Studies On The Detection Of Prunus Necrotic Ringspot Virus With Real-time FluorescenRT-PCR And Hybridization CaptureRT-PCR-ELISA

Primus necrotic Ringspot Virus (PNRSV) is distributed extensively in America and Europe, resulting in serious yield loss in fruit trees, vegetables and other crops. PNRSV has not been reported in China by now, and is an important quarantine virus in China (on the A2 list ). Traditional methods such as serological test are not suited to quarantine detection because of disadvantage such as low sensitivity. Therefore reliable, sensitive, quick and easy handling detection method is necessary to be established.The author reviewed the detection measures of Prunus necrotic Ringspot Virus,and related the research progression of the pathogen detection technology inside and outside .The template Amplication technology include PCR assays ^ NASBA and so on. The cDNA and cRNA probe which labeled with the isotope % biotin or DIG ,the offset probe and the peptide probe can be applied to magnify the signal. PCR-Gene scan assays and PCR Agilent chip chamber combine the template Amplication and the signal magnification. PCR-ELISA and real fluorescence PCR associate the template Amplication and hybridization with the signal magnification. Those biology technologies are developing rapidly and applied to all domains of the biology.A pair of primer, DIG labeled probe and a TaqMan probe based on the conserved nucleotide sequence of CP gene of different PNRSV strains were designed and synthesized. A novel hybridization capture-PCR ELISA was established for PNRSV detection,The main contributions to the development of the method are that a solid primer is successfully bound to PCR tube wall specially for aimed RNA capture in order to raise RNA purification and reduce time consumption. DIG labeled probe hybridization with solid PCR product was performed as well as electrophoresis of liquid product, this method combines RNA purification, PCR amplification and nucleotide probe hybridization detection together and has many advantages including better RNA purity, less time consumption, reliable positive reaction and 10 times sensitivity as RT-PCR gel running detection, reduce false positive result, unpurifiednucleotide requirement, lOug infected plant organism can be detected by solid hybridization.A novel real-time fluorescent RT- PCR was established to detect PNRSV This technique uses a TaqMan probe to monitor in real time amplification of target genes, A Taqman probe labeled by a quenching and fluorecenting was added to the PCR reaction buffer. More and more fluorescent signal can be collected with the PCR reaction carry on . The method is more automatized and much less time consumption (only 3 hours from nucleotide hybridization capture to result found ). with a sensitivity 10 times of RT-PCR's and a detection limit of 4.49fg positive plasmid or 20 positive plasmid molecules. The probe detection and PCR amplification are contemporary, without ethidium bromide staining and gel running and any post-PCR manipulations, so that the contamination risks are reduced.Those two methods were used to detect successfully the Primus necrotic Ringspot Virus from sample delivered by Beijing airport quarantine bureau.The amplified fragment was cloned into the vector of PMD18-T then sequenced. The sequence is 451bp in length and shows98% homologous to isolate gi 1575050 gb U57046.1 PNU57046 deposited in the Gene Bank.Moreover, the two methods were also successfully used to detect Tomato ringspot virus and Fire blight (E. amylovord).