Portable and point-of-care molecular detection of pathogenic Vibrio parahaemolyticus in shrimp

Abstract

In the Mekong Delta regions, Acute Hepatopancreatic Necrosis Disease (AHPND) is a globally serious threat for shrimp farming. AHPND is mainly caused by Vibrio parahaemolyticus carrying the plasmid that encodes the virulence genes, namely, Photorhabdus insect-related (pir). One of the best measures to control the outbreak in shrimp is to rapidly and accurately determine the virulent gene of V. parahaemolyticus. In this study, we developed a novel molecular assay that combines Flinders Technology Associates (FTA) card-embedded tube for nucleic acid extraction and loop-mediated isothermal amplification (LAMP) to detect pirA gene of causative V. parahaemolyticus with a pH-based colorimetric readout. To improve the usability of LAMP assay, a palm-sized three-dimensional (3D) - printed heater operated by batteries was employed to apply heat for the amplification reaction on site. The strategy has high specificity and sensitivity with a limit of detection as low as 102 CFU/mL. The assay can be completed within 75 min at 65 °C. Next, to prove the feasibility of the test for real samples, shrimp collected from shrimp farm was used and spiked with bacteria. The strategy proved the ability for performing three main steps of nucleic acid-based assay for detection including sample extraction, amplification, and detection at the point-of-care for detecting the toxic pirA gene in V. parahaemolyticus. This strategy can also be a cost-effective and rapid tool which can be a potential candidate for on-site disease control in low-resource areas.