The Molecular Identification And Detection Methods Of Potatoleafroll Viruse

PLRV is one of the most important viruses , and with the specific primers which were designed based on the given Potato leaf roll virus coat protein (PLRV CP) gene sequence (627bp), PLRV is detected by reverse transcription polymerase chain reaction (RT-PCR) and Enzyme Enzyme-Linked Immunosorbent Assay (ELISA) .PLRV CP gene sequence is amplified by reverse transcription polymerase chain reaction, using the total RNA of the plant infected by PLRV. The gene is cloned into E. coll DH5 α and sequenced. The sequence is compared with the sequence of the homologous gene of other isolates of PLRV, The result shows high homology with the other isolates (the highest homology can reach 96% of nucleic acid and 98% of amino acid) and shows the virus is PLRV. The CP amino acid sequences of different isolates of PLRV are compared. The result shows: there is little difference on the amino acid sequence among the PLRV isolates, the homology ranges from 95% to 98%. Based on CP amino acid sequence the phylogenic tree of PLRV is established and the isolates are clustered into many groups.The expression vector of PLRV CP gene is constructed in E. coli. DH5 α successfully .By many tests, found some conditions that can't express PLRV CP gene in E. coli. DH5 α and BL21.Simple, quick, sensitive, reliable molecular detection techniques of PLRV are reported in the article: One-step RT-PCR, and Nucleic acid spot hybridization (NASH). The improved nucleic acid spot hybridization is simpler and quicker than the routine nucleic acid spot hybridization. But the sensitivity of improved NASH is as same as the routine NASH's. The improved NASH detection method of PLRV only needs 4-5 hours while the routine one needs about 20hours. It is more sampler and quicker and is same suitable for the detection of a...