Comparison of DNA isolation methods in detection of 16SrV and 16SrXII-A ribosomal group phytoplasmas from grapevine (Vitis vinifera L.)

Haluška, Ivana (2010) Comparison of DNA isolation methods in detection of 16SrV and 16SrXII-A ribosomal group phytoplasmas from grapevine (Vitis vinifera L.). Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

Phytoplasmas (Candidatus 'Phytoplasma') are wall-less bacteria from the class Mollicutes. Their life cycle involves replication in plants and insects. They cause several hundred diseases in various plants, including important agricultural species. Since phytoplasmas cannot be cultivated in vitro, their classification is problematic, but with development of molecular methods, such as nested and real-time polymerase chain reaction (PCR), phytoplasma detection and identification was made possible. Total genomic DNA can be isolated from the plant by various methods.The aim of this thesis was to compare sensitivity of phytoplasma detection by nested and real-time PCR, depending on DNA isolation method. Total nucleic acids of 6 phytoplasma infected grapevine (Vitis vinifera L.) samples were isolated by short CTAB method for TNA isolation, and by commercial kits OmniPrep™ for Plant and DNeasy Plant Mini Kit. Presence of phytoplasmas was detected by nested PCR amplifying 16S rRNA gene and real-time PCR amplifying map gene. Different concentrations of DNA template were used in order to compare the sensitivity of detection. In 5 of the analyzed samples presence of phytoplasmas belonging to 16SrXII-A ribosomal group was detected, while in one sample we detected 16SrV group phytoplasma. Comparison of the results indicated that DNA isolation using short CTAB method gave the highest sensitivity of phytoplasma detection in most of the analyzed samples by both nested and real-time PCR.

Item Type: Thesis (Diploma thesis)
Keywords: phytoplasmas, DNA isolation, PCR, real-time PCR
Supervisor: Šeruga Musić, Martina
Date: 2010
Number of Pages: 57
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Silvana Šehić
Date Deposited: 03 Sep 2014 12:47
Last Modified: 03 Sep 2014 12:47
URI: http://digre.pmf.unizg.hr/id/eprint/2617

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