A rapid and easy method for the DNA extraction from Cryptococcus neoformans

Nucleic acid detection methods such as PCR have become a common tool for Cryptococcus neoformans species complex identification and diagnosis. Although PCR amplification can be performed directly from cultures, prior isolation of DNA is often preferred [1,2].As the DNA extraction process eliminates many unknown interfering substances in the biological material, it plays an important role in ensuring consistent test results. DNA isolation from C. neoformans is difficult due to a thick and resistant capsule that is not readily susceptible to lyses.Therefore, efficiency in the DNA extraction method using phenol, chloroform and isoamylic alcohol requires time and toxic solution manipulation, due to the organic solvents that may be hazardous to the environment and to the technician, and also several washing and centrifugation steps increasing the risk of sample contamination [3]. Several methods have been proposed as an alternative to the use of phenol and chloroform, such as commercial kits for DNA extraction. The use of kits offers a low risk of manipulation and they are faster than conventional protocols, but the amount of DNA recovered from the commercial kits is highly variable [4]The objective of the present study was to compare four DNA extraction protocols from culture of collected strains from 2005-2009. This article summarizes the results of a comparison of the techniques in regards to good amplification and purity of obtained DNA.A total of 150 Tunisian Cryptococcus isolates from clinical and environmental strains and the following standard strains representing each serotype of C. neoformans H99 (serotype A), JEC 21 (serotype D), the hybrid IHEM 13877 (serotype AD), C. gattii Wm 276 (serotype B) and IHEM 4159 (serotype C) were used.Total genomic DNA was extracted from culture (108cells/ml) of Cryptococcus strains by means of the following 4 procedures: Protocol A used extraction with lyticase, phenol-chloroform and isoamylic alcohol; Protocol B used extraction