Academic journal article Iranian Journal of Public Health

Rapid Identification of Alternaria Alternata Fungi,the Cause of Brown Spot Disease by Loop-Mediated Isothermal Amplification

Academic journal article Iranian Journal of Public Health

Rapid Identification of Alternaria Alternata Fungi,the Cause of Brown Spot Disease by Loop-Mediated Isothermal Amplification

Article excerpt

Background: Therefore, in this study rapid identification of A. alternata using Loop-Mediated Isothermal Amplification (LAMP), which is a precise and cost effective method, was developed.

Methods: Phytopathgenic A. alternata strains were isolated from infected citrus trees in Ramsar, Iran. Eight different strains were isolated in PDA and PCA media and were iden- tified as A. alternata using PCR technique and ITS primer. A specific pair of LAMP primers was designed using ACT- toxin gene using PRIMER EXPLORER V4 software. Subse- quently, LAMP reaction was performed with Bst DNA pol- ymerase at constant temperature 60, 65 and 70 °C for 60 min. LAMP reaction was done on all isolated A. alternata in addi- tion of 15 DNA extracted from standard fungi and bacteria, with an exception of A. alternata as a negative control and 5 DNA extracts samples from soil. Fluorescent dye SYBR Green and turbidimetry were employed to detect positive samples.

Results: According to the carried out reactions, all pathogen- ic isolated A. alternata and standard control showed green dye and turbidity caused by amplification of ACT-toxin gene in 60, 65 and 70 °C. …

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