sensitive and cost-effective immunocapture rt-pcr for routine viral detection in large number of...
DESCRIPTION
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 PresentationTRANSCRIPT
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, AR2USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC
3Dept. of Plant Pathology, University of Arkansas, Fayetteville, AR
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
Traditional Diagnostic Technologies
• Observation and examination
• Isolation and inoculation• Serological assays:
- Enzyme- linked Immunosorbent Assay (ELISA)
- Immunofluorescence Assay (IFA)
- Lateral-Flow assay device
Modern Diagnostic Technologies
• Nucleic Acid Hybridization
• PCR, Real-time PCR • Nucleic Acid Sequence-
Based Amplification (NASBA)
• Microarray Technology• Bio-Sensor Technology
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
Thermal Cycle of Polymerase Chain Reaction (PCR)
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.
Combines two widely used detection technologies - ELISA and PCR
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 plant samples with low pathogen titer such as seeds, woody plants and stock plants
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
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.
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-PCR
PepMV CMV PepMV TSWV
10-1 + + + +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
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.
Assay Procedure of
Immunocapture RT-PCR
Kit
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.
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.
AC Diagnostics, Inc.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