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Species-specific duplex PCR for the detection of Pratylenchus penetrans

Lieven Waeyenberge, Niclole Viaene and Maurice Moens UGent (2009) NEMATOLOGY. 11(6). p.847-857
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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
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
journal title
NEMATOLOGY
Nematology
volume
11
issue
6
pages
11 pages
Web of Science type
Article
Web of Science id
000272487300005
JCR category
ZOOLOGY
JCR impact factor
0.937 (2009)
JCR rank
75/126 (2009)
JCR quartile
3 (2009)
ISSN
1388-5545
DOI
10.1163/156854109X428016
language
English
UGent publication?
yes
classification
A1
id
854456
handle
http://hdl.handle.net/1854/LU-854456
date created
2010-02-08 08:38:54
date last changed
2010-02-18 10:05:47
@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},
}

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.