Monday, 24 March 2014

A multiplex PCR assay for rapid detection & characterisation of Vibrio vulnificus & Vibrio parahaemolyticus

Vibrio vulnificus and Vibrio parahaemolyticus (have we mentioned Vibrios before?....) are both commonly found in estuarine and marine environments and are leading causes of seafood poisoning (through ingestion of filter feeding molluscs) and known to cause necrotizing fasciitis, infecting open wounds whilst bathers use infected waters.  Speedy detection of these Vibrios may be beneficial for the public health sector, incorporating both the food industry and environmental monitoring.
V. parahaemolyticus possess a thermolabile haemolysin tlh gene, differentiating it from V. vulnificus which contains the cytolysin vvhA gene.  Additionally, pathogenic strains of V. parahaemolyticus contain a thermostable direct haemolysin (tdh)-encoding or tdh-related (trh)-encoding gene and V. vulnificus pathogenic strains contain a siderophore-encoding gene (viuB) that is linked with iron acquisition and is of particular risk to people with high iron concentrations in their blood.  Bhattacharyya & Hou (2013) appear to have successfully developed a single assay that detects both pathogenic and benign strains of V. vulnificus and V. parahaemolyticus.   The pentaplex PCR (pPCR) assay uses a combination of the above five molecular biomarkers (tlh, tdh,  trh, vvhA and viuB genes) in a single PCR tube that is reportedly able to provide accurate results with up to 98.7% efficiency, within six hours of taking the samples.   
The study used seven reference strains with known genotypes and 293 environmental strains from the Gulf of Mexico.  To validate the results of the assay, results from the pPCR assay were compared with previously published PCR protocols, and efficiency of the pPCR assay was calculated using the number of false-positive bands, false-negative bands and any spurious bands.  51 isolates that did not detect any of the target genes and contained some spurious bands were additionally analysed by 16s rRNA sequencing.  Spurious bands were found in both the pPCR assay and previously published multiplex assays, however the pPCR assay allowed 100% efficiency in detecting the five target genes in the reference strains and 96.6% efficiency in the environmental strains, with the detection limit at 30 cells ml-1.  The authors found that a reduction in the number of PCR cycles from previously published protocols, resolved the problem of spurious bands near the 510- to 550- bp range.   Additionally, 57 °C was the only temperature that allowed all five primers to anneal and amplify the target jeans, with many of the spurious bands disappearing at this temperature. 
This assay appears to be the first of its kind to simultaneously detect and characterise the presence of V. vulnificus and V. parahaemolyticus with high efficiency in as a little as six hours.  Whilst there are some flaws, including not being able to differentiate between the two amplicon products of viuB (504 bp in V. vulnificus) and trh (500 bp in V. parahaemolyticus), this assay is a valuable tool that can provide rapid results pertaining to both seafood and water quality.

Bhattacharyya, N., & Hou, A. (2013). A pentaplex PCR assay for detection and characterization of Vibrio vulnificus and Vibrio parahaemolyticus isolates. Letters in applied microbiology, 57(3), 233-240.


  1. I found this study very interesting; it seems like an important development for a more accurate detection of the strains of both Vibrio species. However I wonder how applicable/cost effective these methods are for testing marine samples in general, since the authors have used only strains of these two species in the study - or have I misunderstood this and they used environmental strains from a diversity of species? Perhaps other marine bacteria closely related to the two mentioned Vibrios could possess the same genes, or are they unique to V. vulnificus and V. parahaemolyticus? The oceans are full of so many diverse bacterial species so I can imagine that there are other that could share the genes tested here. If the genes were unique to only V. vulnificus and V. parahaemolyticus, then this assay could detect pathogenic strains of the two species very accurately and could ensure the safety of water or seafood. Although if this assay is only applicable for the two species, I wonder if the costs for such PCRs would be worth it for commercial seafood farms as they are a normal culturing of the bacteria could show the presence of the species too. Further tests should involve an actual marine water sample to investigate how widespread these genes are among the present bacteria. Nontheless, I think this is a very nice study and highlights the efficiency and accuracy of pPCRs, and the detection of the strains within six hours is very useful as well.

  2. Hi Malin, apologies for the delay in responding. You make some very valid points. Firstly, I think the initial cost of developing the primers for detection of the genes is the most costly aspect and now that they are available, detecting the genes should be fairly inexpensive and reducing in cost all the time. V. vulnificus and V. parahaemolyticus are two of the most common species that are known to cause human disease in both water and food and as far as I'm aware, the genes are diagnositc in identifying those species. Be that as it may, the proteins encoded by these genes have been shown to cause gastroenteritis and able to lyse red blood cells, therefore any species that possess these genes would be detected by the assay and indicate that the water or food was contaminated. I think the test is a good, quick way to indicate whether there is a risk to the public that encompasses two wide areas of human usage - eating & playing!