Comparison of five DNA extraction methods and optimization of a b1 gene nested PCR (nPCR) for detection of Toxoplasma gondii tissue cyst in mouse brain.
Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide. Different techniques have been developed for T. gondii detection. At present, polymerase chain reaction (PCR) has been widely used. However, PCR for identifying T. gondii remains unsatisfactory in many laboratories because of lack of standardization and variations in efficiency. In the present study, we optimized a nested PCR protocol (n-PCR) in order to compare the amplification of T. gondii DNA, after being extracted from mouse brain by five different DNA extraction methods including phenol chloroform, QIAamp DNA minikit, Genomic DNA purification kit and Chelex with or without proteinase K. All DNA extraction methods were able to extract DNA from a single tissue cyst from mouse brain. However, among the five DNA extraction methods, the Chelex without proteinase K appeared to be the most rapid and easiest.
Mahittikorn
Aongart
A
Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
Wickert
Hannes
H
Sukthana
Yaowalark
Y
D000818:Animals; D000953:Antigens, Protozoan; D001921:Brain / Q000469:parasitology / Q000473:pathology*; D003560:Cysts / Q000469:parasitology / Q000473:pathology*; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis*; D005260:Female; D017125:Genes, Protozoan; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D014122:Toxoplasma / Q000235:genetics* / Q000302:isolation & purification; D014124:Toxoplasmosis, Animal / Q000469:parasitology / Q000473:pathology*
D000953:Antigens, Protozoan; D017931:DNA Primers; D016054:DNA, Protozoan