1 / 19

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

kaz
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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)

More Related