Development Of A Quantitative Real-time PCR For Rapid And Sensitive Detection Of Two Virus Species SCBIMV And SCBMOV In Sugarcane

Sugarcane bacilliform viruses?SCBV?which belong to the genus Badnavirus,family Caulimoviridae are one of important viruses infecting sugarcane.The SCBVs occur in the major sugarcane-producing regions of the world and are a serious threat to the yield and quality of sugarcane.It is necessary to develop quick,sensitive and reliable assay of SCBVs for providing key technical supports for the prevention of SCBVs transmission crossing sugarcane-planting regions,disease quarantine,and detection of virus-free seeding.In this study,we designed two set of specific primers and TaqMan fluorescence probes according to the RT/RNase H?reverse transcriptase/RNase H?sequences of the two SCBV species,Sugarcane bacilliform IM virus?SCBIMV,NC₀03031?and Sugarcane bacilliform MO virus?SCBMOV,NC₀08017?,which were released by the International Committee on Taxonomy of Viruses?ICTV?in 2012.Two protocols of real-time quantitative PCR?qPCR?for SCBIMV and SCBMOV species were established with quick,sensitive and reliable characteristics.Two standard curves were performed using a serial concentration?109-10 copies/?L?of pMD18T-IM?SCBIMV?and pSCBV20?SCBMOV?recombinant plasmid DNA as templates.The efficiencies of SCBIMV-qPCR and SCBMOV-qPCR were 99.26%and 101.73%,respectively.The two assays had a detection limit of 100 copies of plasmid DNA and had more 100-1000 times sensitivity than the conventional PCR.Besides,high specificities of the two assays were observed.A total of 176 sugarcane leaf tissue samples from Fuzhou and Yunnan province were collected and parallel detected by conventional PCR,SCBIMV-qPCR,and SCBMOV-qPCR.The conventional PCR results showed that 29.5%?52/176?samples were detected as SCBV-positive,i.e.,41.23%?47/114?for Fuzhou samples and 8.06%?5/62?for Yunnan samples.The virus was detected by SCBIMV-qPCR in 50%of total tested samples,and in 71.05%and 11.29%of Fuzhou and Yunnan samples,respectively.The virus was detected by SCBMOV-qPCR in 46.6%of total tested samples,and in 57.01%and 27.41%for Fuzhou and Yunnan samples,respectively.The findings revealed a higher incidence of SCBV-positive by qPCR than that of conventional PCR.A total of 80 RT/RNase H sequences of SCBV were obtained from the conventional PCR amplified fragments.A neighbor-joining phylogenetic tree was constructed with these sequences in this study and 38 reference sequences representing 18 different SCBV genotypes/strains from NCBI GenBank database.All SCBV isolates were classified into 19 SCBV groups?SCBV-A to-S?.A new phylogenetic group SCBV-S was proposed here.Five SCBV groups,SCBV-E,-F,-L,-N,and-Q were found in Fuzhou samples and three SCBV groups,SCBV-Q,-R,and-S were observed in Yunnan samples.In conclusion,we developed two protocols of efficiency,sensitive and rapid qPCR for SCBIMV and SCBMOV detection in this study.Various SCBV genotypes/strains were obverted in Fuzhou and Yunnan provinces.The findings provided a vital technical supporting for ecological controls of sugarcane diseases caused by SCBVs.