Improvement of the quantitation method for the tdh+ Vibrio parahaemolyticus in molluscan shellfish based on most-probable- number, immunomagnetic separation, and loop-mediated isothermal amplification

Abstract

Vibrio parahaemolyticus is a marine microorganism that can cause seafood-borne gastroenteritis in humans. The infection can be spread and has become a pandemic through the international trade of contaminated seafood. Strains carrying the tdh gene encoding the thermostable direct hemolysin (TDH) and/or the trh gene encoding the TDH-related hemolysin (TRH) are considered to be pathogenic with the former gene being the most frequently found in clinical strains. However, their distribution frequency in environmental isolates is below 1%. Thus, very sensitive methods are required for detection and quantitation of tdh+ strains in seafood. We previously reported a method to detect and quantify tdh+ V. parahaemolyticus in seafood. This method consists of three components: the most-probable-number (MPN), the immunomagnetic separation (IMS) targeting all established K antigens, and the loop-mediated isothermal amplification (LAMP) targeting the tdh gene. However, this method faces regional issues in tropical zones of the world. Technicians have difficulties in securing dependable reagents in high-temperature climates where we found MPN underestimation in samples having tdh+ strains as well as other microorganisms present at high concentrations. In the present study, we solved the underestimation problem associated with the salt polymyxin broth enrichment for the MPN component and with the immunomagnetic bead-target association for the IMS component. We also improved the supply and maintenance of the dependable reagents by introducing a dried reagent system to the LAMP component. The modified method is specific, sensitive, quick and easy and applicable regardless of the concentrations of tdh+ V. parahaemolyticus. Therefore, we conclude this modified method is useful in world tropical, sub-tropical, and temperate zones.

Rapid and Quantitative Detection of Vibrio parahemolyticus by the Mixed-Dye-Based Loop-Mediated Isothermal Amplification Assay on a Self-Priming Compartmentalization Microfluidic Chip

“…Therefore, developing new methods to detect VP rapidly and quantitatively is focused on by scientists, especially in developing countries, such as China. Because of the advantages of isothermal nucleic acid amplification, LAMP for VP detection was first reported in 2008, and subsequently, it was widely developed to target other genes of VP. − However, these traditional LAMP approaches compromised on quantitative analysis and detection of VP with a low concentration, which had to be quantified and pre-enriched through tedious bacteria culture. ,− To address these obstacles, the combination of microfluidics and LAMP is an alternative way, but currently reported methods are still not appropriate for the prospective on-site VP detection. , …”

Review in isothermal amplification technology in food microbiological detection

“…The principle is to make use of the magnetism of magnetic beads and the characteristics of functional groups that can be modified to combine the modified magnetic beads with microorganisms, and the complex will undergo mechanical transfer under the action of a magnetic field, so as to achieve the rapid separation of microorganisms and maintain the activity of microorganisms. IMS-LAMP is to modify specific antibodies on the surface of immunomagnetic beads, concentrate and isolate the target microorganisms in immunomagnetic beads by antigen–antibody reaction, and then detect them by LAMP technology (Escalante-Maldonado et al, 2015 ).…”

Desarrollo y validación del método de amplificación isotérmica mediada en lazo para la detección del virus Zika

“…Últimamente, el método LAMP se ha utilizado en un formato de retro transcriptasa (RT-LAMP) para detectar virus ARN en un solo paso (9) . Además, el reactivo LAMP liofilizado no requiere condiciones especiales durante el almacenamiento, el transporte y la operación, por lo que puede usarse para detectar microorganismos patógenos incluso en países tropicales (10) . Si el reactivo LAMP liofilizado es aplicable a la detección del ZIKV, sería muy útil en varias partes del mundo.…”