Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR

Abstract

We have identified a novel 529 bp fragment that is repeated 200- to 300-fold in the genome of Toxoplasma gondii. This 529 bp fragment was utilised for the development of a very sensitive and specific PCR for diagnostic purposes, and a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice. The 529 bp fragment was found in all 60 strains of T. gondii tested, and it discriminates DNA of T. gondii from that of other parasites. Toxoplasma gondii DNA was detected in amniotic fluid of patients, as well as in various tissues from infected mice. Polymerase chain reaction with the 529 bp fragment was more sensitive than with the 35-copy B1 gene. For the quantitative competitive-PCR, a 410-bp competitor molecule was co-amplified with similar efficiency as the 529 bp fragment. Quantitative competitive-PCR produced a linear relationship between the relative amounts of PCR product and the number of tachyzoites in the range of 10 2–10 4 tachyzoites and 100–3000 tissue cysts. A highly significant correlation between visual counting of brain cysts and quantitative competitive-PCR was obtained in mice chronically infected with Toxoplasma. Thus, quantitative competitive-PCR with the 529 bp fragment can be used as an alternative for the tedious visual counting of brain cysts in experimental animals. With the quantitative competitive-PCR, furthermore, we could confirm the copy number of the 529 bp fragment in tachyzoites and estimate the number of bradyzoites per cyst.