suggests that the primer design was adequate. The primers were specific and intended amplicons were produced. We can conclude the primers were not complementary to one another since primer dimers were not produced.

Many assays have been developed for detection and quantification of V. cholerae (19–24). Though impressive none of these presented any statistical figures (such as coefficient of variation) which would inform us about the reproducibility and repeatability. Many of these assays did not undergo melt curve analysis or the PCR products were not subjected to agarose gel electrophoresis and hence we do not know their status regard the formation of non-specific products. Since they are multiplex in nature, they add to the cost and hence are not suitable for purposes. For instance, during quality control testing of seafood where only quantification is required to see if the levels in food is acceptable to the standards set by the governing bodies.

An extremely impressive multiplex real-time assay has been developed by Bliem and colleagues (38). The assay is multiplex in nature, and hence the use of multiple primers might add to the cost. The assay developed by Bliem and colleagues had inter-assay variance of 2–28% for ompW. But our assay, which utilizes a primer for ompW gene with different sequence, was more precise with inter-assay variance of 1.08–3.79.

Future objectives of our study might include the optimization of this assay to detect and quantify V. cholerae in food, water, and clinical samples. Some samples might contain inhibitory substances that decrease PCR efficiency (35) and hence optimization of the methods involving sample processing, DNA extraction, and assay itself might be required.

PJ and AB are the principal investigators of the project and contributed to the manuscript revision and final version approval to be published. RR conducted the study in the laboratory, performed statistical analysis, and wrote the first draft of the manuscript. ST contributed to revising the manuscript critically for important intellectual content. JF contributed to the study designing, implementation, manuscript reviewing, and revising it critically. The authors have agreed to be accountable for answering questions related to the accuracy and integrity of the work appropriately done.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.