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