| Citrus tristeza is a severe disease for citrus industry worldwide which caused by a virus pathogen, Citrus tristeza virus (CTV). CTV taxonomically belongs to Closterovirus, whose genome is a positive single strand RNA (+ssRNA) consisting of 19296 nucleic acid. Most of the commercial citrus cultivars can be infected by CTV, which would lead to fall of leaf and fruit, decline of plant and degradation of fruit quality. The economic value of the citrus fruits is greatly reduced if infected with CTV, and the plant will die at the last stage. Due to the lack of highly effective chemicals and resistant cultivars, an accurate and rapid diagnostic technology is badly needed for epidemiological monitoring in citrus producing area and quarantine detection of citrus seedling and fruits in custom department. The traditional detection methods such as indicator plant, microscopic detection of inclusionbody, ELISA, DIA and nucleic acid hybridization, can not fulfill the requirements of the detection because they are more or less time-consuming, complicated in operation, less sensitive and too dangerous, while reverse transcription polymerase chain reaction (RT-PCR) has become the most adopted technology for virus detection since PCR was invented. This technology is sensitive, specific and rapid, which can fulfill the requirements of modern plant quarantine.Research purpose: to establish an applicable RT-PCR detection system with internal control and an advanced real-time fluorescent quantitative RT-PCR detection system in order to provide technical support for rapid and accurate identification and monitoring/forecasting of CTV. Research contents and results:①Comparing the different RNA concentration and purity extracted by different extraction methods such as improved CTAB method, extraction kit method, soaking method, minicolumn centrifugal method and so on, the minicolumn centrifugal method could be used to extract qualified RNA from CTV and reduce the false-negative rate for its better performance to exclude PCR inhibitors like plant pigments, protein and polysaccharide.②Two RT-PCR rapid detection systems were optimized and established: one is a conventional RT-PCR system with internal control, and the primers employed in this system were cquctv9/10 (for CTV) and cqucrpII01/02 (for internal control); the other is a real-time fluorescent quantitative RT-PCR system, and the primers employed in this system were cquctv1/2 and a TaqMan probe cquctvp1. The primers/probe in both systems were designed based on the P20 gene sequence of CTV and RNA polymerase II gene sequence, respectively.③After the DNA fragments obtained from RT-PCR being analyzed by sequencing, they were used as template in an in vitro transcription process to obtain massive high quality RNA, which served as harmless positive control and standard samples in the detection systems.④Determine the specificity of both detection systems using common virus and bacteria as controls and the results indicated that both systems had similar specificity; determine the sensitivity of both systems with RNA standard samples and the results showed that the sensitivity of real-time fluorescent quantitative RT-PCR system was 2 fg/25μL, which was higher than conventional RT-PCR for 2 magnitudes.⑤Practical detection with field samples collected from Sichuan province, Chongqing city, Guangxi municipality and Zhejiang province proved that both RT-PCR detection systems were robust and accurate.Research conclusion: rapid, robust and reliable molecular-based detection systems were established with minicolumn centrifugal method and positive standard samples. The whole procedure from sample treatment to detection results cost only 4h in total, which fulfilled the requirements in practical detection. Therefore, the two detection methods could be applied to detect citrus seedlings and monitor the field dynamic changes of CTV.
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