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)

Three species—Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus—account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.

Download Full-text

A Method for Simultaneous Detection of Vibrio cholerae Strains and Drug Resistance Genes in their Genome by Means of Real-Time PCR

Biotekhnologiya ◽

10.21519/0234-2758-2018-34-2-70-79 ◽

2018 ◽

Vol 34 (2) ◽

pp. 70-79 ◽

Cited By ~ 1

Author(s):

A.A. Kritski ◽

◽

N.B. Cheldyshova ◽

S.P. Zadnova ◽

N.A. Plekhanov ◽

...

Keyword(s):

Drug Resistance ◽

Vibrio Cholerae ◽

Real Time ◽

Resistance Genes ◽

Real Time Pcr ◽

Simultaneous Detection

Download Full-text

Occurrence, Seasonal Distribution, and Molecular Characterization of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in Shellfish (Mytilus galloprovincialis and Ruditapes decussatus) Collected in Sardinia (Italy)

Journal of Food Protection ◽

10.4315/0362-028x.jfp-19-021 ◽

2019 ◽

Vol 82 (11) ◽

pp. 1851-1856 ◽

Cited By ~ 1

Author(s):

SONIA LAMON ◽

SIMONETTA G. CONSOLATI ◽

FEDERICA FOIS ◽

MARIA G. CAMBULA ◽

MARGHERITA PES ◽

...

Keyword(s):

Vibrio Cholerae ◽

Molecular Characterization ◽

Vibrio Parahaemolyticus ◽

Mytilus Galloprovincialis ◽

Vibrio Vulnificus ◽

Seasonal Distribution ◽

Ruditapes Decussatus ◽

Pathogenic Vibrios ◽

Vibrio Spp

ABSTRACT In this study, we investigated the occurrence, seasonal distribution, and molecular characterization of pathogenic vibrios in Mediterranean mussels (Mytilus galloprovincialis) and grooved carpet shells (Ruditapes decussatus) from two harvesting areas of Sardinia (Italy). Samples collected before and after depuration were submitted for qualitative and quantitative determination of Vibrio spp. Vibrio spp. isolates were presumptively identified by means of biochemical methods. Identification and virulence profile of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus were performed by molecular methods. The prevalence of Vibrio spp. in M. galloprovincialis and R. decussatus was, respectively, 96 and 77%. The averaged enumeration (mean ± standard deviation) of Vibrio spp. in samples of M. galloprovincialis and R. decussatus collected at the harvesting time was 2.04 ± 0.45 and 2.51 ± 0.65 log CFU/g, respectively. The average contamination levels in samples collected after purification were 2.28 ± 0.58 log CFU/g (M. galloprovincialis) and 2.12 ± 0.67 log CFU/g (R. decussatus). Four potentially pathogenic V. parahaemolyticus isolates (tdh+ or trh+) were recovered from grooved carpet shells samples. No isolate was tdh+/trh+. The presence of potentially pathogenic vibrios in Sardinian waters strengthens the need for rational purification practices under controlled conditions to guarantee the protection of consumers.

Download Full-text

Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus

Journal of Medical Microbiology ◽

10.1099/jmm.0.46759-0 ◽

2007 ◽

Vol 56 (1) ◽

pp. 56-65 ◽

Cited By ~ 22

Author(s):

Dobryan M. Tracz ◽

Paul G. Backhouse ◽

Adam B. Olson ◽

Joanne K. McCrea ◽

Julie A. Walsh ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Rapid Detection ◽

Vibrio Vulnificus ◽

Comparative Sequence Analysis ◽

Molecular Techniques ◽

Array Technology ◽

Species Specific ◽

Pcr Targeting

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.

Download Full-text