Plant virus infections induce tremendous economic losses for agricultural production each year in the U.S. Accurate and reliable diagnosis of the pathogens in plants or seeds is a critical step for control of plant disease in crops. Current ELISA detection methods may not provide the sensitivity needed for samples with low viral concentrations. Although RT-PCR is sensitive, it requires viral sample RNA extraction which is costly and time-consuming, and more importantly, has high risks of cross-contamination. Sample preparation by immunocapture technology eliminates the expensive procedure of sample extraction. The immunocapture RT-PCR developed in our research combines the advantages of the virus immunocapture and the target amplification, and allows the entire assay to be performed in a single PCR reaction in a relatively short time. Development and application of the diagnostic products for detection of plant viruses based on this technology is presented in this study.

Features of Immunocapture RT-PCR Kit:Pre-coated PCR tubes for capturing virus particles which can be directly amplified by RT-PCR. Optimized PCR primers which give robust and reliable test results.Including all RT-PCR assay components and ready to use.Cost-effective, use-friendly, and being suitable for PCR tests of large number of samples.

Immunocapture RT-PCR Kits Developed:

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  

Specific primers/probe:• Develop the specific single-, duo-, multiple- or degenerate primers.• Design and label the probe with a fluorochrome and a quencher.• Synthesize and evaluate the designed primers/probe. 

Conventional or Real-time RT-PCR:• Develop a PCR reaction mixture and run RT-PCR.• Optimize the RT-PCR reaction system with specific cycling profile.• Validate the RT-PCR system for its sensitivity, specificity and reliability.

♦ Combines two widely used detection technologies - ELISA and PCR. ♦ Eliminates expensive and time-consuming sample RNA extraction procedure. ♦ Reduces risks from cross-contaminations in sample process. ♦ Shortens the test time and reduce assay cost for an accurate disease diagnosis. ♦ Makes it possible to routinely conduct RT-PCR assays for large numbers of plant samples.

Benefits of Immunocapture RT-PCR Kit: Getting accurate and consistent result, every time running PCR.  

Saving funds, by capturing vial particles on PCR tube without need of expensive RNA extraction kits.  

Saving times, by eliminating the time-consuming sample RNA extraction procedure.  

Flexible applications, by providing universal PCR protocol to fit different PCR cycling systems.

High sensitivity, using for plant samples with low pathogen titer such as seeds, woody plants and stock plants.

A sensitive, reliable and user-friendly commercial product has been developed based on immunocapture RT-PCR technology. This diagnostic kit includes all assay components and is ready-to-use. Pre-coated PCR tubes for capturing virus particles which can be directly amplified by RT-PCR allows entire assay to be performed in a single PCR tube. Sample is simply ground in a PCR sample extract buffer and added to PCR plate/tubes pre-coated with antibody. After incubation and washing, the captured target viral RNA is ready to be amplified by RT-PCR. Robust and consistent result can be obtained every time running a RT-PCR by using this product because all PCR inhibitors are eliminated through viral immunocapture. This immunocapture RT-PCR product provide one of the most sensitive diagnostic tools for an effective detection of plant viruses in seeds, stock materials or other plant tissues of economically important vegetables, fruits, ornamentals and field crops.

Development of Commercial Products Basing on Immunocapture RT-PCR Technology

Jun Q. Xia1, Chunda Feng2 and Kai-Shu Ling3

1AC Diagnostics, Inc., Fayetteville, AR; 2Dept. of Plant Pathology, University of Arkansas, Fayetteville, AR; 3USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC

Introduction  Immunocapture RT-PCR

 

Product Development 

Summary 

Applications 

Diagnostic Kits 

Alfalfa Mosaic Virus (AMV) Potato Virus X (PVX) Barley Stripe Mosaic Virus (BSMV) Potato Virus Y (PVY) Cherry Leaf Roll Virus (CLRV) Tobacco Etch Virus (TEV) Impatiens Necrotic Spot Virus (INSV) Tobacco Mosaic Virus (TMV) Maize Chlorotic Mottle Virus (MCMV) Tobacco Ringspot Virus (TRSV) Maize Dwarf Mosaic Virus (MDMV) Tomato Aspermy Virus (TAV) Pea Enation Mosaic Virus (PEMV) Tomato Mosaic Virus (ToMV) Peanut Stunt Virus (PSV) Tomato Ringspot Virus (ToRSV) Pepino Mosaic Virus (PepMV) Wheat Streak Mosaic Virus (WSMV) Pepper Mild Mottle Virus (PMMoV)