A highly sensitive molecular diagnosis method for detecting Toxoplasma gondii tachyzoite: a PCR/dot blot hybridization
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This study aimed at finding a fast, sensitive, and efficient protocol for molecular identification of intra-cellular protozoa Toxoplasma (T.) gondii. For molecular detection of T. gondii, we developed a poly-merase chain reaction coupled with dot blot hybridization assay (PCR/DBH). For DBH analysis, the am-plified DNA of T. gondii tachyzoite was labeled by incorporation of digoxigenin. The DBH assay alone was capable of detecting down to 1×104 pg of T. gondii genomic DNA. The PCR alone was capable of detecting down to 1×103 pg of T. gondii genomic DNA, whereas the PCR/DBH assay was capable of detecting down to 1×102 pg of T. gondii genomic DNA, indicating that sensitivity of the PCR/DBH method was approximately 10 to 100 times higher than PCR or DBH alone. Our PCR/DBH assay will be useful for confirming the presence of T. gondii on the samples and differentiating T. gondii infection from other intracellular protozoa infections.
Abstract
This study aimed at finding a fast, sensitive, and efficient protocol for molecular identification of intra-cellular protozoa Toxoplasma (T.) gondii. For molecular detection of T. gondii, we developed a poly-merase chain reaction coupled with dot blot hybridization assay (PCR/DBH). For DBH analysis, the am-plified DNA of T. gondii tachyzoite was labeled by incorporation of digoxigenin. The DBH assay alone was capable of detecting down to 1×104 pg of T. gondii genomic DNA. The PCR alone was capable of detecting down to 1×103 pg of T. gondii genomic DNA, whereas the PCR/DBH assay was capable of detecting down to 1×102 pg of T. gondii genomic DNA, indicating that sensitivity of the PCR/DBH method was approximately 10 to 100 times higher than PCR or DBH alone. Our PCR/DBH assay will be useful for confirming the presence of T. gondii on the samples and differentiating T. gondii infection from other intracellular protozoa infections.
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