title: Evaluation of a PCR probe capture assay for the detection of Toxoplasma gondii. Incorporation of uracil N-glycosylase for contamination control.
abstract: Toxoplasma gondii is a cyst-forming parasite of clinical relevance in humans primarily because of the neurologic abnormalities it can cause. In some clinical circumstances, it is desirable to detect the pathogen directly. We modified a commercially available Toxoplasma polymerase chain reaction (PCR) probe capture assay by incorporating uracil N-glycosylase (UNG) to prevent carryover amplicon contamination. In addition, UNG inactivation and DNA denaturation were accomplished chemically to simplify the DNA hybridization to the capture probe. The incorporation of UNG effectively eliminated carryover contamination; the probe capture assay showed a log increase in detection sensitivity compared with standard agarose gel electrophoresis. To assess sensitivity and possible inhibition of amplification, different sample types were spiked with Toxoplasma organisms. After DNA extraction and PCR amplification, a sensitivity of 2 tachyzoites for the assay was determined in buffered saline, cerebrospinal fluid (CSF), serum, and amniotic fluid; 20 tachyzoites for whole blood; and 200 tachyzoites for brain tissue. An additional 20 human serum and CSF samples submitted for Toxoplasma serologic testing were run by the PCR method. Of these, only an IgM-positive CSF sample was PCR positive. The Toxoplasma PCR probe capture assay showed good sensitivity and was not substantially inhibited by several different clinically relevant samples.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA.
authors.forename: T B
authors.initials: TB
authors.lastname: Martins
authors.forename: D R
authors.initials: DR
authors.lastname: Hillyard
authors.forename: C M
authors.initials: CM
authors.lastname: Litwin
authors.forename: E W
authors.initials: EW
authors.lastname: Taggart
authors.forename: T D
authors.initials: TD
authors.lastname: Jaskowski
authors.forename: H R
authors.initials: HR
authors.lastname: Hill
mesh_terms: D000653:Amniotic Fluid / Q000469:parasitology; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood / Q000134:cerebrospinal fluid; D001769:Blood / Q000469:parasitology; D001921:Brain / Q000469:parasitology; D002555:Cerebrospinal Fluid / Q000469:parasitology; D045647:DNA Glycosylases; D016054:DNA, Protozoan / Q000032:analysis; D005312:Fetal Blood / Q000469:parasitology; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000134:cerebrospinal fluid; D007075:Immunoglobulin M / Q000097:blood / Q000134:cerebrospinal fluid; D009699:N-Glycosyl Hydrolases; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics* / Q000302:isolation & purification*; D051981:Uracil-DNA Glycosidase
chemical_list: D000913:Antibodies, Protozoan; D016054:DNA, Protozoan; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase