Publication: Rapid multiplex polymerase chain reaction for simultaneous detection of Vibrio harveyi, V. parahaemolyticus, and V. vulnificus in pacific white shrimp (Litopenaeus vannamei)
1
0
Issued Date
2016
Resource Type
File Type
application/pdf
ISSN
17556783
Other identifier(s)
2-s2.0-84977635826
Rights Holder(s)
Scopus
Bibliographic Citation
Annals of Tropical Medicine and Public Health. Vol 9, No.4 (2016), p.255-262
Suggested Citation
Thongkao K., Sudjaroen Y., Chaivisuthangkura P. Rapid multiplex polymerase chain reaction for simultaneous detection of Vibrio harveyi, V. parahaemolyticus, and V. vulnificus in pacific white shrimp (Litopenaeus vannamei). Annals of Tropical Medicine and Public Health. Vol 9, No.4 (2016), p.255-262. doi:10.4103/1755-6783.184792 Retrieved from: https://hdl.handle.net/20.500.14740/5337
Author(s)
Abstract
Context: A comparatively small number of species, e.g., Vibrio parahaemolyticus and V. vulnificus, cause disease in both aquatic animals and humans. V. harveyi is marine animal pathogen and rarely causes infections in humans; however, it might become a reservoir of antibiotic-resistant bacteria forms and virulence genes. Aims: 1) to develop rapid multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of V. harveyi, V. parahaemolyticus, and V. vulnificus by using vhhP2, tl, and rpoS genes as the respective target genes and 2) to evaluate specificity and determined detection of multiplex PCR technique. Materials and Methods: The multiplex PCR assay was developed and evaluated for specificity on 36 isolates of V. harveyi, 30 isolates of V. parahaemolyticus, and 14 isolates of V. vulnificus, along with other species of Vibrio and non-Vibrio bacterial isolates. Sensitivity of test was described as detection limit of pathogens in lowest amount of sample (CFU/mL or CFU/g) was determined by diluted DNA extracts of the pure cultures and spiked pacific white shrimp (Litopenaeus vannamei) samples Results: This developed multiplex PCR was proved as an accurate method, which was specific for three Vibrio species. The detection limits of V. harveyi, V. parahaemolyticus, and V. vulnificus in pure cultures and spiked shrimp samples ranged 1.05-4.8 × 103 CFU/mL and 1.9-7 × 104 CFU/g, respectively. Conclusions: This rapid multiplex PCR assay can decrease amount and process of sample preparation, which was time-consuming, and had preferable accuracy. This developed technique will be suitable and useful for food-borne pathogen detection in shrimp and horizontal gene transfer study among different Vibrio species in aquatic animals.
Subject(s)
Bacterial DNA
RNA 16S
Antibiotic resistance
Article
Bacterial gene
Bacterium culture
Bacterium detection
Bacterium isolate
Colony forming unit
Cross reaction
DNA extraction
DNA template
Electrophoresis
Gene targeting
Horizontal gene transfer
Limit of detection
Litopenaeus vannamei
Multiplex polymerase chain reaction
Nonhuman
Real time polymerase chain reaction
Vibrio harveyi
Vibrio parahaemolyticus
Vibrio vulnificus
RNA 16S
Antibiotic resistance
Article
Bacterial gene
Bacterium culture
Bacterium detection
Bacterium isolate
Colony forming unit
Cross reaction
DNA extraction
DNA template
Electrophoresis
Gene targeting
Horizontal gene transfer
Limit of detection
Litopenaeus vannamei
Multiplex polymerase chain reaction
Nonhuman
Real time polymerase chain reaction
Vibrio harveyi
Vibrio parahaemolyticus
Vibrio vulnificus