An Optimized Protocol for DNA Extraction from Wheat Seeds and Loop-Mediated Isothermal Amplification (LAMP) to Detect Fusarium graminearum Contamination of Wheat Grain

International Journal of Molecular Sciences

Kamel Abd-Elsalam¹  Ali Bahkali¹  Mohamed Moslem¹  Osama E. Amin¹ 

[1] King Saud University, College of Science, Botany and Microbiology Department, P.O. Box 2455, Riyadh 1145, Saudi Arabia; E-Mails:

关键词: DNA isolation;    loop-mediated isothermal amplification;    LAMP;    PCR;    seed-borne fungi;   

DOI  :  10.3390/ijms12063459

来源: mdpi

【 摘 要 】

A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In combination with the LAMP assay it is a fast and cost-effective alternative to traditional diagnostic methods for the early detection of toxigenic fusaria in cereals.

【 授权许可】

CC BY   
© 2011 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.