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Molecular inversion probe: a new tool for highly specific detection of plant pathogens.

Lau HY, Palanisamy R, Trau M, Botella JR - PLoS ONE (2014)

Bottom Line: A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided.We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici.MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.

View Article: PubMed Central - PubMed

Affiliation: Plant Genetic Engineering Laboratory, School of Agriculture and Food Sciences, University of Queensland, Brisbane, Queensland, Australia; Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia.

ABSTRACT
Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. The MIP assay was able to detect the presence of the pathogen in infected Arabidopsis thaliana plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.

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Quantification of Foc genomic DNA in infected A. thaliana using real-time PCR.The average amount of Foc genomic DNA present in the infected plant samples were quantified using the 2 primer sets (Foc-F1/Foc-R1 and Foc-F2/Foc-R2) in the real-time PCR.
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pone-0111182-g006: Quantification of Foc genomic DNA in infected A. thaliana using real-time PCR.The average amount of Foc genomic DNA present in the infected plant samples were quantified using the 2 primer sets (Foc-F1/Foc-R1 and Foc-F2/Foc-R2) in the real-time PCR.

Mentions: The standard curves of the two primer sets (Foc-F1/Foc-R1 and Foc-F2/Foc-R2) resulting from linear regression are shown in Fig. S2a and S2b, respectively. Both standard curves indicated a good correlation between the amount of DNA template and the Ct values (R2∶0.98 and 0.99, respectively). Melting curve analysis of the PCR amplicons was also performed to confirm successful amplification indicating a maximum Tm of 82.5°C and 85°C, respectively (Fig. S2c and S2d). The amount of Foc genomic DNA in all stages of infection was then determined based on the Ct values and averages of the quantified concentrations from the two primer sets (Fig. 6). Infection in stages 1 and 2 showed very low amounts of Foc genomic DNA while a very significant increase was observed in stage 3 that kept steadily growing until stage 5 (Fig. 6).


Molecular inversion probe: a new tool for highly specific detection of plant pathogens.

Lau HY, Palanisamy R, Trau M, Botella JR - PLoS ONE (2014)

Quantification of Foc genomic DNA in infected A. thaliana using real-time PCR.The average amount of Foc genomic DNA present in the infected plant samples were quantified using the 2 primer sets (Foc-F1/Foc-R1 and Foc-F2/Foc-R2) in the real-time PCR.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4208852&req=5

pone-0111182-g006: Quantification of Foc genomic DNA in infected A. thaliana using real-time PCR.The average amount of Foc genomic DNA present in the infected plant samples were quantified using the 2 primer sets (Foc-F1/Foc-R1 and Foc-F2/Foc-R2) in the real-time PCR.
Mentions: The standard curves of the two primer sets (Foc-F1/Foc-R1 and Foc-F2/Foc-R2) resulting from linear regression are shown in Fig. S2a and S2b, respectively. Both standard curves indicated a good correlation between the amount of DNA template and the Ct values (R2∶0.98 and 0.99, respectively). Melting curve analysis of the PCR amplicons was also performed to confirm successful amplification indicating a maximum Tm of 82.5°C and 85°C, respectively (Fig. S2c and S2d). The amount of Foc genomic DNA in all stages of infection was then determined based on the Ct values and averages of the quantified concentrations from the two primer sets (Fig. 6). Infection in stages 1 and 2 showed very low amounts of Foc genomic DNA while a very significant increase was observed in stage 3 that kept steadily growing until stage 5 (Fig. 6).

Bottom Line: A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided.We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici.MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.

View Article: PubMed Central - PubMed

Affiliation: Plant Genetic Engineering Laboratory, School of Agriculture and Food Sciences, University of Queensland, Brisbane, Queensland, Australia; Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia.

ABSTRACT
Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. The MIP assay was able to detect the presence of the pathogen in infected Arabidopsis thaliana plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.

Show MeSH
Related in: MedlinePlus