In Silico Analyses of Primers Used to Detect the Pathogenicity Genes of Vibrio cholerae

In Vibrio cholerae, the etiological agent of cholera, most of the virulence genes are located in two pathogenicity islands, named TCP (Toxin-Co-regulated Pilus) and CTX (Cholera ToXins). For each V. cholerae pathogenicity gene, we retrieved every primer published since 1990 and every known allele in order to perform a complete in silico survey and assess the quality of the PCR primers used for amplification of these genes. Primers with a melting temperature in the range 55–60°C against any target sequence were considered valid. Our survey clearly revealed that two thirds of the published primers are not able to properly detect every genetic variant of the target genes. Moreover, the quality of primers did not improve with time. Their lifetime, i.e. the number of times they were cited in the literature, is also not a factor allowing the selection of valid primers. We were able to improve some primers or design new primers for the few cases where no valid primer was found. In conclusion, many published primers should be avoided or improved for use in molecular detection tests, in order to improve and perfect specificity and coverage. This study suggests that bioinformatic analyses are important to validate the choice of primers.


Fig. S1: Lifetime of valid and invalid published PCR primers used to amplify
V. cholerae pathogenicity genes. For each primer, total number of primers is plotted against the number of years it was still cited in the literature after its first publication. Dotted curve: invalid primers, full curve: valid primers (124 invalid primers and 57 valid primers with a 1year lifetime are not shown). Note the large number of primers used one or two years only (5 valid and 4 invalid primers). Tms were computed for each variant gene sequence and each primer using OHM. Accession number, number of other sequences identical to or contained in the sequence used in this tree, species names, and Tms are indicated (forward and reverse primers in columns from left to right). For strain, biovar and serotype information, see Table S9. Tms are symbolized by a color code described in the box on the right. A predicted Tm lower than the lowest temperature mentioned in the box is symbolized by a grey square. White boxes when a partial sequence does not encompass a primer. Please note that OHM tends to underestimate the Tm of primers (as compared to other tools).
ctxA: the forward and reverse primers amplify every allele of the gene and are specific of V. cholerae. hapA: the forward and reverse primers amplify every allele of the gene and are specific of V. cholerae. However, a slight variation of Tm is observed for the reverse primer and the sequence CP001236, and a lower Tm (50.8°C) should be used for the detection.
hlyA: the forward and reverse primers detect every known sequence of V. harveyi but cannot amplify every allele of the gene in V. cholerae (AF194418, D58374, AY427780).
ompU: the forward and reverse primers amplify every allele of the gene in V. cholerae, but also detect some sequences of V. mimicus (e.g. DQ356331).
ompW: the high variation of Tms (from 46 to 54°C) for the second couple is probably too large for a proper detection. Only the first couple is probably useful to amplify ompW in V. cholerae. sto: the couple of primer amplifies only one allele (M85198) of the gene in V. cholerae. tcpA: the reverse primer is the primer targeting the classical strain of V. cholerae. The one targeting the El tor strain was not studied because of its localization outside the CDS. As shown in this figure, the forward and reverse primers cannot amplify every allele of the gene in V. cholerae, but will detect two sequences of V. mimicus (AF315787, DQ356010). The Tms of the forward primer vary from 43.8 to 51.4°C, according to the method of computation of Tm, with a high Tm (51.4°C) for the sequences of V. mimicus. For the reverse primers, the Tms vary from 52.1 to 52.5°C. The efficiency of this primer cannot be appreciated for sequences of V. mimicus because only partial sequences are available for the target region.
tcpI: the forward and reverse primers amplify every allele of the gene.
toxR: the primers of the first (Nandi et al. 2000) and the second (Halpern et al. 2005) published couples amplify every allele of the gene in V. cholerae. However, a variation of Tm is observed for the foward primer of the second couple for sequence CP001485. The lowest Tm (45.8°C) should be used for the detection, but this condition can favor the amplification of sequences of V. harveyi. Indeed, because of the partial sequences, it is not possible to estimate the efficiency of couples for the outgroup sequences, and therefore their specificity toward V. cholerae. As a result, toxR is not a validated genetic marker for the detection of V. cholerae. zot: the forward and the reverse primers amplify every allele of the gene in V. cholerae. A variation of Tm is observed for the foward primer and sequence AF220606. A low Tm (47.7°C) should be used for the detection. The specificity of this couple cannot be estimated because of the partial sequence of V. mimicus. The zot gene is therefore not validated for detecting V. cholerae.

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Table S1: Summary of results obtained from the analysis of the complete list of sequences retrieved for V. cholerae pathogenicity genes.
Columns 1: pathogenicity island, 2: most frequently used gene name, 3: total number of sequences retrieved, 4: total number of unique sequences, 5: number of alternate gene and protein names found, 6: published and 7: valid primers retrieved from the literature.              S10: List of softwares available for the design of primers. "Speciality" describes if softwares can design primers for specific molecular methods and "Input" reports the several types of format compatible with each software. "Parameter settings" presents common parameters of softwares and the methods used to compute melting temperature (Tm).

Gene Sequences
"Option" shows if a program supports degenerated positions (IUPAC code), checks specificity (BLAST of primers) and coverage (BLAST of input sequences) of primers, and allows using target sequences and non-target sequences. qPCR: quantitative PCR, RFLP: