Recipient Organization
Lucigen Corporation
2905 Parmenter Street
Middleton,WI 53562
Performing Department
RESEARCH AND DEVELOPMENT
Non Technical Summary
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's proprietary thermostable OmniAmp® 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'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
Animal Health Component
50%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
Goals / Objectives
The goal of this project proposal is to develop an easy to use "sample-to-answer" molecular diagnostic device for penside detection of viral pathogens associated with PRDC that can be used to perform diagnostic assays in field settings. This assay will be based on LAMP technology and performed on an automated molecular detection platform. The results of the assay will be available within 40 minutes, allowing multiple samples to be tested in a short period of time.
Project Methods
Aim 1. Optimization of sample preparation method. In Phase I, we developed a buffer formulation for simple and rapid sample prep and showed its feasibility for extraction of nucleic acid from serum and oral fluid samples. In Phase II, we will further optimize the extraction procedure for detection of all three targeted pathogens (PRRSv, PCV-2, and SIV) from different sample matrices such as serum, oral fluid, and semen.In order to develop a simple and easy to use sample extraction method, sample matrices will be spiked with different concentrations of virus cultures (PCV-2 or SIV or PRRSv) and extracted using the extraction method developed in Phase I. Because of heterogeneity of different sample matrices, it is difficult to formulate a universal extraction buffer and method. To overcome this problem, a sample type-specific buffer formulation and extraction method will be developed. For each sample type the buffer formulation will be optimized by varying the concentration of components. Extraction conditions such as sample volume, buffer concentration, lysis time and temperature, will also be optimized to match the sensitivity of a PCR "gold-standard".Once developed, the extraction method will be used to test pre-characterized positive and negative clinical samples (20 each per virus). The samples will then be extracted and the lysate will be used as template in LAMP for each targeted virus and the results will be compared with those of real time-PCR.Criteria for success: Method will be considered suitable for extraction if it meets following benchmarks:1. Simple, easy to use, and rapid (total time taken for extraction < 5 min.).2. Extracted nucleic acid does not inhibit amplification in LAMP reaction.3. Matches sensitivity and specificity of "gold standard" method.Aim 2. Integration of LAMP assay with the diagnostic platform. The extraction method development and designing of the LAMP primers, as part of Aim 1 of this study, will be integrated into the SCM and reaction cartridge so that the reaction can be carried out on diagnostic platform.The SCM will be filled with the required volume (1-2 ml) of extraction buffer. In the reaction cartridge, the reaction wells will be filled with target specific LAMP primers (1 target/well), whereas the Master Mix well will be filled with the required volume of LAMP reagents (including OmniAmp polymerase). To ensure the room temperature stability of reagents, all components in the reaction cartridge will be dried. The reaction cartridge has a sample heating port for the heat lysis of the sample, which is introduced into the cartridge from the SCM.The device software will be modified so that the reaction wells can be maintained at the required temperatures. Device deigns allow for carrying out reactions at four different temperatures in a single run. As determined in the feasibility study, the optimum temperature for PRRSv and SIV LAMP is 72°C and for PCV-2, it is 70°C. However, it is possible that reaction temperature might need to be optimized further.Extraction from different matrices can require different lysis temperatures, time of incubation, and optimized extraction buffer, therefore each SCM will be bar coded for identification using the built-in bar code reader. This information will allow device software to control the time and temperature of "sample heating well" within preset limits as determined in Aim 1 for a given sample type. MS-2 bacteriophage will be added to the "sample-heating" well as a positive control for both extraction and amplification. Lucigen has already developed the LAMP method for MS-2, and these primers will be added to one of the reaction wells.For optimization of reaction conditions on the diagnostic device, such as lysis time and reaction temperature, the performance of reaction cartridge will be evaluated by testing different dilutions of viruses (SIV, PCV-2, PRRSv-NA, and PRRSv-EU) in extraction buffer.Aim 3. Specificity and sensitivity of the PRDC detection system. Sensitivity of the assay will be determined by testing different dilutions of each virus. For this, 10-fold serial dilutions of each virus will be prepared in different sample matrices (serum, semen, or oral fluid) and added to the SCM and tested in the reaction cartridge. Specificity will be determined by testing serum and oral fluid samples spiked with other swine pathogens, such as porcine respiratory coronavirus, porcine enterovirus, porcine parvovirus, porcine cytomegalovirus, PPV, PEDv and TGEV. These viruses will be obtained from MVDL or KSU.Criteria for success: PRDC molecular detection system will be considered acceptable if it meets the following benchmarks:1. Target amplification within 40 min.2. High specificity with no amplification of any of the non-targeted viruses.3. Sensitivity equal to or better than real time PCR.Aim 4.Clinical validation of PRDC molecular detection system. Once all the reaction conditions have been optimized, we will test a set of known positive and negative samples (serum, oral fluid, semen) on PRDC molecular detection systems. In the past, we have obtained clinical samples from Minnesota Veterinary Diagnostic Lab, Veterinary Diagnostic Labs at Kansas State University and South Dakota State University and for Phase II we will approach these labs for the clinical samples. These samples are usually tested in diagnostic labs, by real time PCR or ELISA, for detection of these pathogens. We will obtain these samples along with associated gold standard test results and use them for our evaluation study.For a given virus and sample type, we plan to test 25 positive and 25 negative samples. At Lucigen, these samples will be tested using the PRDC molecular detection system using the workflow as described earlier and outlined below. Briefly, the sample will be added to the SCM using a swab. The SCM will be inverted and attached to the reagent cartridge, and the bulb squeezed to introduce the extraction buffer containing sample into the cartridge. The SCM is removed, the cartridge placed in the instrument, and the lid closed. By pressing the "start" icon on screen, the run willinitiate. After 40 minutes of incubation, results will be displayed on the screen.Overall criteria for success:1. The PRDC LAMP detection system is simple, easy to use, and rapid (total time for result about 40 minutes).2. Sensitivity and specificity of the assay is equal to PCR-based testing.3. Rapid sample preparation method achieves similar results as standard sample preparation methods.4. The detection system has high clinical sensitivity/specificity and high PPV.