Development of a Simple and Easy to Use Diagnostic Assay for Rapid Detection of Virulent Strains of Aeromonas hydrophila (vAh)

Development of a Simple and Easy to Use Diagnostic Assay for Rapid Detection of VirulentStrains of Aeromonas hydrophila (vAh)Project SummaryIn the U.S. commercial production of channel Catfish (Ictalurus punctatus) is the leadingaquaculture industry. However in recent years the catfish industry has suffered huge economiclosses due to an epidemic of motile Aeromonas septicemia (MAS) caused by a highly virulentstrain of Aeromonas hydrophila (vAh). The key to stopping the spread of infection is the rapididentification of vAh so that containment strategies can be implemented in time. Currently theonly available diagnostic assays for vAh include a qPCR assay or an assay based on the growthof A. hydrophila on a unique carbon source (myo-inositol). In either case these diagnostic assaysrequire multiple days for a confirmatory diagnosis and no field-based diagnostic assay exists. Toaddress this unmet need we propose to develop a rapid easy-to-use diagnostic method forvAh detection at the point of care (POC). This diagnostic test will be designed to differentiatebetween virulent and non-virulent strains of A. hydrophila. Once developed this technology willbe leveraged to develop diagnostic kits for POC detection of other aquatic pathogens.This test will be based on a novel loop mediated isothermal amplification (LAMP) method"Duplex-LAMP" which allows detection of 2 targets in a single reaction. This method usesfluorophore-labeled primers for real-time detection of amplification products in a LAMP assay.Use of 2 different fluorophores allow detection of both targets in a single reaction. LAMP assayswill be performed using Varigen Biosciences's proprietary thermostable Bst polymerase. Theproposed LAMP assay will be performed on a commercially available benchtop instrument® AmpliFire (www.agdia.com). This instrument is suitable for use at POC as it has a smallfootprint is easy to use and is battery-operated. Results of this assay will be available within 30minutes (including sample preparation and amplification) with minimal hands-on time andwithout the need for any additional equipment. Results will be displayed on instrument screen aspositive or negative or non-conclusive minimizing errors caused by user interpretation.A Duplex LAMP assay for rapid detection of vAh strains and their differentiation from non- vAh strains will be developed. Performance of this LAMP assay will be compared with referencereal time PCR method. For use at POC a simple and rapid sample preparation method forextraction of nucleic acid from samples (fish skin swabs) will also be developed. For ease of useat POC and increase shelf life of reagents complete reaction formulation will be lyophilized.Diagnostic sensitivity and specificity of duplex LAMP assay will be determined by testing skinand anal swab samples collected from fish suspected of MAS infection. In total 200 clinicalsamples 100 suspected positive and 100 suspected negative will be tested. Successfulcompletion of this project will lead to:• Development of a vAh diagnostic assay for rapid detection at POC• Total assay time of 30 minutes or less including sample preparation.• Lyophilized reagents for storage at ambient temperature.• Platform technology that can be used to develop diagnostic assay for additional pathogens.The development and availability of this diagnostic kit will allow on-site testing of samples.As a result the time required for testing will be significantly reduced and laboratories will saveon shipping costs. These aspects will help producers by enabling them to rapidly respond to vAhoutbreaks increasing production and profits. This detection kit will be made available directlythrough Varigen Biosciences (www.varigenbiosciences.com) and its marketing partner VWR.