We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the BI gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the BI gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (105/ml) in 1 mi of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The BJ gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the BJ gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

The complete sequence of P30, the major surface Ag of the protozoan parasite, Toxoplasma gondii, has been deduced through the cloning and analysis of its gene. Using polyclonal serum specific for P30, we have isolated a P30 cDNA clone from a lambda gt11 cDNA expression library derived from tachyzoites of T. gondii (RH strain). This clone produces a beta-galactosidase fusion protein which reacts with several anti-P30 mAb. In addition, polyclonal anti-serum raised to the fusion protein reacts with purified P30 protein and exclusively with P30 in a whole cell lysate of T. gondii. This cDNA clone was used to isolate near full-length cDNA molecules and a cosmid clone containing the P30 gene. Sequence analysis of the cDNA reveals a single open reading frame with coding capacity for 34.7 kDa of primary translation product (consistent with the apparent Mr of P30 on SDS-acrylamide gels) including a presumptive hydrophobic signal sequence. The P30 primary translation product also has a carboxy-terminal hydrophobic tail which is predictive of a posttranslational cleavage and modification with a glycolipid anchor. We have identified the apparent 5' and 3' ends of the P30 mRNA transcript which is extremely abundant, 1500 nucleotides in length, and polyadenylated. The P30 gene is single copy and contains no introns.

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