RRC ID 80232
Author Saetang J, Sukkapat P, Palamae S, Singh P, Senathipathi DN, Buatong J, Benjakul S.
Title Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus.
Journal Biosensors (Basel)
Abstract Vibrio parahaemolyticus is usually found in seafood and causes acute gastroenteritis in humans. Therefore, a detection method of pathogenic V. parahaemolyticus is necessary. Multiplex PCR combined with lateral flow dipstick (LFD) assay was developed to detect pathogenic V. parahaemolyticus. Biotin-, FAM-, and Dig-conjugated primers targeting thermolabile hemolysin (TLH) and thermostable direct hemolysin (TDH) genes were used for multiplex PCR amplification. The condition of the method was optimized and evaluated by agarose gel electrophoresis and universal lateral flow dipstick. The specificity assay was evaluated using strains belonging to seven foodborne pathogen species. The sensitivity of the method was also evaluated using DNA in the concentration range of 0.39-100 ng/reaction. The artificial spiking experiment was performed using 10 g of shrimp samples with an enrichment time of 0, 4, and 8 h with 101, 102, and 103 CFU of V. parahaemolyticus. The developed multiplex PCR-LFD assay showed no non-specific amplification with a limit of the detection of 0.78 ng DNA/reaction visualized by agarose gel electrophoresis and 0.39 ng DNA with LFD assay. The artificial spiking experiment demonstrated that this method could detect pathogenic V. parahaemolyticus at 10 CFU/10 g shrimp samples following a 4 h of enrichment. Multiplex PCR-LFD assay was therefore established for detecting pathogenic V. parahaemolyticus with high sensitivity and specificity and might be a useful tool to develop a detection kit used in the food safety sector.
Volume 13(7)
Published 2023-6-30
DOI 10.3390/bios13070698
PII bios13070698
PMID 37504096
PMC PMC10377466
MeSH Bacterial Toxins* DNA Hemolysin Proteins / genetics Humans Multiplex Polymerase Chain Reaction Vibrio parahaemolyticus* / genetics
Resource
General Microbes JCM 20190