Rosa C, Polek M, Falk B W, Rowhani A
Department of Plant Pathology, University of California, Davis 95616.
California Citrus Tristeza Eradication Agency, Tulare 93224.
Plant Dis. 2007 Sep;91(9):1089-1095. doi: 10.1094/PDIS-91-9-1089.
Reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for the detection of Citrus tristeza virus (CTV; genus Closterovirus) and Citrus psorosis virus (CPsV; genus Ophiovirus) in citrus trees. Real-time TaqMan RT-PCR was also developed for the detection of CTV. Three different sample preparation methods were compared. The total RNA extraction method by Qiagen was found to be more reliable than the other two methods consisting of crude plant sap extraction and total nucleic acid trapping on a silica bed. Of 287 samples tested for CTV, 210 samples tested positive by RT-PCR and 198 samples by enzyme-linked immunosorbent assay (ELISA). Furthermore, the results from monthly tests of a selected number of field-grown CTV-infected trees showed that RT-PCR detected the virus in 100% of the infected trees in winter and summer, whereas ELISA did not. The one-tube RT-PCR detection was developed for CPsV and was more sensitive than ELISA. Notably, three of 10 CPsV isolates were not detected by ELISA. As demonstrated here, our approach allows the efficacious detection of different viruses in citrus plants using a minimal amount of tissue during all seasons. The molecular methods described could be used in citrus certification programs and to test trees in nurseries and commercial orchards.
已开发出逆转录聚合酶链反应（RT-PCR）检测方法，用于检测柑橘树中的柑橘衰退病毒（CTV；长线形病毒属）和柑橘鳞皮病毒（CPsV；蛇形病毒属）。还开发了实时TaqMan RT-PCR用于检测CTV。比较了三种不同的样品制备方法。发现Qiagen的总RNA提取方法比其他两种方法更可靠，后两种方法分别是粗植物汁液提取和在硅胶床上捕获总核酸。在检测的287个CTV样品中，210个样品经RT-PCR检测呈阳性，198个样品经酶联免疫吸附测定（ELISA）检测呈阳性。此外，对选定数量的田间种植的CTV感染树进行的月度检测结果表明，RT-PCR在冬季和夏季能检测出100%的感染树中的病毒，而ELISA则不能。已开发出用于CPsV的单管RT-PCR检测方法，其比ELISA更灵敏。值得注意的是，10个CPsV分离株中有3个未被ELISA检测到。如本文所示，我们的方法能够在所有季节使用最少的组织量有效检测柑橘植物中的不同病毒。所描述的分子方法可用于柑橘认证项目以及检测苗圃和商业果园中的树木。
