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 speciesspecific 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.Vibrio parahaemolyticus is found worldwide in estuarine waters and is the leading cause of gastroenteritis from seafood in the United States, with most infections resulting from the consumption of raw or mishandled seafood (2, 29). The tlh (thermolabile hemolysin) gene is a species-specific marker for V. parahaemolyticus (42,26), while the tdh (thermostable direct hemolysin) (33, 34) and trh (thermostable-related hemolysin) (35) genes are pathogenicity markers for V. parahaemolyticus. Pathogenic V. parahaemolyticus bacteria in the environment and food samples typically comprise 0.3 to 3% of the total V. parahaemolyticus population (4,8,20,22,43). DNA-based assays targeting these genes were developed for the detection and enumeration of V. parahaemolyticus bacteria (7,10,21,26,27,30,38,44). However, the ability of the PCR assays to detect low levels of pathogenic V. parahaemolyticus bacteria in the presence of a high background of nonpathogenic V. parahaemolyticus bacteria was not reported by the authors (10,30,44). The preferential amplification of nonpathogenic members of the total V. parahaemolyticus population due to the muchhigher copy number of the target could preclude the detection of pathogenic strains pres...

Deadliest Catch?

“…For example, Jessica Nordstrom and colleagues at FDA's Gulf Coast Seafood Laboratory and the Alaska Department of Environmental Conservation developed a multiplex, real-time PCR assay that allowed them not only to detect the presence of Vibrio spp. bacteria in oysters but also to identify and quantify pathogenic V. parahaemolyticus strains in a background of excess nonpathogenic species (8). (V. parahaemolyticus is related to the microbe that causes cholera and can trigger gastrointestinal problems such as watery diarrhea, abdominal cramping, nausea, and vomiting.…”

Detection of PCR inhibition in food and feed with a synthetic plasmid

“…Real-time PCR can be used to identify human pathogens, to quantify genetically modified organisms (GMOs), or to check correct labelling of food and feed (Renault et al 2004;Deer et al 2010). Even though qPCR is a very sensitive and precise technique, it can be hindered by the presence of the so-called PCR inhibitors or less common PCR enhancers (Hoorfar et al 2004;Hartman et al 2005;Nordstrom et al 2007). Those substances, when present in the PCR mixture, interfere with the PCR via diverse mechanisms and can result in under-or overestimation of the content of the target DNA sequence in the sample (Hartman et al 2005;Nordstrom et al 2007).…”

“…Even though qPCR is a very sensitive and precise technique, it can be hindered by the presence of the so-called PCR inhibitors or less common PCR enhancers (Hoorfar et al 2004;Hartman et al 2005;Nordstrom et al 2007). Those substances, when present in the PCR mixture, interfere with the PCR via diverse mechanisms and can result in under-or overestimation of the content of the target DNA sequence in the sample (Hartman et al 2005;Nordstrom et al 2007). Inhibiting substances can originate from the sample itself or be introduced during its processing (Bickley & Hopkins 1999;Hartman et al 2005;Cook et al 2013).…”

“…One of the possible approaches is to perform a serial dilution of the sample, which is however quite time-consuming and requires a large quantity of PCR reagents. Another possibility is to use an amplification control consisting in adding a known amount of an exogenous nucleic acid into the reaction mixture; the analysed amount of this control is compared with the added amount and the efficiency of the PCR reaction and presence of possible inhibitors can be evaluated (Hoorfar et al 2004;Hartman et al 2005;Nordstrom et al 2007).…”

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay Combined with Enrichment Culture for Rapid Detection of Very Low Numbers of Vibrio parahaemolyticus in Seafood Samples

“…Therefore, it may be appropriate to target tlh/ldh gene for the detection of V. parahaemolyticus. 11,[15][16][17] The loop-mediated isothermal amplification (LAMP) method described by Notomi et al is more advantageous than the PCR assay because it is more rapid, easier to perform and does not necessarily require expensive thermal cycler machine. 18) In the LAMP method it is possible to amplify DNA fragments within 90 min by using only water bath or heat block.…”