Differentiation of Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus using real-time TaqMan PCR. 2010

L C Papayiannis, and T A Iacovides, and N I Katis, and J K Brown
Agricultural Research Institute, Nicosia 1516, Cyprus. l.papayiannis@arinet.ari.gov.cy

During the past four decades, Tomato yellow leaf curl disease has become one of the major constraints in tomato production worldwide. In the Mediterranean basin, several isolates from two major Begomovirus species are involved in outbreaks and persistent epidemics. A real-time TaqMan PCR assay was developed and evaluated for the rapid and multiplex detection and differentiation of two begomoviruses often found in mixed infections in the region, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV). This assay was 1000-fold more sensitive than conventional PCR assays described previously, allowing the use of simple template preparation methods and eliminating the need for total nucleic acid purification. The viral DNA template was obtained by spotting sap extract derived from TYLCV or TYLCSV infected tissues on a nylon membrane or by directly using crude plant extracts in the real-time reaction cocktail. Preliminary results showed that this method can successfully detect and discriminate virus species from infected tomato, bean, pepper and different weed species obtained from the Mediterranean basin, the USA and Japan, allowing the simple, fast and cost-effective testing of a large number of samples in certification schemes. The assay can also be used for the detection of these two begomovirus species in their whitefly vector biotypes of the Bemisia tabaci (Gennadius) species group.

UI MeSH Term Description Entries
D007303 Insect Vectors Insects that transmit infective organisms from one host to another or from an inanimate reservoir to an animate host. Insect Vector,Vector, Insect,Vectors, Insect
D009757 Nylons Polymers where the main polymer chain comprises recurring amide groups. These compounds are generally formed from combinations of diamines, diacids, and amino acids and yield fibers, sheeting, or extruded forms used in textiles, gels, filters, sutures, contact lenses, and other biomaterials. Polyamides,Dermalon,Ethilon,Nylon,Polyamide,Supramid,Dermalons,Ethilons,Supramids
D010935 Plant Diseases Diseases of plants. Disease, Plant,Diseases, Plant,Plant Disease
D010936 Plant Extracts Concentrated pharmaceutical preparations of plants obtained by removing active constituents with a suitable solvent, which is evaporated away, and adjusting the residue to a prescribed standard. Herbal Medicines,Plant Extract,Extract, Plant,Extracts, Plant,Medicines, Herbal
D002212 Capsicum A plant genus of the family SOLANACEAE. The hot peppers yield CAPSAICIN, which activates VANILLOID RECEPTORS. Several varieties have sweet or pungent edible fruits that are used as vegetables when fresh and spices when the pods are dried. Bell Pepper,Cayenne Pepper,Chili Pepper,Chilli Pepper,Green Pepper,Jalapeno Pepper,Paprika,Pimiento,Red Pepper,Capsicum annuum,Cayenne,Chile Pepper,Hot Pepper,Sweet Pepper,Bell Peppers,Cayenne Peppers,Chile Peppers,Chili Peppers,Chilli Peppers,Green Peppers,Hot Peppers,Jalapeno Peppers,Pepper, Bell,Pepper, Cayenne,Pepper, Chile,Pepper, Chili,Pepper, Chilli,Pepper, Green,Pepper, Hot,Pepper, Jalapeno,Pepper, Red,Pepper, Sweet,Peppers, Bell,Peppers, Cayenne,Peppers, Chile,Peppers, Chili,Peppers, Chilli,Peppers, Green,Peppers, Hot,Peppers, Jalapeno,Peppers, Red,Peppers, Sweet,Red Peppers,Sweet Peppers
D006430 Hemiptera A large order of insects characterized by having the mouth parts adapted to piercing or sucking. It is comprised of four suborders: HETEROPTERA, Auchenorrhyncha, Sternorrhyncha, and Coleorrhyncha. Aleurodoidea,Cicadas,Cicadelloidea,Cicadoidea,Coccoidea,Fulgoroidea,Leafhoppers,Psyllids,Psylloidea,Scale Insects,Treehoppers,Whiteflies,Homoptera,Aleurodoideas,Cicada,Cicadelloideas,Cicadoideas,Coccoideas,Fulgoroideas,Hemipteras,Homopteras,Insect, Scale,Insects, Scale,Leafhopper,Psyllid,Psylloideas,Scale Insect,Treehopper,Whitefly
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D013698 Templates, Genetic Macromolecular molds for the synthesis of complementary macromolecules, as in DNA REPLICATION; GENETIC TRANSCRIPTION of DNA to RNA, and GENETIC TRANSLATION of RNA into POLYPEPTIDES. Genetic Template,Genetic Templates,Template, Genetic
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain

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