Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Fusarium...
1
Results Sep 2014 Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Fusarium oxysporum f. sp. ciceris – wilt pathogen of chickpea This work is undertaken as part of R Ghosh, A Nagavardhini and M Sharma Introducon Rapid detecon of pathogens in chickpea has become a pre-requisite to facilitate accurate disease diagnosis and surveillance for beer management strategy. Among the diseases affecng chickpea, Fusarium wilt [Fusarium oxysporum f. sp. ciceris (Foc)] can cause 100% yield losses in suscepble culvars. In this study a method based on the Loop-Mediated Isothermal Amplificaon (LAMP) assay for the detecon of Foc was developed and sensivity and specificity of the assay was evaluated. Objecves • Development of LAMP assay for detecon of Fusarium oxysporum f. sp. ciceris infecng chickpea. • Evaluaon of sensivity and specificing of LAMP assay. Materials and methods Figure 1. Schematic representation of position and sequence of primer sets. Figure 2. LAMP detection using HNB as a visual indicator (A) and agarose gel electrophoresis (B). Figure 3. Optimization of LAMP reaction time and temperature. Figure 4. Specificity of the LAMP assay for Foc detection. Figure 5. Comparative sensitivity of LAMP and conventional PCR analysis for detection of Foc. Conclusion This is the first report of the use of LAMP assay for the detecon of Foc. It is a promising assay for extensive applicaon and rapid diagnosis of Foc infecon in the laboratory and will be very useful for monitoring the disease complex and for suggesng further management strategies in chickpea. Acknowledgements: The funding support by Department of Science and Technology and Department of Biotechnology, GoI to conduct this study is acknowledged. DST & DBT • LAMP primers based on the EF-1alpha gene were designed using the LAMP designer soſtware program (Figure 1). • The Foc specific LAMP reacon was standardized for opmum temperature and me. • LAMP specificity was determined by performing the assay with DNA of Foc and of six other plant-pathogenic fungi (Fusarium acuminatum, Fusarium udum, Fusarium solani, Rhizoctonia batacola, Alternaria alternata and Phytophthora cajani). • Total 50 isolates of Foc collected from diverse geographical locaons and represenng Foc races in India were subjected to LAMP and convenonal Polymerase Chain Reacon (PCR). The LAMP assay • A simple colorimetric assay using a metal ion indicator, namely hydroxynaphthol blue (HNB) was used for detecon of Foc using LAMP. • A posive LAMP reacon was indicated by a sky blue color; the color remained violet for negave reacons (Figure 2 A). • Characterisc ladder like bands were evident in the agarose gel electrophoresis if the product was present (Figure 2 B). • Posive results were obtained at 60 min (Figure 3, A & B) and 63°C (Figure 3, C & D) both in HNB and convenonal PCR. Evaluaon of LAMP assay • All the 50 Foc isolates represenng diverse geographical locaons in India showed posive reacon as indicated by a sky blue color. The results were consistent with PCR method. Specificity and sensivity of the LAMP assay • No posive LAMP reacon on gel electrophoresis (Figure 4 A) or HNB visualisaon (Figure 4 B) was observed on other plant pathogenic fungi Fusarium acuminatum, Fusarium udum, Fusarium solani, Rhizoctonia batacola, Alternaria alternata and Phytophthora cajani) indicang the specificity of the LAMP assay for Foc. • The detecon limit of this LAMP assay for Foc was 0.1 fg μL −1 of genomic DNA per reacon, while that of convenonal PCR was pg μL −1 (Figure 5).
-
Upload
icrisat -
Category
Government & Nonprofit
-
view
74 -
download
2
Transcript of Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Fusarium...
Results
Sep 2014
Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Fusarium oxysporum f. sp. ciceris – wilt pathogen of chickpea
This work is undertaken
as part of
R Ghosh, A Nagavardhini and M Sharma
IntroductionRapid detection of pathogens in chickpea has become a pre-requisite to facilitate accurate disease diagnosis and surveillance for better management strategy. Among the diseases affecting chickpea, Fusarium wilt [Fusarium oxysporum f. sp. ciceris (Foc)] can cause 100% yield losses in susceptible cultivars. In this study a method based on the Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Foc was developed and sensitivity and specificity of the assay was evaluated.
Objectives• Development of LAMP assay for detection of Fusarium oxysporum f. sp. ciceris infecting chickpea.• Evaluation of sensitivity and specificing of LAMP assay.
Materials and methods
Figure 1. Schematic representation of position and sequence of primer sets.
Figure 2. LAMP detection using HNB as a visual indicator (A) and agarose gel electrophoresis (B).
Figure 3. Optimization of LAMP reaction time and temperature.
Figure 4. Specificity of the LAMP assay for Foc detection.
Figure 5. Comparative sensitivity of LAMP and conventional PCR analysis for detection of Foc.
ConclusionThis is the first report of the use of LAMP assay for the detection of Foc. It is a promising assay for extensive application and rapid diagnosis of Foc infection in the laboratory and will be very useful for monitoring the disease complex and for suggesting further management strategies in chickpea.
Acknowledgements: The funding support by Department of Science and Technology and Department of Biotechnology, GoI to conduct this study is acknowledged.
DST & DBT
• LAMP primers based on the EF-1alpha gene were designed using the LAMP designer software program (Figure 1).
• The Foc specific LAMP reaction was standardized for optimum temperature and time.
• LAMP specificity was determined by performing the assay with DNA of Foc and of six other plant-pathogenic fungi (Fusarium acuminatum, Fusarium udum, Fusarium solani, Rhizoctonia bataticola, Alternaria alternata and Phytophthora cajani).
• Total 50 isolates of Foc collected from diverse geographical locations and representing Foc races in India were subjected to LAMP and conventional Polymerase Chain Reaction (PCR).
The LAMP assay• A simple colorimetric assay using a metal ion
indicator, namely hydroxynaphthol blue (HNB) was used for detection of Foc using LAMP.
• A positive LAMP reaction was indicated by a sky blue color; the color remained violet for negative reactions (Figure 2 A).
• Characteristic ladder like bands were evident in the agarose gel electrophoresis if the product was present (Figure 2 B).
• Positive results were obtained at 60 min (Figure 3, A & B) and 63°C (Figure 3, C & D) both in HNB and conventional PCR.
Evaluation of LAMP assay• All the 50 Foc isolates representing diverse
geographical locations in India showed positive reaction as indicated by a sky blue color. The results were consistent with PCR method.
Specificity and sensitivity of the LAMP assay• No positive LAMP reaction on gel electrophoresis
(Figure 4 A) or HNB visualisation (Figure 4 B) was observed on other plant pathogenic fungi Fusarium acuminatum, Fusarium udum, Fusarium solani, Rhizoctonia bataticola, Alternaria alternata and Phytophthora cajani) indicating the specificity of the LAMP assay for Foc.
• The detection limit of this LAMP assay for Foc was 0.1 fg μL−1 of genomic DNA per reaction, while that of conventional PCR was pg μL−1 (Figure 5).
