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

2. Detection of Plant Viruses 1.Plant virus infections induce tremendous economic losses. 2.Accurate and reliable detection of plant viruses is critical. 3.Current ELISA detection methods may not provide the sensitivity needed. 4.RT-PCR requires costly and time-consuming viral sample RNA extraction.

3. Immunocapture RT-PCR PCR tubes/plate Coat antibody on PCR tube Virus particle captured by antibody Virus RNA released from particle RT-PCR amplification Immunocapture incubation ♦ 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 1X8 PCR strips pre-coated with specific antibody PCR buffers - sample extraction and washing Positive and negative controls Instruction 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

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. Alfalfa Mosaic Virus (AMV) Potato Virus X (PVX) Barley Stripe Mosaic Virus (BSMV)Potato Virus Y (PVY) Cherry Leaf Roll Virus (CLRV)Tobacco Etch Virus (TEV) Impatiens Necrotic Spot Virus (INSV)Tobacco Mosaic Virus (TMV) Maize Chlorotic Mottle Virus (MCMV)Tobacco Ringspot Virus (TRSV) Maize Dwarf Mosaic Virus (MDMV)Tomato Aspermy Virus (TAV) Pea Enation Mosaic Virus (PEMV)Tomato Mosaic Virus (ToMV) Peanut Stunt Virus (PSV)Tomato Ringspot Virus (ToRSV) Pepino Mosaic Virus (PepMV)Wheat Streak Mosaic Virus (WSMV) Pepper Mild Mottle Virus (PMMoV)

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