Molecular Diagnostic Device for Penside Detection of Viral Pathogens Associated with Porcine Respiratory Disease Complex

Respiratory tract infection in pigs, commonly referred to as "Porcine Respiratory Disease Complex (PRDC)", is a major challenge for the swine industry as it causes significant production and economic losses to producers worldwide. Timely detection of causative agents is required to minimize the spread of infection and reduce economic loses. Current diagnostic methods such as ELISA and PCR are not suitable for field use because of the need for expensive equipment, highly-trained personnel, and a specialized laboratory. At present, no rapid molecular diagnostic kit is available for veterinary use in the field or in small clinics that lack infrastructural support. To address this unmet need, we propose to develop an easy to use "sample-to-answer" molecular diagnostic device for penside detection of the three viral pathogens associated with PRDC: porcine reproductive and respiratory syndrome virus (PRRSv), swine influenza virus (SIV), and porcine circovirus type 2 (PCV-2).The proposed assay will be based on loop mediated isothermal amplification (LAMP) using Lucigen & #39;s proprietary thermostable OmniAmp & reg; polymerase, and performed on a simple, easy to use automated molecular detection platform currently being developed by Lucigen. Total assay time will be 40 minutes with minimal hands-on time and without need of any additional equipment, such as pipettes, centrifuge etc. Results will be displayed on-screen as positive or negative for a specific pathogen, minimizing error & #39;s caused by user interpretation. This diagnostic device has been designed keeping in mind that people without any specialized training can use it to perform the assay. Once developed, this technology will be used to rapidly develop diagnostic assays for point of care detection of additional veterinary pathogens.During Phase I, we demonstrated feasibility of LAMP assay to detect PRRSv (NA and EU), PCV-2, and SIV (H1N1 and H3N2). The sensitivity of the assays was equivalent to respective real time PCR assays. To facilitate penside testing, we also developed a rapid sample preparation method for extraction of nucleic acid from serum and oral fluid samples. For Phase II studies, our goal is to further optimize the sample preparation method and integrate it with the molecular detection platform being developed. The proposed molecular diagnostic device has three components:1). a sample collection module containing extraction buffer.2). a reaction cartridge.3). the instrument.The cartridge contains the port for nucleic acid extraction, a port for dried LAMP master mix and 16-wells which can be filled with target-specific primers. The cartridge is placed into the instrument which automatically performs all the necessary steps such as sample extraction, thermal incubation and fluorescent detection to generate a positive or negative result for the specific target, or an invalid run.Successful completion of this project will lead to:? Development of molecular diagnostic device for penside detection of PRRSv, SIV, and PCV2.? Total assay time of 40 minutes including sample preparation.? Detection of up to 3 pathogens (PRRSv, SIV, and PCV-2) in a single run.? Dried reagents for storage at ambient temperature.? Simple, easy to use device, with digital read-out and no user interpretation required.? A technology platform that can be used for detection of other pathogens.Development of this innovative molecular diagnostic device will significantly improve the ability of producers to monitor the health of their animals in real time without incurring significant testing costs. Use of this technology will be economically beneficial to producers as this will allow them to test samples penside, saving them money in terms of testing and shipping costs as well as time. Also, enhanced access to in-the-field diagnosis of pathogens will improve