Rapid Field-appropriate Diagnostics for 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 one of the major challenges for the swine industry, as PRDC causes significant economic losses to swine producers worldwide. Rapid detection of causative agents is the key to management of PRDC, as it allows quick implementation of a control plan to minimize the potential spread of infection. 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 diagnostic kit is available for veterinary use in the field or in small veterinary clinics that offer minimal infrastructural support. To address this unmet need, we propose to develop a rapid and easy-to-use point of care (POC) diagnostic method for detection of viral pathogens associated with PRDC. Initially, a test kit will be developed to detect three viral pathogens: porcine reproductive and respiratory syndrome virus (PRRSv), swine influenza virus (SIV), and porcine circovirus type 2 (PCV2). These viruses are considered as primary pathogens of PRDC, and each can cause porcine disease on its own. Once developed, this technology will be used to rapidly develop diagnostic kits for POC detection of many other veterinary pathogens. This test will be based on loop mediated isothermal amplification (LAMP) using a proprietary thermostable polymerase, PyroScript. This enzyme is the only known thermostable enzyme containing both strand displacement activity for LAMP-based amplification and reverse transcriptase (RT) activity. Pyroscript is therefore uniquely suitable for the LAMP reaction, as it can be used to amplify both DNA and RNA without any additional steps or reagents in the testing protocol. Since LAMP is isothermal it does not require specialized instruments. Further, LAMP is much faster than PCR and is resistant to inhibitors in crude sample preparations. The primary goal of the proposed study is to develop a diagnostic kit that is capable of detecting PRRSv, SIV, and PCV2 from clinical samples within 30 minutes, including sample preparation time. Part of this goal is to develop a simple method of sample preparation to extract nucleic acid (DNA/RNA) from clinical samples. Reaction tubes will contain dried reagents to allow specific detection of pathogen. The assay will be carried out in an heating block at constant temperature followed by detection on a lateral flow strip for easy interpretation of results. Successful completion of this project will lead to (1) Development of diagnostic kit for identification of PRRSv, SIV, and PCV2 in 30 minutes or less. (2) Dried reagents for storage of kit at ambient temperature. (3)Platform technology that can be used to develop diagnostic kits for additional pathogens. Development of this rapid and easy-to-use diagnostic kit will improve ability of producers to monitor the health of their animals in real time without incurring significant testing costs. Enhanced in-the-field detection of pathogens will enable field veterinarians to provide rapid diagnoses, even in resource-limited settings.