In:
Journal of Clinical Microbiology, American Society for Microbiology, Vol. 38, No. 11 ( 2000-11), p. 4121-4125
Abstract:
The protozoan Toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of T. gondii . Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the T. gondii B1 gene. After 40 PCR cycles, the cycle threshold values (C T ) indicative of the quantity of the target gene were determined. Typically, a C T of 25.09 was obtained with DNA from 500 tachyzoites of the T. gondii RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05 T. gondii tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a C T of 〈 40 and were scored as positive for this test. These results were consistent with those obtained through our nested-PCR control experiments. We have developed a rapid, sensitive, and quantitative real-time PCR for detection of T. gondii . The advantages of this technique for the diagnosis of toxoplasmosis in a clinical laboratory are discussed.
Type of Medium:
Online Resource
ISSN:
0095-1137
,
1098-660X
DOI:
10.1128/JCM.38.11.4121-4125.2000
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2000
detail.hit.zdb_id:
1498353-9
SSG:
12
Bookmarklink