>Some time ago, I suggested that people could post abstracts of
>diagnostics papers here, in order to stimulate more on-topic
>discussion. Here is an abstract from my own laboratory, of a paper
>published in the Proceedings of the Diagnostics in Crop Protection
>conference held in Warwick, UK earlier this month.
>>STUFF DELETED
>>Dr Kevin O'Donnell "Nature is not cruel, only
>Diagnostics and Molecular Biology pitilessly indifferent."
>SASA - Richard Dawkins
>Edinburgh
Here is another abstract from the same conference. Comments
gratefully received - look forward to seeing any posts.
---------------------------------------------------------------------
USE OF THE POLYMERASE CHAIN REACTION TO DISCRIMINATE POTATO CYST
NEMATODE AT THE SPECIES LEVEL
V. Mulholland1, L. Carde1, K.J. O'Donnell1, C.C. Fleming2 and T.O.
Powers3
1 Diagnostics & Molecular Biology Section, SASA, East Craigs,
Edinburgh EH12 8NJ, UK.
2 Dept of Applied Plant Science, DANI, Newforge Lane, Belfast, UK.
3 Dept of Plant Pathology, University of Lincoln, Nebraska, USA.
Potato cyst nematode (PCN) is responsible for significant loss in the
potato production of the EC. The use of PCN-contaminated land for
further potato crops is dependent on the species that infests the
field. Two species of PCN are endemic in the UK; Globodera pallida
and Globodera rostochiensis. As there are no potato varieties fully
resistant to G. pallida, land contaminated with this species of PCN
can be unavailable for potato crops for a longer period. The present
identification techniques include microscopic examination of the cyst
contents, and the use of isoelectric focusing gels.
A polymerase chain reaction (PCR) technique has been developed based
on allele-specific amplification. This method amplifies a region
within the internally transcribed spacer (ITS) region of the
ribosomal RNA genes. A species-specific primer was designed for each
species, binding to different regions of the ITS. Each of these
primers is designed to mismatch with the other species at the 3' end.
A third primer, which binds perfectly to both species is located
downstream of the binding sites of the species-specific primers.
Amplification using the Stoffel fragment of Taq DNA polymerase of G.
pallida extracts gives rise to a 391 bp fragment, while G.
rostochiensis produces a 238 bp fragment. Mixed populations will
result in the production of both fragments. The DNA products are
visualised following agarose gel electrophoresis. This technique
allows identification of PCN species within 3 hours.
---------------------------------------------------------------------
Vincent Mulholland
Higher Scientific Officer,
Diagnostics & Molecular Biology Section,
Scottish Agricultural Science Agency,
East Craigs,
Edinburgh EH12 8NJ,
U.K.
E-Mail: mulholl at sasa.gov.uk
Tel: (0131) 244 8845
Fax: (0131) 244 8912
