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Species-specific duplex PCR for the detection of Pratylenchus penetrans

(2009) NEMATOLOGY. 11(6). p.847-857
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Abstract
ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.
Keywords
RIBOSOMAL DNA, POTATO CYST-NEMATODE, MOLECULAR-IDENTIFICATION, PRIMERS, GLOBODERA-PALLIDA, species-specific primers, rDNA sequences, molecular, ITS, POLYMERASE-CHAIN-REACTION, FRAGMENT-LENGTH-POLYMORPHISM, SPP., RDNA, DIFFERENTIATION

Citation

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Chicago
Waeyenberge, Lieven, Niclole Viaene, and Maurice Moens. 2009. “Species-specific Duplex PCR for the Detection of Pratylenchus Penetrans.” Nematology 11 (6): 847–857.
APA
Waeyenberge, L., Viaene, N., & Moens, M. (2009). Species-specific duplex PCR for the detection of Pratylenchus penetrans. NEMATOLOGY, 11(6), 847–857.
Vancouver
1.
Waeyenberge L, Viaene N, Moens M. Species-specific duplex PCR for the detection of Pratylenchus penetrans. NEMATOLOGY. 2009;11(6):847–57.
MLA
Waeyenberge, Lieven, Niclole Viaene, and Maurice Moens. “Species-specific Duplex PCR for the Detection of Pratylenchus Penetrans.” NEMATOLOGY 11.6 (2009): 847–857. Print.
@article{854456,
  abstract     = {ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.},
  author       = {Waeyenberge, Lieven and Viaene, Niclole and Moens, Maurice},
  issn         = {1388-5545},
  journal      = {NEMATOLOGY},
  keyword      = {RIBOSOMAL DNA,POTATO CYST-NEMATODE,MOLECULAR-IDENTIFICATION,PRIMERS,GLOBODERA-PALLIDA,species-specific primers,rDNA sequences,molecular,ITS,POLYMERASE-CHAIN-REACTION,FRAGMENT-LENGTH-POLYMORPHISM,SPP.,RDNA,DIFFERENTIATION},
  language     = {eng},
  number       = {6},
  pages        = {847--857},
  title        = {Species-specific duplex PCR for the detection of Pratylenchus penetrans},
  url          = {http://dx.doi.org/10.1163/156854109X428016},
  volume       = {11},
  year         = {2009},
}

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