Paper abstract

Vince Mulholland mulholl at sasa.gov.uk
Wed Apr 17 08:15:14 EST 1996


>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.




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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.

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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








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