Molecular Identification And Detection Methods Of Tobacco Viruses

Based on the coat protein (CP) gene sequence (480nt)of Tobacco mosaic virus (TMV), a 442bp fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) with the specific primers. The product was cloned into E. coli JM109 and sequenced. Comparing the sequence with the homologous gene sequence of other TMV isolates, high homology was observed (the highest was 99.8% with Yunnan isolate AJ239099). The nucleotide sequence identity among the published sequences of various isolates and strains of TMV ranged from 73. 1% to 100%, the difference was obvious. Phylogenic tree of TMV was established on these nucleotide sequences. In this way, the 15 isolates were separated into three easily identifiable groups.With the specific primers designed on the coat protein (CP) gene sequence of Cucumber mosaic virus (CMV), a 776bp fragment was amplified by RT-PCR using the total RNA of the CMV infected tobacco leaves. Then the PCR product was cloned into E. coli JM109 and sequenced. Compared the sequence with analogous sequences of other CMV isolates. The highest homology is 98. 6% between it and AF523342. Analyzing the phylogenic tree established on the nucleotide sequences, 43 isolates were separated into two main subgroups. In the subgroup I , the difference among them is still apparent, the identity of 21 isolates ranged between 90.3 to 98. 6%.Three 808bp fragments of different area isolates were obtained by RT-PCR with specific primers of the coat protein gene of Potato virus Y (PVY)using the total RNA of the PVY infected leaves as template, then was cloned into E. coli JM109. The sequences of the fragments was determined and compared with analogous published sequences of various PVY isolates. The homologies are 93.4-96.6%, 92.0-94.0%, 91.9-94.0% between them and the isolates of subgroup III,respectively. Phylogenic tree of PVY was established on CP gene nucleotide sequences of 55 isolates. All isolates were separated into three subgroups.The One-step RT-PCR detection methods of the three viruses using their specific primers are described. The results showed that these methods were sensitive, quick and specific. Digoxigenin-labelled cDNA probes were synthesized by PCR, and applied in Nucleic acid spot hybridization (NASH). The improved NASH procedure saved time and cut costs to aboutYO. 4 per sample without reducing sensitivity, but enabling the processing of a large number of samples.According to the detection methods of the three viruses,NASH detection Kits were established. The result of usage of the kits showed that they were stability, sensitivity and speciality.