Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters

Abstract

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

FIG. 1.

Standard curves of three target genes in multiplex and simplex PCR. (A) Standard curve for the multiplex assay as described in Materials and Methods; the r² value was 0.99 for the standard curve of each target. (B, C, and D) Standard curves for each of the targets when run as a single target with the same reaction conditions as described in Materials and Methods. Results for the tlh, tdh, and trh targets are shown in panels B, C, and D, with r² values of 0.98, 0.99, and 0.99, respectively. Each standard curve was run in triplicate; each replicate is plotted. V. parahaemolyticus with all three target genes from an exponential-phase culture was diluted in PBS, boiled, and used as a template for all experiments, with concentrations of 0.9 × 10⁵ to 9 × 10⁵ CFU/reaction, except that the tlh simplex assay was tested with 0.9 × 10⁴ to 9 × 10⁴ CFU/reaction. rxn, reaction.

FIG. 2.

Reporting of the IAC without and with sample matrix inhibition. (A) Consistent reporting (C_T of 19 to 21) of the IAC in the presence of 0.9 × 10⁵ to 9 × 10⁵ CFU/reaction of V. parahaemolyticus targets is demonstrated. (B and C) Shifts and complete inhibition of the IAC, respectively, in some Alaskan oyster samples are shown. The large number of reactions without any shift emphasizes the overall lack of inhibition seen in the Alaskan oyster samples.