<i>In Silico</i> Analyses of Primers Used to Detect the Pathogenicity Genes of <i>Vibrio cholerae</i>

Abstract

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.

Whole Genome Sequencing Reveals Antimicrobial Resistance and Virulence Genes of Both Pathogenic and Non-Pathogenic B. cereus Group Isolates from Foodstuffs in Thailand

“…Moreover, BTyper may detect the presence of a toxin gene, while the PCR result may be negative [ 29 ]. A negative PCR result may be due to variant sequences causing poor binding of the published primers to the DNA sequence [ 32 ]. Therefore, cytotoxicity assays of B. cereus group isolates, along with their virulence factor gene profiles, may offer more information about potential harm to human health.…”

Development and Evaluation of Real Time PCR for Detection and Identification of C. jejuni and C. coli from Human Food

“…The quality of CadF primers may be one of the reasons affected the uorescence level of C. coli obtained despite the doubled amount of primers introduced in the ampli cation mixture. Gardès et al (2012) strongly recommended the bioinformatic analyses of primers, before their use, to validate the choice and thus avoid or improve their specify use in molecular detection tests.…”