Evaluation of new primers for detecting toxigenic vibrio cholerae by multiplex PCR

Vibrio cholerae is the etiological agent of cholera that has emerged as an endemic disease in different regions of the world in recent years. Traditional microbial culture and microscopy methods are considered to be the best standard for diagnosing V. cholerae infection. These methods, however, delay any available confirmatory answer by days. Molecular methods have the potential to provide sensitive, accurate, and rapid analysis of V. cholerae infection. We have developed a multiplex PCR assay to detect virulence and toxigenic-associated (VTA) genes (ctxA, tcpA, and ompW). To evaluate PCR specificity, additional bacteria from the enterobacteriaceae family (Salmonella typhi, Shigella dysantry, and entrotoxigenic E. coli) and Aeromonas hidrophyla were examined in this study. Specificity tests were evaluated using the genome dilution method. Importantly, the results show that our PCR specificity method represents the best tool for the rapid detection of VTA genes because of its simplicity, cost effectiveness, and accuracy. This multiplex PCR method can be used for examining the existence of VTA genes in patient samples, and therefore will distinguish V. cholerae from other vibrios and bacteria. This method is able to detect 10-100 colony forming units (CFUs) of V.Cholerae and 8.5-85 picograms (pg) of genomic DNA. The multiplex PCR method is also more specific and sensitive than other methods, validating it as an appropriate and sensitive tool for detecting the presence of toxigenic and pathogenic V. cholerae. Introduction Diarrheal diseases are among the greatest world healthcare problems. Cholera is one of the most serious diarrheal diseases, causing an epidemic with Vibrio cholerae O1 or O139 serogroups.1 Primary virulence factors of V.cholerae are cholera toxin and toxin-co-regulated pilus, which are encoded by the ctxAB and tcpA genes, respectively.2,3 CtxA and tcpA are uncommon in environmental strains,4 yet are responsible for the pathogenesis in V.cholerae serogroups O1 and O139.5-7 Biochemical methods for the detection of V.cholerae are timeconsuming and can require 2 to 7 days for assessment. Cholera detection requires urgent testing, and efficiency is a critical factor for determining the usefulness of any detection method. Moreover, special expertise is required to process these tests, but such expertise is not available in all laboratories.8 Thus, rapid detection tests are functional and necessary for creating a better detection method for cholera infection. These tests are fast and sensitive techniques for the primary diagnosis and control of pathogenicity, and employ several biotype-specific genes to diagnose the presence of V.cholerae, such as ctxA and tcpA. The aim of this study was to develop such an assay for the rapid detection of pathogenic and toxogenic V. cholerae bacteria. To develop a standard, accurate, and rapid method for the detection of V. cholerae, we designed the Multiplex PCR method for genes ctxA, tcpA, and ompW. Materials and Methods Bacterial strains V. cholerae O1 and non-O1 stains, Salmonella typhi, Shigella dysenteriae, Aeromonas hydrophila, and Enterotoxigenic Escherichia coli (ETEC) were all included in this study and were provided by the Bu-Ali reference laboratory in Iran. DNA extraction All strains were cultured in pepton soy broth and incubated at 37oC overnight. Genomic DNA was extracted using the DNA Pure Extraction Kit (Bioneer, South Korea) according to the manufacturer’s protocol. Target sequences and primers Three target sequences were used that encoded cholera toxin (ctxAB), toxin coregulated pilus (tcpA), and outer membrane protein (ompW). The primers used were ctxA, tcpA, ompW (NC-012583, NC-009457, NC-012583), respectively, and are shown in Table 1. PCR amplification for uniplex and multiplex assays Three uniplex PCR assays were carried out in 50 μL reaction volumes consisting of 20 mM Tris-HCl (pH 8.4), 1 unit of Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, California, USA), 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Invitrogen, Carlsbad, California, USA), 2 mM MgCl2, 25 pmol of each primer, and 50 ng of DNA template. Multiplex PCR reactions were performed with the simultaneous addition of primers for the three genes in the same mixture. Thermocycling conditions consisted of an initial denaturation at 94°C for 4 min, followed by 35 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 50°C, and 1 min of extension at 72°C. The final extension was carried out at 72°C for 5 min. Microbiology Research 2011; volume 3:e1 Correspondence: Jalil F. Mehrabadi, MARS Bioinformatics Institute, Tehran, Iran. E-mail: jalil.fallah@gmail.com


Introduction
Diarrheal diseases are among the greatest world healthcare problems.Cholera is one of the most serious diarrheal diseases, causing an epidemic with Vibrio cholerae O1 or O139 serogroups. 1 Primary virulence factors of V.cholerae are cholera toxin and toxin-co-regulated pilus, which are encoded by the ctxAB and tcpA genes, respectively. 2,36][7] Biochemical methods for the detection of V.cholerae are timeconsuming and can require 2 to 7 days for assessment.Cholera detection requires urgent testing, and efficiency is a critical factor for determining the usefulness of any detection method.Moreover, special expertise is required to process these tests, but such expertise is not available in all laboratories. 8Thus, rapid detection tests are functional and necessary for creating a better detection method for cholera infection.These tests are fast and sensitive techniques for the primary diagnosis and control of pathogenicity, and employ several biotype-specific genes to diagnose the presence of V.cholerae, such as ctxA and tcpA.The aim of this study was to develop such an assay for the rapid detection of pathogenic and toxogenic V. cholerae bacteria.To develop a standard, accurate, and rapid method for the detection of V. cholerae, we designed the Multiplex PCR method for genes ctxA, tcpA, and ompW.

Bacterial strains
V. cholerae O1 and non-O 1 stains, Salmonella typhi, Shigella dysenteriae, Aeromonas hydrophila, and Enterotoxigenic Escherichia coli (ETEC) were all included in this study and were provided by the Bu-Ali reference laboratory in Iran.

DNA extraction
All strains were cultured in pepton soy broth and incubated at 37ºC overnight.Genomic DNA was extracted using the DNA Pure Extraction Kit (Bioneer, South Korea) according to the manufacturer's protocol.

PCR amplification for uniplex and multiplex assays
Three uniplex PCR assays were carried out in 50 μL reaction volumes consisting of 20 mM Tris-HCl (pH 8.4), 1 unit of Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, California, USA), 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Invitrogen, Carlsbad, California, USA), 2 mM MgCl 2 , 25 pmol of each primer, and 50 ng of DNA template.Multiplex PCR reactions were performed with the simultaneous addition of primers for the three genes in the same mixture.Thermocycling conditions consisted of an initial denaturation at 94°C for 4 min, followed by 35 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 50°C, and 1 min of extension at 72°C.The final extension was carried out at 72°C for 5 min.For assessment and specificity determination of the designed primers, genomic DNA of V. cholerae O1 and non-O1, ETEC, S. typhi, Sh.dysenteriae, and A. hydrophila was used as templates in uni-and multiplex PCR reactions.

Sensitivity test
For detection of V. cholerae genomic DNA, serial dilutions of DNA were made ranging from 100 ng to 10 pg.PCR was performed at each dilution value.

PCR of ctxA, tcpA, and ompW genes
CtxA, tcpA, and ompW genes were amplified by PCR using genomic DNA of both V. cholerae O1 and non-O1 serogroups.The amplicons related to V. cholerae O1 ctxA, tcpA and ompW genes were 219 bp, 295 bp, and 558 bp, respectively.The PCR reaction with the V. cholerae non-O1 ompW gene was the only reaction that resulted in an amplicon of the same size (Figure 1).

Specificity test for ctxA, tcpA, and ompW genes
To confirm the specificity of the designed primers, genomic DNA of S. typhi, Sh. dysenteriae, A. hydrophila, and ETEC was used as a template.The results showed that the designed primers were specific for these genes, since no additional amplicons were detected on gel electrophoresis.

Sensitivity test for ctxA, tcpA, and ompW genes
Although the infective dose of V. cholerae is approximately 10 8 bacteria, performing a sensitivity test by PCR is imperative for determining the robustness of the test.Genomic DNA of V. cholerae O1 was extracted and diluted from 100 ng to 10 pg, and PCR was performed at each concentration.The minimum concentration of genomic DNA at which ctxA, tcpA, and ompW could be detected were 8.5, 85, and 85 pg, respectively.Moreover, the ompW gene was detected when the DNA was diluted to 14 pg in the in the V. cholerae non-O1 sample (Figure 2).

Triplex PCR of V. cholerae O1 and non-O1 serogroups
Using all three sets of primers and V. cholerae O1 genomic DNA in a single PCR assay, the ctxA, tcpA, and ompW genes were amplified.The amplicon sizes were 219 bp, 295 bp, and 588 bp for the ctxA, tcpA, and ompW genes, respectively (Figure 1).The same triplex PCR reaction was carried out for detection of three genes in the V. cholerae non-O1 strain, but only showed a 588 bp amplicon, which corresponded to ompW.

Discussion
The standard methods in microbiology that are traditionally used to detect V. cholerae infection have several disadvantages.Therefore, a rapid and more efficient method is needed.We employed an updated nucleic acid based approach based on PCR assays, which simplified bacteria detection.It has been shown that toxicon-regulated pili are used by V. cholerae for intestinal colonization, and the main mediator is the regulatory ctxA gene. 9Therefore, an important standard gene for the detection of toxigenic V. cholerae is ctxA. 10,11andi et al. designed rapid detection methods using ompW and toxR and analyzed 254 V. cholerae samples. 12Samples 229 and 239 were shown to have the ompW and toxR genes, respectively.This was reported for the O1, O139, non-O1, and non-O139 serogroups, but was undetectable in other Vibrio serogroups. 12n that study, similar to our research, the ompW gene was 588 bp in length.In our particular test, ctxA was used for V. cholerae detection.
Panikar et al. applied DNA microarrays and Multiplex PCR to detect pathogenic V. cholerae. 13In that particular study, 10 genes were used for diagnosing the Vibrio family.For V. volnificus, rrh and viuB were detected with specific primers and probes.Moreover, for V. parahaemolyticus, the tlh, tdh, trh, and ORF8 genes were used for bacterial detection; for V. cholerae, the ompU, toxR, and tcpI genes were used for detection; and for El Tor and the classical biotype, the hlyA gene was used for bacterial detection.The specificity for this method was 100%.Diagnosis in non-broth culture was 100-1000 CFU/mL, while it was 1 CFU/mL in broth culture.The genes ompW, ctxA, and tcpA were used to detect V. cholerae stains. 13ubala et al. applied Multiplex Real Time PCR to amplify rtxA, epsM, tcpA, and mshA genes as a method for detecting toxigenic V. cholere. 14In this study, the tcpA gene was not detected in the non-O1 and O1 serogroups.Non-Vibrio was not detected in the specificity analysis of primers, but the tcpA gene was detected in V. cholerae serogroups O1 and non-O1. 14atsaki Hoshino and his group applied Multiplex PCR to detect toxigenic Vibrio O1 and O139 using the ctxA and rfb genes. 15Of 121 patient waste samples, 38 samples tested positive by this method, and importantly, the method sensitivity was 100% and specificity was 95.5% compared to the bacteriologic culture method.Detection in this study was reported for V. cholerae O1 at 65 CFUs and for O139 at 200CFUs. 15alsgaard et al. analyzed the virulence of V. cholerae non-O1 and non-O139 by amplifying genes ctxA and tcpA via PCR. 16In this study, ctxA and tcpA genes were detected in the V. cholerae serogroup O1.
In our research study, the ompW gene was used to detect all Vibrio species.Multiplex PCR was applied for rapid and specific detection of Vibrio containing the toxigenic gene as well as other Vibrio of gram-negative bacteria.In addition to using the cholera toxin gene to specifically identify V. cholerae pathogenic strains, we applied the tcpA and ompW genes for detection of other V. cholerae species.Method sensitivity was analyzed by diluting V. cholerae bac-