Abstract
Real-time PCR with fluorogenic hydrolysis probes (5' nuclease assay) is increasingly used for the detection of pathogens for diagnostic purposes. Nevertheless, the size of the probes, usually 25–40 nucleotides, might limit their use to detect pathogens with high genome variability between isolates, where an identical sequence cannot be found without multiple mismatches. In this report, we describe a 5' nuclease assay, to detect Apple stem pitting virus, based on the use of a shorter probe which is chemically modified with a minor groove binder in order to increase duplex stability and raise the melting temperature to a value suitable for real-time analysis. The short size of the probe, which is critical to target a conserved cluster sequence of 14 nucleotides in the RNA polymerase gene, circumvents the genome variability of the virus. The assay correlates at 96 percent with gel analysis and is more reliable than biological indexing to detect Apple stem pitting virus field isolates. It is fast and fully compatible with automation, and therefore particularly suitable for plant certification.
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References
Bugos RC, Chiang VL, Zhang XH, Campbell WH, Podila GK and Campbell WR (1995) RNA isolation from plant tissues recalcitrant to extraction in guanidine. Biotechniques 19: 734–737
Bustin SA (2000) Absolute quantification of mRNA using realtime reverse transcription polymerase chain reaction assays. Journal of Molecular Endrocrinology 25: 169–193
Fang X, Li JJ, Perlette J, Tan W and Wang K (2000) Molecular beacons: Novel fluorescent probes. Analytical Chemistry 1: 747–753
Desvignes JC (1999) Virus diseases of fruit trees. Centre Technique Interprofessionnel des fruits et légumes, Paris, France
Holland PM, Abramson RD, Watson R and Gelfand DH (1991) Detection of specific polymerase chain reaction products by utilizing 5′ → 3′ exonuclease activity of Thermus aquaticus DNA polymerase. In: Proceedings of the National Academy of Sciences, USA 88: 7276–7280
Jelkmann W (1994) Nucleotide sequence of Apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex-and carlaviruses. Journal of General Virology 75: 1535–1542
Jelkmann W and Keim-Konrad R (1997) Immuno-capture polymerase chain reaction and plate-trapped ELISA for the detection of Apple stem pitting virus. Journal of Phytopathology 145: 499–503
Klerks MM, Leone G, Linder JL, Schoen CD and van den Heuvel JFJM (2001) Rapid and sensitive detection of Apple stem pitting virus in apple trees through RNA amplification and probing with fluorescent molecular beacons. Phytopathology 91: 1085–1091
Kummert J, Vendrame M, Steyer S and Lepoivre P (2000) Development of routine RT-PCR tests for routine certification of fruit tree multiplication material. OEPP/EPPO Bulletin 30: 441–448
Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB and Hedgpeth J (2000) 3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperature. Nucleic Acids Research 28(2): 655–661
Kutyavin IV, Lukhtanov EA, Gamper, HB and Meyer RB (1997) Oligonucleotides with conjugated dihydropyrroloindole tripeptides: Base composition and backbone effects on hybridization. Nucleic Acids Research 25(18): 3718–3723
Lie YS and Petropoulos CJ (1998) Advances in quantitative PCR technology: 5′ nuclease assay. Current Opinion in Biotechnology 9: 43–48
Livak K, Flood SJA, Marmaro J, Giusti W and Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods and Applications 4: 357–362
Livak K, Marmaro J and Flood S (1996) Guidelines for designing TaqMan fluorogenic probes for 5′ nuclease assays. Perkin-Elmer Research News 57: 1–5
MacKenzie DJ, McLean MA, Mukerij S and Green M (1997) Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant Disease 81: 222–226
Nemchinov L, Hadidi A and Faggioli F (1998) PCR-detection of Apple stem pitting virus from pome fruit hosts and sequence variability among viral isolates. Acta Horticulturae 472: 67–73
Schwarz K and Jelkmann W (1998) Detection and characterization of European Apple stem pitting virus isolates of apple and pear by PCR and partial sequence analysis. Acta Horticulturae 472: 75–85
Stouffer RF (1989) Apple stem pitting virus. In: Fridlund PR (ed) Virus and Virus-like Diseases of Pome Fruit and Simulating Noninfectious Disorders (pp 138–144) Washington State University, Pullman
Thelwell N, Millington S, Solinas A, Booth J and Brown T (2000) Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Research 28(19): 3752–3761
Tyagi S and Kramer R (1996) Molecular beacons: Probes that fluoresce upon hybridization. Nature Biotechnology 14: 303–308
Walburger DK, Afonina IA and Wydro R (2001) An improved real time PCR method for simultaneous detection of C282Y and H63D mutations in the HFE gene associated with hereditary hemochromatosis. Mutation Research 432: 69–78
Whitcombe D, Theaker J, Guy SP, Brown T and Little S (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nature Biotechnology 17: 804–807
Yoshikawa N, Matsuda H, Oda Y, Isogai M and Takahashi T (2001) Genome heterogeneity of Apple stem pitting virus in apple trees. Acta Horticulturae 550: 285–290
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Salmon, M.A., Vendrame, M., Kummert, J. et al. Rapid and Homogenous Detection of Apple stem pitting Virus by RT-PCR and a Fluorogenic 3' Minor Groove Binder-DNA Probe. European Journal of Plant Pathology 108, 755–762 (2002). https://doi.org/10.1023/A:1020849304902
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DOI: https://doi.org/10.1023/A:1020849304902