31Jan 2017

MICROBIAL DNA EXTRACTION FROM INFECTED SEED POTATO FOR MOLECULAR DIAGNOSTIC APPLICATIONS

  • Department of Biotechnology, Faculty of Agriculture & Plantation Management,Wayamba University of Sri Lanka.
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Transmission of tuber borne diseases is a major cause of yield reduction in potato cultivation. Molecular level diagnosis of the microbial pathogens is a challenge to obtain a sufficient quantity of DNA from infected potato tubers. Therefore, this study focused to find a simple protocol to extract microbial DNA from the infected seed potato. Three different DNA extraction protocols were performed and the extracted DNA was tested for the quantity and quality of DNA using agarose gel electrophoresis. Next Generation Sequencing of 16S rDNA and ITS1 region based amplification was used for the detection of bacterial and fungal pathogens in infected samples using Ion Torrent sequencing platform.Both the enrichment and the culture independent boiling methods of extraction yielded sufficient amount of total genomic DNA from the infected tuber samples.In each method, DNA yield varied depending on the type and severity of infection.The extracted DNA matched with the stringent quality control standards for sequencing on Ion Torrent PGM and amplified both 16S rDNA and ITS1 regions with clear distinctive and reproducible banding patterns in agarose gels.Both bacterial and fungal pathogens were detected with these modified extraction protocols. However, enrichment method was found to be suitable in detecting pathogens even in latent infections. In conclusion, these DNA extraction protocols provided simple, inexpensive and effective methodology in detecting microbial diversity in infected potato tubers.

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[R. W. P. M. Rajapaksha and K. Vivehananthan. (2017); MICROBIAL DNA EXTRACTION FROM INFECTED SEED POTATO FOR MOLECULAR DIAGNOSTIC APPLICATIONS Int. J. of Adv. Res. 5 (Jan). 2605-2611] (ISSN 2320-5407). www.journalijar.com

R.W.P.M. RAJAPAKSHA
WAYAMBA UNIVERSITY OF SRI LANKA

DOI:

Article DOI: 10.21474/IJAR01/3050      
DOI URL: http://dx.doi.org/10.21474/IJAR01/3050

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