Detection Of Secoviridae And Arabis Mosaic Virus By RT-PCR

Secoviridae is of importance on the economic value,which comprise 66 determinant virus and 3 tentative members.Ten viruses were determined as quarantine pests in China.A semi-nested RT-PCR method for detecting Secoviridae and a RT-PCR method for detecting Comovirus were established using degenerate PCR.The current RT-PCR methods for detecting Arabis mosaic virus were evaluated,and a new RT-PCR method was established in this research.A SYBR Green I real-time RT-PCR method was also established for the detection of Arabis mosaic virus.The coat protein.gene sequences of ArMV were amplified by RT-PCR and to analyze the genetic diversity.To establish a semi-nested RT-PCR method for detecting Secoviridae,three degenerate primers were screened out from designed primers according to the conserved regions of RdRp gene.This method was successful for PCR amplification of 27 isolates of 20 virus species of Secoviridae,including nine virus species of Comovirus,two virus species of Fabavirus and nine virus species of Nepovirus.Sequencing primers were added to the 5' end of degenerate primers to facilitate sequencing of PCR products.The partial RdRp sequences of BLMoV and BBS V were determined by this method for the first time.The phylogenetic analysis reveals that the phylogenetic relationship among virus species is highly consistent with current classification of Secoviridae.And all isolates were closely related with the corresponding virus in phylogenetic tree.The specificity assays showed that the method did not amplify a specific product from host plants.We detected BBWV-2 from Radix Pseudostellariae for field application.An RT-PCR method for detecting Comovirus was established using degenerate primers designed according to the conserved regions of cp gene.It was successful for PCR amplification of ten isolates of seven virus species of Comovirus,but did not amplify any PCR product from other Comovirinae virus or host plants.It was demonstrated to be a highly versatility and specific PCR assay.The addition of an A/T-rich sequence at the 5' termini of degenerate primers led to an increase in sensitivity up to 100 folds.Sequencing primers were added to the 5' end of degenerate primers to facilitate sequencing of PCR products.The sensitivity was enhanced 1000 folds when PCR was conducted by using two pairs of primers(ComoV-2-F2-M4/ComoV-2-Rl-M1 and M4/M1)at the ratio of 1:10.The nucleotide sequence analysis showed that isolates had very high homology to the corresponding virus except CPSMV(PV-0050).An evaluation of theoretical analysis and experimental research on six RT-PCR methods(a novel method in this research)for detecting Arabis mosaic virus was analyzed.Theoretical analysis showed that the difference of Tm between ArMV-370-F and ArMV-370-R was more than 5 ?.Sequence alignments indicated that primer ArMV-2-350F,ArMV-2-350R and ArMV-364-R showed the highest sequence identity with the reported sequences.An expected fragment was amplified in all 25 ArMV isolates from narcissus,lilies and tulips by RT-PCR with primers ArMV-2-350F/ArMV-2-350R or primers ArMV-364-F/ArMV-364-R,while other primers for method didn't amplify the expected fragment completely.The specificity assays showed that the primers ArMV-2-350F/ArMV-2-350R did not amplify a specific product from other Comovirinae virus or host plants.The sensitivity of primers ArMV-2-350F/ArMV-2-350R and primers ArMV-364-F/ArMV-364-R made no difference.As a result,the novel RT-PCR with the primers ArMV-2-350F/ArMV-2-350R and the previous RT-PCR with the primers ArMV-364-F/ArMV-364-R had good versatility,specificity and sensitivity,and it could be used to detect ArMV in daily inspection.The coat protein gene of 21 ArMV isolates(narcissus,lilies and tulips)from Netherlands during 2011 and 2013 were amplified by RT-PCR and obtained the complete cp gene sequences(1515 bp).Sequence analysis showed that cp gene in 21 ArMV isolates shared 85.8%-99.9%nucleotide and 92.1%-100.0%amino acid identities.The coat protein gene in 21 ArMV isolates shared 71.6%-73.1%nucleotide and 75.9%-78.3%amino acid identities with the reported sequence of Butterbur isolate,and shared 89.7%-99.2%amino acid identity with the other reported sequences.The phylogenetic analysis showed three groups existed in all isolates,and the Butterbur isolate was placed in group ? alone.12 ArMV isolates from narcissus were placed in group ?,however,8 ArMV isolates from lilies and tulips were placed in group ?.It suggested that the ArMV isolate had genetic diversity even if came from the same country.A S YBR Green ? real-time RT-PCR method was established for the detection of Arabis mosaic virus according to the conserved regions of cp gene.The coefficient correlation(R2)of standard curve was 0.9972,and its amplification efficiency was 87.2%.The sensitivity was 1.0×102 copies/?L-1.0×101 copies/?L,which was 10-100 more sensitive than that of the conventional PCR.The specific showed a good specificity.Using the method to detect isolates from narcissus,lilies and tulips,the results showed all isolates displayed amplification curve,and melting curve showed one specific peak.It was demonstrated to be a good reproducibility,highly sensitive,specific and rapid PCR assay for the detection of Arabis mosaic virus.