Performing Department
(N/A)
Non Technical Summary
The proposed AqPathDx kit enables rapid, pondside detection of the most common catfish and tilapia pathogens in two separate panels in less than 30 minutes using a novel multiplex molecular diagnostic assay. The AqPathDx kit is based on multiplex loop-mediated isothermal amplification (mLAMP) method, which allows real-time detection of up to 4 targets in a single reaction. This assay will be performed on a benchtop isothermal instrument, and the results will be displayed on the screen as positive, negative, or non-conclusive.AqPathDx kit performance will be compared with that of the reference method. A simple and rapid sample preparation method will also be developed, and complete reaction formulation will be lyophilized for ambient storage. The analytical performance of the AqPathDx kit will be evaluated by testing a cohort of 300 positive and 200 negative samples.
Animal Health Component
30%
Research Effort Categories
Basic
5%
Applied
30%
Developmental
65%
Goals / Objectives
Major goals of this Phase II study are:• Development of a diagnostic kit (AqPathDx) for the detection of multiple pathogens at pondside.• Total assay time of 30 min, including sample preparation.• Development of a simple and easy-to-use sample preparation method.• Evaluate diagnostic sensitivity and specificity of AqPathDx kit.• Lyophilizereagents for storage at ambient temperature.• Platform technology for rapid detection of other pathogens.
Project Methods
Aim 1. Design LAMP primers. LAMP primers specific for the detection of Aeromonas sp.,Edwardsiella sp., Flavobacterium sp.,Streptococcus sp. and Tilapia lake virus (TiLV) will be designed.Aim 2. Develop monoplex LAMP assay. Primer designs will be tested in a LAMP assay usingan Isothermal Master Mix produced by Varizymes. Reaction conditions will be optimized fortarget amplification in <30 min. The sensitivity and specificity of the LAMP assay will bedetermined and compared to real-time PCR/RT-PCR methods specific to each pathogen.Aim 3. Develop and optimize mLAMP (AqPathDx) assay. Selected primer sets from Aim 2will be used to develop mLAMP. The primer ratio will be optimized to ensure maximumsensitivity of mLAMP assay and no non-specific amplification. Sensitivity and specificity willbe determined and compared with real-time PCR/RT-PCR methods specific to each fishpathogen.Aim 4. Optimize sample preparation method. Asimpleand rapid sample preparation method for testing skin swab samples will be developed and optimizedfor the detection of all targeted pathogens(Aeromonas sp., Edwardsiella sp., Flavobacterium sp., Streptococcus sp. and TiLV) fromdifferent sample matrices such as skin swabs, swabs collected from gills, and water samples fromproduction tank/rearing. Success will be measured by equivalent clinical sensitivity andspecificity to standard extraction methodology on the same sample cohort.Aim 5. Lyophilize mLAMP reagents: A complete reaction formulation sufficient for a singlereaction will be dispensed and lyophilized in tubes to have shelf-stable reagents. Amplificationwill be carried out at an optimized temperature (as determined in Aim 3), and the performance oflyophilized reagents will be compared to that of wet reagents as well as reference method.Aim 6. Clinically validate AqPathDx kit. Once all the reaction conditions have been optimized,a set of known positive and negative samples will be tested using the AqPathDx test kit. For eachtest panel (catfish or tilapia), we plan to test at least 300 positive and 200 negative samples, with a minimum of 50 positive and 25 negative samples for each pathogen. Results obtained will be compared with the reference method to determine the clinical sensitivity/specificity of the test.