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
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 SequenceBased 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 RTBlock, Dye and Enzyme.
• Run thermal cycling and
fluorescence signal detection with
a Real PCR System.
Sensitivity of
Immunocapture
Real-Time RTPCR of Pepino
Mosaic Virus
(PepMV) and
Tomato Spotted
Wilt Virus
(TSWV)
Comparative Sensitivity of Real-Time RT-PCR and Virus
Infectivity Using Serially Diluted Leaf Tissue Extract
Dilution
10-1
10-2
10-3
10-4
10-5
10-6
10-7
Virus Infectivity on
Plant
Immunocapture Realtime RT-PCR
PepMV
CMV
PepMV
TSWV
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
“+” = Positive reaction; “-“ = Negative reaction
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 userfriendly, 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 costeffective.
• 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.
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)