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1、Virus vectoring ability of a Xiphinema americanum population,By:Charlie TaMentor:Dr.Inga ZasadaUnited States Department of Agriculture:Agricultural Research Services,Justification,Plant-parasitic nematodes cause$100 billion in crop loss annually worldwide;$10 billion in the U.S.(blueberries,red rasp
2、berries,and wine grape industry)Plants affected by X.americanum or nepoviruses become(s)necrotic,yield is reduced,and plant mortality can occur Currently few methods exist to control nematodes or remediate the diseases they transmitRegulations by the U.S.Environmental Protection Agency will soon lim
3、it/ban pre-plant fumigation which has traditionally been used to eradicate virus-transmitting nematodes,Xiphinema americanum(Dagger Nematode),Microscopic roundworm(s)that parasitize plantsMigratory ectoparasiteAcquire and transmit nepoviruses such as Tomato Ringspot Virus(ToRSV)and Tobacco Ringspot
4、Virus(TRSV)with their odontostyle,head,tail,Nepoviruses,Nematode-transmitted virus with polyhedral particlesType IV virus under the Baltimore classification system(positive sense single-stranded RNA that directly translates into protein)Acquisition of virus occurs during feeding and binds to the sur
5、face ofthe odontostyleViruses are lost when nematodes molt,odontostyle,Molecular Protocols,A RT-qPCR can be used for the detection of ToRSV in X.americanum at low concentration levels.Virus detection using RT-qPCR allows for a detailed study of nematode-virus interactions.,The coloration occurs due
6、to adding p-nitrophenyl phosphate.,http:/homepage.usask.ca/vim458/virology/studpages2007/Maura_Tim/For%20Maura%20-%20Virology%20website%20assignment/elisa.jpg,1,2,3,4,5,Steps:,Reverse Transcriptase-Quantitative Polymerase Chain Reaction(RT-qPCR),Enzyme-Linked Immunosorbent Assay(ELISA),Hypothesis,Co
7、uld a RT-qPCR method be developed to enable detection of small concentrations of ToRSV?,Prediction,A RT-qPCR method will be proficient in detecting low concentrations of viruses.X.americanum acquires ToRSV within a week of feeding on a virus infected host.This time period allows for additional virus
8、 particles to be acquired by the nematode,Objectives,Develop and optimize the efficiency of a RT-qPCR to detect ToRSVQuantify acquisition and saturation level of ToRSV in X.americanum,Probe:ACA AAT AAA AGG CAC TGC GTG TCT CG,Reverse:TCG AAT TTA AAT GCA GAC TCA AGA TAT G,Forward:GGA GAC GAT AAA TCC T
9、AT GTG GG,Methodology,Develop an internal positive control(IPC)for RT-qPCR by examining homogeneity of the internal transcribed spacer(ITS)region 1 of X.americanumDesign IPC to similar length as the ToRSV primer/probe set for multiplex purposesAnalyze the two sets for cross reaction and non target R
10、NA with each other.Examine the thermodynamic compatibility using hybridization software and cross referencing sequence data available on GenbankValidate RT-qPCR method with known virus infected samples.Ensures that our samples have nematodes,Objective I:Development,Data,IPC unsuccessfulIndividual ge
11、netic diversity in the group X.americanum,Data,Chromatograph illustrating the heterogeneity within the ITS1 region of rDNA for a single individual X.americanumA single signal becomes multiple signals;We observed this with individuals other than Xiphinema as well Literature suggest phylogenetic studi
12、es on nematodes is a common problem,Data,10-1,10-2,10-5,10-3,10-4,10-7,10-6,10-8,10-9,10-10,A 1 to 10 dilution series of ToRSV from leaves.10-1 to 10-10 all amplified,Objective I:Efficiency,Data,Detection of ToRSV in roots 11,9,6,and 5 weeks,Threshold values of ToRSV from amplification plot,Data,Dil
13、ution series of ToRSV in RootsDetection of ToRSV in roots was lower than ToRSV in leaves10-1 to 10-4 amplified,10-1,10-2,10-3,10-4,Objective II:,http:/image.made-in-,http:/www.medicine.virginia.edu/research/cores/biomolec/images/rt-pcr.jpg,http:/www.tari.gov.tw/tarie/photos/introduction/introduction
14、_PPD_17.jpg,http:/,http:/2010.igem.org/wiki/images/thumb/c/cb/IC_Assay_3_sept.jpg/300px-IC_Assay_3_sept.jpg,Obstacles,X.americanumHave low fecundityDelicate and sensitive to disturbancesInoculation and recovery of nematodes were lowRNA extraction was poorIPC did not work,Future Studies,Develop and o
15、ptimize a RT-qPCR method to detect TRSV in X.americanumDetermine the persistency and duration of ToRSV/TRSV within X.americanum by using the developed primers/probes for RT-qPCRLink the genetic variability of X.americanum populations to virus vectoring capabilities as a means to facilitate the development of diagnostic tools,Acknowledgements,Dr.Inga ZasadaAmy PeetzDr.Bob MartinKaren KellerNola MosierRuth PriceDr.Kevin AhernHoward Hughes Medical InstituteCripps Scholarship,
