title: Development of Loop-Mediated Isothermal Amplification (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris - wilt pathogen of chickpea.
abstract: BACKGROUND
  Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt is a devastating pathogen of chickpea. In chickpea, various soil borne pathogens produce (s) similar symptoms, therefore cannot be distinguished easily at field level. There is real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks.
  
  
  RESULTS
  In this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeting the elongation factor 1 alpha gene sequence for visual detection of Foc. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue (HNB) was added before amplification, samples with Foc DNA developed a characteristic sky blue colour but those without DNA or with the DNA of six other plant pathogenic fungi did not. Results obtained with LAMP and HNB were confirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for Foc was 10 fg of genomic DNA per reaction, while that of conventional PCR was 100 pg.
  
  
  CONCLUSIONS
  In conclusion, it was found that a LAMP assay combined with HNB is simple, rapid, sensitive, and specific. The LAMP assay does not require specialized equipment, hence can be used in the field for the rapid detection of Foc. This is the first report of the use of LAMP assay for the detection of Foc. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of Foc, with the potential to be standardized as a detection method for Foc in endemic areas and will be very useful for monitoring the disease complex in the field further suggesting the management strategies.
authors.affiliation: International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, PO 502324, Andhra Pradesh, India. r.ghosh@cgiar.org.
authors.forename: Raju
authors.initials: R
authors.lastname: Ghosh
authors.affiliation: International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, PO 502324, Andhra Pradesh, India. a.nagavardhini@cgiar.org.
authors.forename: Avuthu
authors.initials: A
authors.lastname: Nagavardhini
authors.affiliation: International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, PO 502324, Andhra Pradesh, India. a.sengupta@cgiar.org.
authors.forename: Anindita
authors.initials: A
authors.lastname: Sengupta
authors.affiliation: International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, PO 502324, Andhra Pradesh, India. mamta.sharma@cgiar.org.
authors.forename: Mamta
authors.initials: M
authors.lastname: Sharma
mesh_terms: D001483:Base Sequence; D029863:Cicer / Q000382:microbiology; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry*; D004271:DNA, Fungal / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D005670:Fusarium / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005838:Genotype; D057230:Limit of Detection; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020648:Peptide Elongation Factor 1 / Q000235:genetics*; D010935:Plant Diseases / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D009282:Naphthalenesulfonates; D020648:Peptide Elongation Factor 1; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate