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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZING, DETECTING, AND ELIMINATING PATHOGENS FOR THE SAFE INTRODUCTION OF PLANT GENETIC RESOURCES Title: A Reliable and Inexpensive Method of Nucleic Acid Extraction for the PCR-Based Detection of Diverse Plant Pathogens

Authors
item Li, Ruhui
item Mock, Raymond
item Huang, Qi
item Abad, Jorge - APHIS
item Hartung, John
item Kinard, Gary

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 11, 2008
Publication Date: November 1, 2008
Citation: Li, R., Mock, R.G., Huang, Q., Abad, J., Hartung, J.S., Kinard, G.R. 2008. A Reliable and Inexpensive Method of Nucleic Acid Extraction for the PCR-Based Detection of Diverse Plant Pathogens. Journal of Virological Methods. 154:48-55.

Interpretive Summary: Many microorganisms including viruses, viroids, phytoplasma and bacteria can infect and cause diseases in crops, and the best strategy to control these pathogens is rapid identification and detection in quarantine, breeding programs, certification and production. Molecular techniques such as polymerase chain reaction (PCR)-based tests can be very fast and sensitive, and are now used widely to detect genetic materials (DNA or RNA) of these pathogens. A significant challenge in using these techniques, however, is the need to prepare plant samples or extracts containing these genetic materials but free of other compounds that occur in plant cells, such as proteins and sugars. These contaminants can interfere with the pathogen detection technique. Different methods have been described and commercial kits are available for sample preparation, but most of them are labor-intensive, time-consuming and costly or specific to only one type of genetic materials, and therefore, not suitable for testing numerous of samples for multiple pathogens in different crops. Here we report a modified method that works well for sample preparation for PCR-based detection of pathogens containing genetic materials of either DNA or RNA. It uses a mechanical device to grind samples, and therefore, allows large numbers of samples to be processed and reduces potential contamination between samples. The supplies and chemicals needed are inexpensive and readily available. The procedure performed as well as and, in some cases, better than the more expensive commercially available kits in preparation of DNA and RNA samples suitable for pathogen detection by the molecular methods mentioned above. This protocol has proven valuable in detecting a wide range of plant pathogens including viruses, viroids, phytoplasma and bacteria from diverse woody and herbaceous crops. This information will be useful to those who work in quarantine and certification programs that need to test valuable plant material for pathogens.

Technical Abstract: A reliable extraction method is described for the preparation of total nucleic acids from several plant genera for subsequent detection of plant pathogens by PCR-based techniques. By the combined use of a modified CTAB (cetyltrimethylammonium bromide) extraction protocol and a semi-automatic homogenizer (FastPrep® instrument), the method allows rapid sample processing and minimizes potential cross-contamination. The method was applied to sample preparation for PCR-based detection of different RNA and DNA viruses, viroids, phytoplasmas and bacterial pathogens from a wide range of infected host plants. The procedure is cost-effective and the qualities of the nucleic acid preparations are equal to or exceed those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of large numbers of samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.

Last Modified: 12/20/2014
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