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A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat

Tana, M.K. and Ghalayini, A. and Sharma, I. and Yi, J.P. and Shivas, R. and Priest, M. and Wright, D. (2009) A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat. Australasian Plant Pathology, 38 (2). pp. 101-109.

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Article Link(s): http://dx.doi.org/10.1071/AP08088

Publisher URL: http://www.publish.csiro.au

Abstract

A molecular assay with enhanced specificity and sensitivity has been developed to assist in the surveillance of Karnal bunt, a quarantineable disease with a significant impact on international trade. The protocol involves the release of DNA from spores, PCR amplification to enrich Tilletia-specific templates from released DNA and a five-plex, real-time PCR assay to detect, identify and distinguish T. indica and other Tilletia species (T. walkeri, T. ehrhartae, T. horrida and a group comprising T. caries, T. laevis, T. contraversa, T. bromi and T. fusca) in wheat grains. This fluorescent molecular tool has a detection sensitivity of one spore and thus bypasses the germination step, which in the current protocol is required for confirmation when only a few spores have been found in grain samples. The assay contains five dual-labelled, species-specific probes and associated species-specific primer pairs in a PCR mix in one tube. The different amplification products are detected simultaneously by five different fluorescence spectra. This specific and sensitive assay with reduced labour and reagent requirements makes it an effective and economically sustainable tool to be used in a Karnal bunt surveillance program. This protocol will also be valuable for the identification of some contaminant Tilletia sp. in wheat grains.

Item Type:Article
Corporate Creators:Department of Employment, Economic Development and Innovation (DEEDI), Agri-Science, Crop and Food Science, Plant Science
Business groups:Agri-Science, Crop and Food Science
Additional Information:© Australasian Plant Pathology Society.
Keywords:Polymerase chain reaction (PCR); Karnal bunt surveillance; Tilletia walkeri; differentiation; internal transcribed spacer (ITS).
Subjects:Science > Science (General)
Plant pests and diseases > Individual or types of plants or trees > Wheat
Science > Biology > Genetics
Plant pests and diseases > Pest control and treatment of diseases. Plant protection > Inspection. Quarantine
Deposited On:14 Apr 2009 06:48
Last Modified:26 Oct 2011 04:22

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