Development And Application Of Real Time PCR Approach For Quantification Of Six Plant Viruses

Detection of plant virus is very important part in plant diseases research. A large variety of identification procedures is available. As documented, direct observation, Western blot, SPA-ELISA (square protein A enzyme-linked immunosorbant assay) and DIBA (dot-immunobinding assay), have been used for plant virus detection. Among those, direct observation and Western blot are not suitable for large scale and routine diagnosis in agricultural production due to time-consuming (14-18 days for infected rice showing disease symptoms), high labor intensity, low sensitivity and specificity (only multi-antibody of rice stripe disease specific protein (sp) could be detected in Western blot). SPA-ELISA and DIBA have been taken to forecast epidemics of rice stripe disease, but the specificity is low and careful controls are necessary. Hence, areliable diagnosis is required to detect plant virus.Real Time PCR assay were developed for the specific detection of Rice stripe virus, Barely yellow dwarf viruses, Wheat dwarf virus and Cucumber green mottle mosaic virus. The assay detects a region encoding a highly conserved CP gene. And the method had benn applicated for preliminary research of these viruses. Concrete contents are as follows:1. One-step real-time RT-PCR methods using TaqMan probe are described for quantitative detection of RSV in both rice tissue and single small brown planthopper (SBPH) (Laodelphax striatellus Fallen). Primers and probe for specific detection of RSV were designed within the conserved region identified within the coat protein gene sequence. Sensitivity assay showed that the detection limit of the assay was 20 copies and the standard curve had a linear range from 20 to 2×10~5 copies with a good reproducibility. An internal control assay designed to target the rice ubiquitin 5 (UBQ5) gene was included in detecting RSV in different infected rice tissue. Simultaneously, real-time RT-PCR assay was applied to detect RSV coat protein gene in viruliferous single SBPH. The results showed that the numbers of RSV CP gene in different tissues were fluctuant. RSV coat protein gene accumulation was higher in rice leaf and SBPH thoraco-abdominal tissue than in stem and head respectively. By the end-point dilution comparison, one-step real-time RT-PCR was close 104-times more sensitive than Indirect-ELISA (indirect enzyme linked immunosorbant assay) assays published previously for RSV detection.2. One-step real-time RT-PCR methods using TaqMan probe are developed for quantitative detection of BYDV-GAV, GPV and PAV. Primers and probe for specific detection of BYDV were designed within the conserved region identified within the coat protein gene sequence. Sensitivity assay showed that the detection limit of the assay was 50 copies and the standard curve had a linear range from 50 to 5×10~5 copies with a good reproducibility.3. A sensitive Real Time PCR methods using TaqMan probe are developed for quantitative detection of WDV wheat strain in suspected viruliferous wheat. Primers and probe for specific detection were designed within the conserved region identified within the coat protein gene sequence and long intergenic region. Sensitivity assay showed that the detection limit of the assay was 30 copies and the standard curve had a linear range from 30 to 3×106 copies with a good reproducibility. The results showed that Real Time PCR was more sensitive than DAS-ELISA and traditional PCR assays published previously for WDV detection. Simultaneously, real-time PCR assay was applied to detect WDV DNA in single Psammotettix striatus (Linnaeus) from field.4. One Step Real Time RT-PCR methods are developed for quantitative detection of CGMMV in watermelon seeds tissue. Primers and TaqMan probe specific for the CGMMV CP gene were designed by aligming the sequences of known CGMMV strain. Sensitivity assay showed that the detection limit of the assay was 50 copies and the standard curve had a linear range from 50 to 5×10~5 copies with a good reproducibility. Simultaneously, real-time RT-PCR assay was applied to detect CGMMV coat protein gene in different watermelon seeds tissues. The results showed that the numbers of CGMMV CP gene in different tissues were fluctuant. CGMMV coat protein gene accumulation was higher in cotyledon than endotesta and exopleura.