Efficiency Comparison of Three Methods for Extracting
Genomic DNA of the Pathogenic Oomycete
Pythium insidiosum
Tassanee Lohnoo MSc*, Nujarin Jongruja PhD*, Thidarat Rujirawat MSc*,
Wanta Yingyon*, Tassanee Lerksuthirat MSc****, Umporn Nampoon BSc*,
Yothin Kumsang MSc**, Pornpit Onpaew MSc***, Piriyaporn Chongtrakool PhD***,
Angsana Keeratijarut MSc****, Tristan T Brandhorst PhD*****, Theerapong Krajaejun MD***
Affiliation :
* Research Center, Ramathibodi Hospital, Bangkok, Thailand
** Department of Radiology, Ramathibodi Hospital, Bangkok, Thailand
*** Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok, Thailand
**** Multidiscipinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand
***** Department of Pediatrics, University of Wisconsin Medical School, University of Wisconsin-Madison, Wisconsin, USA
Background : The fungus-like organism Pythium insidiosum is the causative agent of a life-threatening tropical infectious
disease, pythiosis, which has high rates of morbidity and mortality. A lack of reliable diagnostic tools and effective treatments
for pythiosis presents a major challenge to healthcare professionals. Unfortunately, surgical removal of infected organs
remains the default treatment for pythiosis. P. insidiosum is an understudied organism. In-depth study of the pathogen at
the molecular level could lead to better means of infection control. High quality genomic DNA (gDNA) is needed for
molecular biology-based research and application development, such as: PCR-assisted diagnosis, population studies,
phylogenetic analysis, and molecular genetics assays.
Objective : To evaluate quality and quantity of the P. insidiosum gDNA extracted by three separate protocols intended for
fungal gDNA preparation.
Material and Method: Seven P. insidiosum isolates were subjected to gDNA extraction by using conventional-extraction,
rapid-extraction, and salt-extraction protocols.
Results : The conventional protocol offered the best gDNA in terms of quality and quantity, and could be scaled up. The
rapid-extraction protocol had a short turnaround time, but the quality and quantity of the gDNA obtained were limited. The
salt-extraction protocol was simple, rapid, and efficient, making it appealing for high throughput preparation of small-scale
gDNA samples.
Conclusion : Compared to rapid-extraction protocol, both conventional-extraction and salt-extraction protocols provided
a better quality and quantity of gDNA, suitable for molecular studies of P. insidiosum. In contrast to the other two methods,
the salt-extraction protocol does not require the use of hazardous and expensive materials such as phenol, chloroform, or
liquid nitrogen.
Keywords : DNA extraction, Genomic DNA, Oomycete, Pythiosis, Pythium insidiosum
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