Development of a multiplex real-time PCR method for pathogenic Vibrio parahaemolyticus detection (tdh+ and trh+)

A real-time PCR method targeting the toxR gene of Vibrio parahaemolyticus was developed to quantify the number of V. parahaemolyticus cells, including those of both the hemolysin-producing and nonproducing strains. The specificity of the primer and probe set was confirmed using 25 strains of V. parahaemolyticus and 30 strains of other microbial species. We determined the threshold cycle number using the real-time PCR and the number of V. parahaemolyticus cells by plate count using serially diluted pure culture and developed a standard curve for quantification. Standard curves for V. parahaemolyticus in seawater and seafood were established using artificially inoculated samples. The threshold cycle number and the number of V. parahaemolyticus cells were correlated with 101 to 107 CFU/ml in pure culture, seawater, and shellfish homogenate. The real-time PCR method developed in this study was compared with the most-probable-number method in seafood samples that were naturally contaminated. The differences in the number of V. parahaemolyticus cells as determined by the culture method and the PCR method were less than 10-fold.

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Application of a novel pathogenicity marker in a multiplex real-time PCR method to assess total and pathogenic Vibrio vulnificus in food and environmental samples

Food Control ◽

10.1016/j.foodcont.2013.07.007 ◽

2014 ◽

Vol 35 (1) ◽

pp. 274-283 ◽

Cited By ~ 5

Author(s):

Alejandro Garrido-Maestu ◽

María-José Chapela ◽

Belén Román ◽

Juan M. Vieites ◽

Ana G. Cabado

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Vibrio Vulnificus ◽

Pathogenic Vibrio ◽

Pcr Method

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Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. in raw shrimp

Food Control ◽

10.1016/j.foodcont.2014.11.007 ◽

2015 ◽

Vol 51 ◽

pp. 31-36 ◽

Cited By ~ 56

Author(s):

Zhaohuan Zhang ◽

Lili Xiao ◽

Yang Lou ◽

Mengtong Jin ◽

Chao Liao ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Simultaneous Detection ◽

Salmonella Spp ◽

Pcr Method ◽

Raw Shrimp

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

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2010.08.016 ◽

2010 ◽

Vol 143 (3) ◽

pp. 190-197 ◽

Cited By ~ 30

Author(s):

A. Robert-Pillot ◽

S. Copin ◽

M. Gay ◽

P. Malle ◽

M.L. Quilici

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Pathogenic Vibrio ◽

Reliable Quantification

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Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

Anti-Infective Agents ◽

10.2174/2211352518999200629165108 ◽

2020 ◽

Vol 18 ◽

Author(s):

Pegah Shakib ◽

Mohammad Reza Zolfaghari

Keyword(s):

Streptococcus Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Cross Sectional Study ◽

Laboratory Culture ◽

Cross Sectional ◽

Negative Results ◽

False Negative Results ◽

Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

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Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

Author(s):

Trevor G. Phister ◽

David A. Mills

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

Rrna Gene ◽

Dekkera Bruxellensis ◽

Pcr Assay ◽

Specific Pcr ◽

Spoilage Yeast ◽

Pcr Method ◽

26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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