Date Published: June 6, 2016
Publisher: Springer Berlin Heidelberg
Author(s): S. Umesha, H. M. Manukumar, Sri Raghava.
Food contaminated with fungal pathogens can cause extremely harmful effects to human even when present in low concentrations. Researchers now pay more attention towards rapid DNA extraction for the quick screening, which is highly demanded in diverse research field. Molecular description of many fungal species is identified by different molecular characteristics. Hence, the efficient isolation of genomic DNA and amplification using PCR is a prerequisite for molecular characterization. Here, we used an improved Sodium dodecyl sulfate-Cetyltrimethyl ammonium bromide-Chloroform-isoamyl alcohol method by combining Sodium dodecyl sulfate with cetyl methylammonium bromide without addition of proteinase K, RNase K, and β-mercaptoethanol. To analyze the quality of recovered DNA, this method was compared with the other four routine methods. The present method has been chosen in the study as a preferred method because of easy adaptation to routine laboratories/food industries considering its rapid, sensitivit,y and cost effectiveness involved in the method.
In under-developed countries, one of the leading causes of illness and death is due to food-borne pathogens, which accounts approximately up to 1.8 million people annually (Bisha and Brehm-Stecher 2010). To ensure food safety, rapid detection of pathogenic organisms causing food-borne illness is a basic requirement. Plating methods have been replaced by more rapid and sensitive methods, such as Fluorescence In-Situ Hybridization (FISH) (Chattopadhyay et al. 2013), Enzyme Linked Immuno-Sorbent Assays (ELISA) (Naravaneni and Jamil 2005), Polymerase Chain Reaction (PCR) (Jaykus 2003), and Real-Time PCR (RT-PCR) (Wolffs et al. 2004). However, the prerequisite for all these methods is a high-quality DNA from the pathogen. Various procedures are being used in these contests, but these protocols are mainly suited for specific groups with known morphologies and not for versatile fungal groups. Therefore, DNA extraction is a very critical step, as it eliminates unwanted interfere substances and ensures consistency in the nucleic acid test results (Bolano et al. 2001).
Due to the presence of cell wall in fungi, it interferes and hinders the efficiency of DNA extraction from the conventional extraction methods (Maaroufi et al. 2004). After several repetitions, we have optimized rapid and inexpensive method to isolate of fungal DNA by slight modifications in the existing CTAB buffer constitution and steps involved in DNA extraction (Rogers 1989). The yield and purity of genomic DNA obtained from all four extraction methods are depicted in Table 1. Different species of microorganisms having its own varied membrane structural organization with unique sets of protein to carry out the specialized functions (Arachea et al. 2012). In the present study, four different detergents-based protocol for disruption of membrane structure and removal of proteins (irreversibly from the cell) have been compared. The high DNA yield was obtained in S-CCI protocol (645.45 μg/g sample for Trichoderma) followed by CTABPCI, TPCI, MW, and IM in descending order was represented in Table 1. It is evident from the data that protocol IM recovered very less yield compared to the other methods. In Fig. 1, the highest yield of DNA extracted by different methods was depicted.Table 1Genomic DNA yield from different fungal pathogens using different extraction methodsSl. noMethodPathogensYield (μg DNA/g sample)Purity (A260/A280)1CTABPCIAspergillus niger301.251.90Aspergillus flavus246.951.67Bipolaris cyanodontis210.521.92Fusarium oxysporum239.572.01Penicillium250.452.10Trichoderma280.001.96Fusarium equiseti219.951.87Acremonium strictum195.302.00Colletotrichum gloeosporioides236.201.98Aspergillus fumigatus210.301.652TPCIAspergillus niger108.851.75Aspergillus flavus33.971.94Bipolaris cyanodontis158.622.01Fusarium oxysporum97.271.84Penicillium151.001.99Trichoderma200.452.10Fusarium equiseti51.972.21Acremonium strictum167.522.10Colletotrichum gloeosporioides172.131.69Aspergillus fumigatus171.851.853MWAspergillus niger34.671.40Aspergillus flavus189.052.10Bipolaris cyanodontis110.251.78Fusarium oxysporum70.321.75Penicillium244.501.98Trichoderma189.672.10Fusarium equiseti185.671.89Acremonium strictum46.751.78Colletotrichum gloeosporioides105.521.70Aspergillus fumigatus197.391.504IMAspergillus niger113.211.84Aspergillus flavus211.321.98Bipolaris cyanodontis78.052.20Fusarium oxysporum127.602.01Penicillium184.721.45Trichoderma220.421.79Fusarium equiseti152.601.99Acremonium strictum67.752.00Colletotrichum gloeosporioides211.001.89Aspergillus fumigatus235.471.985S-CCIAspergillus niger306.471.99Aspergillus flavus596.151.93Bipolaris cyanodontis314.802.01Fusarium oxysporum390.022.00Penicillium359.471.70Trichoderma645.451.83Fusarium equiseti354.922.01Acremonium strictum253.531.72Colletotrichum gloeosporioides305.071.69Aspergillus fumigatus249.361.78Different fungal cultures were used for extraction of DNA by CTABPCI, TPCI, MW, and IM compared to modified S-CCI method in this study. Yield and purity of established S-CCI method were compared to other methods. Each values of yield and purity mentioned are average of triplicate assay carried out for all the fungal pathogens usedFig. 1Yield of DNA in different DNA extraction methods. Different fungal genomic DNA was prepared by CTABPCI, TPCI, MW, IM, and modified S—CCI methods. Highest DNA yield was obtained by the S-CCI method from Aspergillus niger, Trichoderma, Penicillium, and Aspergillus fumigates compared to the other methods of DNA extraction. Among different DNA extraction methods, the S-CCI method showed maximum yield of DNA