Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26°C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r =−0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus–specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

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In-house validation of novel multiplex real-time PCR gene combination for the simultaneous detection of the main human pathogenic vibrios (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus)

Food Control ◽

10.1016/j.foodcont.2013.09.026 ◽

2014 ◽

Vol 37 ◽

pp. 371-379 ◽

Cited By ~ 20

Author(s):

Alejandro Garrido-Maestu ◽

María-José Chapela ◽

Elvira Peñaranda ◽

Juan M. Vieites ◽

Ana G. Cabado

Keyword(s):

Vibrio Cholerae ◽

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Vibrio Vulnificus ◽

Simultaneous Detection ◽

Gene Combination ◽

Pathogenic Vibrios

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Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

Applied and Environmental Microbiology ◽

10.1128/aem.72.3.2031-2042.2006 ◽

2006 ◽

Vol 72 (3) ◽

pp. 2031-2042 ◽

Cited By ~ 85

Author(s):

Linda N. Ward ◽

Asim K. Bej

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Detection Methods ◽

Tissue Homogenate ◽

Oyster Tissue ◽

Reading Frame ◽

Initial Inoculum ◽

Hemolysin Gene ◽

Pure Cultures

ABSTRACT We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 104 CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 109 CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

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Multiple Displacement Amplification Enables Large Scale Clonal Analysis after Retroviral Gene Therapy.

Blood ◽

10.1182/blood.v108.11.5469.5469 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5469-5469

Author(s):

Stephanie Bleier ◽

Patrick Maier ◽

Frederik Wenz ◽

W. Jens Zeller ◽

Stephanie Laufs ◽

...

Keyword(s):

Gene Therapy ◽

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Large Scale ◽

Integration Site ◽

Multiple Displacement Amplification ◽

Quantitative Real Time Pcr ◽

Integration Site Analysis ◽

Reliable Quantification

Abstract Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole genome amplification named multiple displacement amplification (MDA) with respect to the even and accurate representation of retrovirally transduced genomic DNA. We were able to show that MDA is a suitable method to subsequently specify engraftment efficiencies by quantitative real-time PCR as the retroviral integrations are amplified the same way and by the same probability as all other parts of the genome. We validated the method by analyzing a dilution series containing retrovirally transduced DNA and untransduced background DNA and retroviral integrations found in primary material from a retroviral transplantation model by quantitative real-time PCR. The representation of the portion of retroviral DNA in the amplified samples was 0.9-fold (range 0.2 – 2.1-fold) of the portion determined in the original genomic DNA. Furthermore, the succession of the combination of MDA and integration site analysis by ligation-mediated PCR showed an increase in the sensitivity of the method as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show that MDA enables large scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow up analysis in gene therapy studies even from smallest amounts of starting material.

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