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Sensitive and Cost-effective Immunocapture RT-PCR for Routine Viral Detection in Large Number of Plant Samples

Sensitive and Cost-effective Immunocapture RT-PCR for Routine Viral Detection in Large Number of Plant Samples. Jun Q. Xia 1, Kai-Shu Ling 2 , and Chunda Feng 3 1 AC Diagnostics, Inc., Fayetteville, AR 2 USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC

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Sensitive and Cost-effective Immunocapture RT-PCR for Routine Viral Detection in Large Number of Plant Samples

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  1. Sensitive and Cost-effective Immunocapture RT-PCR for Routine Viral Detection in Large Number of Plant Samples • Jun Q. Xia1, Kai-Shu Ling2, and Chunda Feng3 • 1AC Diagnostics, Inc., Fayetteville, AR • 2USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC • 3Dept. of Plant Pathology, University of Arkansas, Fayetteville, AR

  2. Importance of Plant Pathogen Detection Early and accurate diagnosis of plant pathogens is a crucial step in: • management of plant diseases • prevention of further spread of pathogens • maintenance of the safety of agricultural products and the human food supply

  3. Traditional Diagnostic Technologies • Observation and examination • Isolation and inoculation • Serological assays: - Enzyme- linked Immunosorbent Assay (ELISA) - Immunofluorescence Assay (IFA) - Lateral-Flow assay device

  4. Modern Diagnostic Technologies • Nucleic Acid Hybridization • PCR, Real-time PCR • Nucleic Acid Sequence-Based Amplification (NASBA) • Microarray Technology • Bio-Sensor Technology

  5. Procedure: Sandwich ELISA • (1) Coat plate with capture antibody • (2) Incubate plate with sample and plate washing • (3) Add antibody-enzyme conjugate (DAS) or primary antibody (TAS) • (4) Add anti antibody-enzyme conjugate • (5) Color reaction with substrate

  6. Thermal Cycle of Polymerase Chain Reaction (PCR)

  7. Real-Time PCR TaqManTM System • An oligonucleotide probe is labeled at the 5’ end with a fluorochrome and at the 3’ end with a quencher. • The TaqManTM probe is degraded by the Taq DNA polymerase and the fluorescent chromophore is released. • The level of fluorescence is measured at each cycling point by Real-time PCR machine.

  8. Combines two widely used detection technologies - ELISA and PCR

  9. Advantages of Immunocapture Real-time RT-PCR • Eliminate pre- and post-PCR manipulation • Reduce risks from contamination • Shorten the test time and reduce assay cost • Can be used for a large number of samples • Improve assay sensitivity and use for plantsamples with low pathogen titer such as seeds, woody plants and stock plants

  10. Development of Immunocapture RT-PCR- Antibody-Coated PCR tube • Prepare Specific antibody • Coat antibody on PCR tube • Use the antibody-coated PCR tube for viral capture • Or post-coat and stabilize the coating antibody in PCR tube • Wash, dry and store the antibody-coated PCR tube for later use

  11. Development of Immunocapture RT-PCR- Specific primers/probe • Develop the specific single-, duo-, multiple- or degenerate primers. • Design and label the probe with a fluorochrome and a quencher. • Add a PCR Master Mix plus RT-Block, Dye and Enzyme. • Run thermal cycling and fluorescence signal detection with a Real PCR System.

  12. Sensitivity of Immunocapture Real-Time RT-PCR of Pepino Mosaic Virus (PepMV) and Tomato Spotted Wilt Virus (TSWV)

  13. Comparative Sensitivity of Real-Time RT-PCR and Virus • Infectivity Using Serially Diluted Leaf Tissue Extract

  14. Multiplex Immunocapture Real-time RT-PCR for Simultaneously Detecting Two Viruses

  15. Immunocapture RT-PCR Kit ♥ Pre-coated PCR tubes for capturing virus. ♥ Optimized PCR primers which give robust and reliable test results. ♥ Including all PCR assay components and ready to use. ♥ Cost-effective and user- friendly, and being suitable for PCR tests of large number of samples.

  16. Assay Procedure of Immunocapture RT-PCR Kit

  17. Beneficial to Agri-Diagnostics • Researchers, diagnosticians, extension pathologists, private consultants, government inspectors and regulatory officers. • Routine application at diagnostic and research laboratories. • Seed certification programs; Pathogen elimination programs; Plant diagnostic network systems; State and federal government plant quarantine programs.

  18. Conclusions • Immunocapture PCR is developed by combining two widely used diagnostic technologies-ELISA and PCR. • This technology is sensitive, reliable , simple and cost-effective. • It is possible for PCR technology to be used in routine detection of pathogen for large number of plant samples . • The technology benefits agricultural research and disease diagnosis communities.

  19. Acknowledgments Funding support provided by the USDA NIFA SBIR Phase-I Grant project and now a Phase II Project AC Diagnostics, Inc. We Believe in Agri-diagnostics! (www.acdiainc.com)

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