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Journal of Clinical Microbiology, November 2000, p. 4121-4125, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Real-Time PCR for Quantitative Detection of Toxoplasma gondii

Mei-Hui Lin,1 Tse-Ching Chen,2 Tseng-tong Kuo,2 Ching-Chung Tseng,3 and Ching-Ping Tseng1,*

School of Medical Technology, Chang Gung University,1 and Department of Pathology, Chang Gung Memorial Hospital,2 Tao-Yuan 333, Taiwan, Republic of China, and Divisions of Basic Sciences and of Restorative and Prosthodontic Sciences, College of Dentistry, New York University, New York, New York 10010-40863

Received 22 June 2000/Returned for modification 27 July 2000/Accepted 8 August 2000

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 (CT) indicative of the quantity of the target gene were determined. Typically, a CT 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 CT 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.

* Corresponding author. Mailing address: School of Medical Technology, Chang Gung University, 259 Wen-Hwa 1st Rd., Kwei-Shan, Tao-Yuan, 333 Taiwan. Phone: 011-886-3-328-3016, ext. 5202. Fax: 011-886-3-328-9355. E-mail: ctseng{at}mail.cgu.edu.tw.

Journal of Clinical Microbiology, November 2000, p. 4121-4125, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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