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Paper Information

Journal:   IRANIAN JOURNAL OF PUBLIC HEALTH   AUGUST 2014 , Volume 43 , Number SUPPLEMENT 2; Page(s) 51 To 51.
 
Paper: 

RAPID IDENTIFICATION OF ALTERNARIA ALTERNATA FUNGI, THE CAUSE OF BROWN SPOT DISEASE BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION

 
 
Author(s):  MORADI AMIR*, MOGHIMI HAMID, HAMEDI JAVAD
 
* DEPARTMENT OF MICROBIAL BIOTECHNOLOGY, SCHOOL OF BIOLOGY AND CENTER OF EXCELLENCE IN PHYLOGENY OF LIVING ORGANISMS, COLLEGE OF SCIENCE, UNIVERSITY OF TEHRAN, TEHRAN, IRAN
 
Abstract: 

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 identified as A. alternata using PCR technique and ITS primer. A specific pair of LAMP primers was designed using ACTtoxin gene using PRIMER EXPLORER V4 software. Subsequently, LAMP reaction was performed with Bst DNA polymerase at constant temperature 60, 65 and 70oC for 60 min. LAMP reaction was done on all isolated A. alternata in addition 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 pathogenic isolated A. alternata and standard control showed green dye and turbidity caused by amplification of ACT-toxin gene in 60, 65 and 70oC. No amplification was detected in 75oC. In contrast, none of standard fungi DNA samples showed positive result in this reaction. Moreover, positive and negative results were observed for 2 and 3 soil samples, respectively. The obtained results showed that the LAMP designed primers for ACT-toxin gene as well as the introduced approach can be applied to detect the Phytopathgenic A. alternata with high precision in the short time.
Conclusion: Results presented in this study showed that ACT-toxin gene is highly specific and designed LAMP primers can be used in molecular diagnosis of A. alternata in DNA sample, which extracted from fungi and even from soil.

 
Keyword(s): MOLECULAR IDENTIFICATION, BROWN SPOT DISEASE, ALTERNARIA ALTERNATA
 
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