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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 - PowerPoint PPT Presentation

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Detection of Plant Pathogens: Modern Technologies and Applications

Sensitive and Cost-effective Immunocapture RT-PCR for Routine Viral Detection in Large Number of Plant SamplesJun Q. Xia1, Kai-Shu Ling2, and Chunda Feng31AC Diagnostics, Inc., Fayetteville, AR2USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC3Dept. of Plant Pathology, University of Arkansas, Fayetteville, ARImportance of Plant Pathogen Detection

Early and accurate diagnosis of plant pathogens is a crucial step in: management of plant diseasesprevention of further spread of pathogensmaintenance of the safety of agricultural products and the human food supply2Traditional Diagnostic TechnologiesObservation and examinationIsolation and inoculationSerological assays:- Enzyme- linked Immunosorbent Assay (ELISA)- Immunofluorescence Assay (IFA)- Lateral-Flow assay device

3Modern Diagnostic TechnologiesNucleic Acid Hybridization PCR, Real-time PCR Nucleic Acid Sequence-Based Amplification (NASBA)Microarray TechnologyBio-Sensor Technology

4Procedure: 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

5Thermal Cycle of Polymerase Chain Reaction (PCR)

6Real-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. 7

Combines two widely used detection technologies - ELISA and PCR8Advantages of Immunocapture Real-time RT-PCREliminate 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 plant samples with low pathogen titer such as seeds, woody plants and stock plants

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Development of Immunocapture RT-PCR- Antibody-Coated PCR tubePrepare 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

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.11duplication here

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

DilutionVirus Infectivity on PlantImmunocapture Real-time RT-PCRPepMVCMVPepMVTSWV10-1++++10-2++++10-3++++10-4--++10-5--++10-6---+10-7---++ = Positive reaction; - = Negative reaction Comparative Sensitivity of Real-Time RT-PCR and Virus Infectivity Using Serially Diluted Leaf Tissue Extract

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

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.

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Assay Procedure of Immunocapture RT-PCR KitBeneficial 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.ConclusionsImmunocapture 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.

AC Diagnostics, Inc.We Believe in Agri-diagnostics!

(www.acdiainc.com)AcknowledgmentsFunding support provided by the USDA NIFA SBIR Phase-I Grant project and now a Phase II Project19Good afternoon. My name is Deana Ivy and I would like to introduce AC Diagnostics. AC Diagnostics was founded in 2004 by Dr. John Xia, a highly accomplished plant pathologist.

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