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

Abstract

In the Mekong Delta region, Acute Hepatopancreatic Necrosis Disease (AHPND) is a globally serious threat to shrimp farming. AHPND is mainly caused by Vibrio parahaemolyticus carrying a plasmid that encodes virulence genes, namely, Photorhabdus insect-related (pir). One of the best measures to control outbreaks in shrimp is to rapidly and accurately determine V. parahaemolyticus carrying virulence gene. In this study, we developed a fully-integrated molecular diagnostic device that combines a Flinders Technology Associates (FTA) card-embedded tube for nucleic acid extraction and loop-mediated isothermal amplification (LAMP) to detect the pirA gene of causative V. parahaemolyticus, with a pH-based colorimetric readout realized by the use of phenol red. To improve the usability of the 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 showed high specificity and sensitivity, with a limit of detection as low as 10² CFU mL⁻¹. 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 a shrimp farm were used and spiked with bacteria. The strategy was proven to be capable of performing three main steps of molecular diagnosis such as sample extraction, amplification, and detection at the point of care for detecting the toxic pirA gene in V. parahaemolyticus. This strategy can also serve as a cost-effective and rapid tool and can be a potential candidate for on-site disease control in low-resource areas.