Novel Immunocapture RT-PCR Kits for Detection of Plant Viruses

1. Novel Immunocapture RT-PCR Kits for Detection of Plant Viruses Jun Q. Xiaand Chunda Feng AC Diagnostics, Inc., Fayetteville, AR We Believe in Agri-Diagnostics! (www.acdiainc.com)

2. Detection of Plant Viruses • Plant virus infections induce tremendous economic losses. • Accurate and reliable detection of plant viruses is critical. • Current ELISA detection methods may not provide the sensitivity needed. • RT-PCR requires costly and time-consuming viral sample RNA extraction.

3. Immunocapture RT-PCR • Virus particle captured by antibody • Virus RNA released from particle • RT-PCR amplification • Immunocapture incubation • Coat antibody on PCR tube PCR tubes/plate • ♦ Combines two widely used detection technologies - ELISA and PCR. • ♦ Eliminates costly and time-consuming sample RNA extraction procedure. • ♦ Reduces risks from cross-contaminations in sample process . • ♦ Shortens the test time and reduce assay cost for an accurate disease diagnosis. • ♦ Makes it possible to routinely conduct RT-PCR assays for large numbers of • plant samples.

4. Product Development • Antibody-Coated PCR tube: • Coat antibody on PCR tube • Post-coat and stabilize the coating antibody in PCR tube • Specific primers/probe: • Develop the specific single-, duo-, multiple- or degenerate primers • Design and label the probe with a fluorochrome and a quencher • Conventional or Real-time RT-PCR: • Optimize the RT-PCR reaction system • Validate the RT-PCR system for its sensitivity, specificity and • reliability.

5. Immunocapture RT-PCR Kit • ☺Pre-coated PCR tubes for capturing virus particles • which can be directly amplified by RT-PCR • ☺Optimized PCR primers which give bust and • reliable test results • ☺Include all components and ready to use • ☺Cost-effective and use-friendly, and being suitable • or PCR tests of large number of samples

6. Kit Formats Immunocapture Kit Immunocapture RT-PCR Kit 1X8 PCR strips pre-coated with specific antibody PCR buffers - sample extraction and washing Positive and negative controls RT-PCR primers, Master Mix (Optional) Reverse Transcriptase, and Taq Polymerase (Optional) Instruction • 1X8 PCR strips pre-coated with specific antibody • PCR buffers - sample extraction and washing • Positive and negative controls • Instruction

7. Assay Procedure of Immunocapture RT-PCR Kit

8. Immunocapture RT-PCR Kits Developed: Immunocapture Kits: Immunocapture kits are currently available for detection of more than 50 plant viruses at AC Diagnostics Inc.

9. Benefits by Using Immunocapture RT-PCR Kit • Getting accurate and consistent result, every time running a RT-PCR. • Saving funds, by capturing vial particles on PCR tube without need of expensive RNA extraction kits. • Saving times, by eliminating the time-consuming sample RNA extraction procedure. • Flexible applications, by providing universal PCR protocol to fit different PCR cycling systems. • High sensitivity, using for plant samples with low pathogen titer such as seeds, woody plants and stock plants.

10. Acknowledgments Funding support provided by the USDA NIFA SBIR Phase-I Grant project and now a Phase II Project • Novel Immunocapture RT-PCR Kits for Detection of Plant Viruses Jun Q. Xiaand Chunda Feng AC Diagnostics, Inc., Fayetteville, AR We Believe in Agri-Diagnostics! (www.acdiainc.com)