DNA extraction and PCR assays for detection of Toxoplasma gondii.
For detection of Toxoplasma gondii we compared the sensitivity of two different DNA extraction methods and three different PCR assays. Sensitivities of DNA extraction by QIAamp DNA mini Kit or MagNa pure followed by PCR, nested PCR and oligochromatography or Light Cycler PCR using either SYBR green chemistry or TaqMan probe were compared. No significant difference between extraction methods was found using pure T. gondii tachyzoites. Spiked blood samples, 10(4) to 10 parasites per sample, generated no difference in sensitivity between the two DNA extraction methods when analysed by nested PCR detected by oligochromatography or analysed by Light Cycler PCR TaqMan. In spiked blood samples Light Cycler PCR SYBR green was unable to detect the parasite and a reduction in sensitivity was observed with the TaqMan assay. Conventional PCR was more sensitive when DNA was extracted from the spiked samples using the QIAamp DNA mini Kit. Conventional and nested PCR were found to be more sensitive than Light Cycler PCR TaqMan using the QIAamp DNA mini Kit. It was not possible to use Light Cycler PCR SYBR green in blood samples. Conventional PCR was more sensitive for detection of T. gondii in spiked blood samples using QIAamp DNA mini Kit DNA extraction, suggesting that the choice of DNA extraction method may affect PCR assays differently.
Edvinsson
Benjamin
B
Department Laboratory Medicine, F82, Karolinska Institute, Huddinge University hospital, Stockholm, Sweden. benjamin.edvinsson@labmed.ki.se
Jalal
Shah
S
Nord
Carl Erik
CE
Pedersen
Babill Stray
BS
Evengård
Birgitta
B
D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D017125:Genes, Protozoan; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics* / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis / Q000469:parasitology
D016054:DNA, Protozoan