Detection Of Citrus Psorosis Virus By RT-PCR

Citrus Psorosis is a widespread and serious damaging disease of citrus in many parts of the world, including Argentina, Brazil, Italy, Spain and America et al. The casual agent named CPV (citrus psorosis virus) or CPsV (citrus psorosis-associated virus) belongs to genus Ophiovirus, is a multicomponent ssRNA virus with a coat protein of approximately 48kDa. Psorosis is graft-transmissible and primarily spread by propagation of infected buds, seedlings. With the introduction of citrus varieties from abroad, there is the risk of introduction of CPV. Therefore, to develop a rapid and accurate diagnosis method based on the detection of the pathogen is urgently demanded for plant quarantine that is a primary method to prevent Chinese citrus industry from being disserved by this severe disease.Several diagnosis methods have become available to detect CPV at present, such as biological indexing (indictor plant indexing and cross protection test), ELISA, molecular blot and RT-PCR et al. However there isn't any relevant report in China yet. In this research we have investigated the virus resources of CPV maintained in Citrus institute of CAAS, optimized a rapid mini-preparing protocol for virus RNA extraction by comparing several existing nucleotide extraction methods, finally developed a rapid, highly sensitive and specific RT-PCR diagnosis method, then applied it in the analysis of one-year-distribution of CPV in different parts of infected Dweet tangor plant. The conclusions are as follows:1. Three total nucleotide extraction methods including dipping method, Trizol method and rapid mini-extraction method had been compared. The results showed that, the A₂₆₀/A₂₈₀ value of extract of dipping method was average 1.2579, followed unsatisfactory PCR effect; The Trizol method yielded RNA of high purity with the average A₂₆₀/A₂₈₀ value of extract 1.7666, followed good PCR effect, however this method is unsuitable for RNA preparing from a large amount of samples...