ISHS Acta Horticulturae 657: XIX International Symposium on Virus and Virus-like Diseases of Temperate Fruit Crops - Fruit Tree Diseases
REAL-TIME RT-PCR OF PPV WITH R.A.P.I.D. A FIELD-HARDENED PCR UNIT FOR IN-FIELD DETECTION |
| Authors: | V.A. Mavrodieva, L. Levy |
| Keywords: | Quarantine, Prunus, Plum pox virus, diagnosis |
| Abstract:
Plum pox virus (PPV) is a regulated plant virus, subject to state and federal quarantine in the United States. PPV was first detected in North America in fall of 1999 in Pennsylvania (Levy et al., 2000) and is the subject of an eradication program. Accurate and timely diagnosis of regulatory plant pathogens is the first step in their control. Most of the existing methods for pathogen detection and identification, such as morphology and serology, are time consuming and/or not adequately sensitive. Conventional PCR is highly specific and sensitive but requires gel analysis. Real-time PCR is becoming more widely used for diagnostic purposes due to its format flexibility, speed, and sensitivity. Portable real-time PCR instruments enable real-time PCR application for on-site plant pathogen detection in quarantine zones, ports of entry, or survey sites. PPV PCR diagnosis in our laboratory is done by IC-RT-PCR using 2 pairs of primers: universal 3-NCR primers (Levy and Hadidi, 1994) and P1-P2 primers (Wetzel et al., 1991). We adapted and optimized the 3-NCR PCR to develop a one-step real-time IC-RT-PCR using SYBR Green for fluorescent detection of PPV. Combining anti-PPV polyclonal antibody for immunocapture with universal 3-NCR primers allows detection of all 4 known groups of PPV strains D, M, SoC and El Amar. The IC step can be done for 2 hours at 37ēC without compromising PCR sensitivity and the entire PCR detection can be completed in 5-6 hours. Further, we designed and evaluated three TaqMan probes for PPV real-time RT-PCR detection using the 3-NCR and P1/P2 primer sets that also showed high sensitivity and specificity. | |
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