Direct detection and genotyping of Toxoplasma gondii in meat samples using magnetic capture and PCR.
Different transmission routes, including the ingestion of undercooked meat, can result in Toxoplasma gondii infection in humans. The development of effective prevention strategies is hampered by a lack of quantitative information on the contamination level of different types of meat. Therefore, we developed a method for detection and quantification of T. gondii. The method involved preparation of crude DNA extract from hundred gram samples of meat, magnetic capture of T. gondii DNA and, quantitative real-time PCR targeting the T. gondii 529-bp repeat element. The detection limit of this assay was approximately 230 tachyzoites per 100 g of meat sample. There was a linear relation between the number of parasites added to the samples and Cp-values. Results obtained with the PCR method were comparable to bioassay results for experimentally infected pigs, and to serological findings for sheep. In addition, the T. gondii in 50% of the positive sheep samples could be genotyped by sequencing of the GRA6 gene, after isolation of the gene by magnetic capture. Two subtypes of GRA6 type II were identified in the 16 samples from sheep. For seven samples, the identification of T. gondii as type II was confirmed by microsatellite typing. The PCR method can be used as an alternative to bioassay for detection and genotyping of T. gondii, and to quantify the organism in meat samples of various sources.
Opsteegh
Marieke
M
National Institute for Public Health and the Environment, Bilthoven, The Netherlands. marieke.opsteegh@rivm.nl
Langelaar
Merel
M
Sprong
Hein
H
den Hartog
Laurien
L
De Craeye
Stéphane
S
Bokken
Gertie
G
Ajzenberg
Daniel
D
Kijlstra
Aize
A
van der Giessen
Joke
J
D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis; D005506:Food Contamination / Q000032:analysis; D016266:Food Parasitology; D005838:Genotype; D057230:Limit of Detection; D008280:Magnetics / Q000379:methods; D008460:Meat / Q000469:parasitology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012756:Sheep; D013552:Swine; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*
D016054:DNA, Protozoan