A rapid and simple molecular diagnostic test for penside detection of Porcine circovirus (PCV2)

Porcine circovirus type 2 (PCV2) infections in pigs is a major challenge for the swine industry as it causes significant production and economic losses to producers worldwide. Timely detection of PCV2 in herds is important in order to minimize the spread of infection and reduce economic losses. Current diagnostic methods such as ELISA and PCR are not suitable for field use because of the need for expensive equipment, trained technicians, and a specialized laboratory. To address this unmet need, we have developed an easy to use “sample-to-answer” molecular diagnostic test for penside detection of PCV2 directly from oral fluid samples.

This assay is based on loop mediated isothermal amplification (LAMP) amplification of ORF2 gene. For amplification, primers are mixed with an isothermal buffer, fluorescent dye, and target, followed by incubation for 40 minutes under isothermal conditions. Initially, serial 10-fold dilutions of PCV2 genomic DNA were tested to determine sensitivity in a clean system. Fluorescent dye in the reaction mixture allowed real-time monitoring of amplification products and post reaction thermal melt analysis to confirm the correct amplification product. Furthermore, complete formulation (buffer, enzyme, dye, and primers) sufficient for single reactions were lyophilized in 0.2 ml PCR tubes. An easy to use sample preparation method, which involves dilution of sample (oral fluid) in a lysis buffer followed by incubation at 90°C for 5 min., was also developed. The performance of this sample preparation method was evaluated by testing oral fluid samples spiked with different titers of PCV2 virus. To determine the clinical performance of this method, 20 samples (12 positive and 8 negatives) were tested.

The analytical sensitivity of the assay using purified DNA was demonstrated to be about 30 copies of DNA, which is comparable to real time PCR. Using the newly developed sample preparation method, a sensitivity of 500 virus/test within 40 minutes was achieved. When tested using clinical samples, the sensitivity and specificity of this assay were found to be 89% and 100%, respectively.

Results indicate that this new LAMP assay is a rapid and effective method for detection of PCV2 directly from oral fluid samples. This assay can be run on a simple and easy to use, portable isothermal amplification unit and results are avilble within 40 minutes, making it ideal for penside diagnostic testing.

Disclosure of Interest: B. Converse Conflict with: Lucigen corp, J. Benzine Conflict with: Lucigen corp, K. Brown Conflict with: Lucigen corp, K. Yoon: None Declared, S. Goyal: None Declared, D. Mead Conflict with: Lucigen corp, Y. Chander Conflict with: Lucigen corp.