Development of multienzyme isothermal rapid amplification (MIRA) combined with lateral-flow dipstick (LFD) assay to detect species-specific tlh and pathogenic trh and tdh genes of vibrio parahaemolyticus.

Authors: PARK, SEONGBIN - Mississippi State University; ZHANG, YAN - Mississippi State University

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2024
Publication Date: 1/6/2024
Citation: Park, S., Zhang, Y. 2024. Development of multienzyme isothermal rapid amplification (MIRA) combined with lateral-flow dipstick (LFD) assay to detect species-specific tlh and pathogenic trh and tdh genes of vibrio parahaemolyticus.. Pathogens. 13(1):57. https://doi.org/10.3390/pathogens13010057.
DOI: https://doi.org/10.3390/pathogens13010057

Interpretive Summary: Vibrio parahaemolyticus can cause severe gastroenteritis in humans who eat contaminated seafood. Traditionally, detecting this bacterium and its harmful genes (tlh, trh, tdh) involves complex lab tests like PCR and qPCR. This study developed a simpler, faster method using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD) for field use. The new test identifies V. parahaemolyticus in 20 minutes at 30-45°C and can detect tiny amounts of DNA and bacteria, similar to qPCR sensitivity. Tests showed it effectively identified the bacterium in oysters, proving MIRA-LFD a quick and sensitive tool for detecting harmful genes in V. parahaemolyticus.

Technical Abstract: Vibrio parahaemolyticus causes severe gastroenteritis in humans after consuming contaminated raw or undercooked seafood. A species-specific marker, the thermolabile hemolysin (tlh) gene, and two pathogenic markers, thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes, have been used to identify V. parahaemolyticus and determine its pathogenicity using both PCR and qPCR assays. To enable testing in field conditions with limited resources, this study aimed to develop a simple and rapid method to detect the species-specific (tlh) and pathogenic (trh and tdh) genes of V. parahaemolyticus using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD). The amplification of the tlh, trh, and tdh genes could be completed within 20 min at temperatures ranging from 30 to 45 'C (p < 0.05). The test yielded positive results for V. parahaemolyticus but produced negative results for nine Vibrio species and eighteen foodborne pathogenic bacterial species. MIRA-LFD could detect 10 fg of DNA and 2 colony-forming units (CFU) of V. parahaemolyticus per reaction, demonstrating a sensitivity level comparable to that of qPCR, which can detect 10 fg of DNA and 2 CFU per reaction. Both MIRA-LFD and qPCR detected seven tlh-positive results from thirty-six oyster samples, whereas one positive result was obtained using the PCR assay. No positive results for the trh and tdh genes were obtained from any oyster samples using MIRA-LFD, PCR, and qPCR. This study suggests that MIRA-LFD is a simple and rapid method to detect species-specific and pathogenic genes of V. parahaemolyticus with high sensitivity.