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Keywords:

  • Diagnosis;
  • PCR;
  • Toxoplasma gondii

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References

DNA was extracted with a modified Qiagen DNA Mini Kit method from 20 clinical samples and was amplified by PCR using specific primers for the T. gondii B1 gene. T. gondii was detected correctly in 18 of the 20 clinical samples in < 5 h, with a detection limit of two parasites/sample. The results were in good agreement with those obtained by a more complicated and time-consuming procedure involving two-step nested PCR and either liquid hybridisation or colorimetric detection using internal probes.

The diagnosis of toxoplasmosis is based mainly on the isolation and cultivation of parasite cells, or on detection by serology, but these methods are rather insensitive and time-consuming. Nested PCR and PCR-ELISA have been used to complement serodiagnosis [1–4], but although these approaches are comparatively sensitive, they are still complicated and time-consuming [5–7]. In the present report, a simple PCR method to detect Toxoplasma gondii-specific DNA directly from clinical samples is described. The method can be applied to amniotic fluid, vitreous fluid, cerebrospinal fluid, blood samples and tissue specimens.

The B1 gene of T. gondii is a 35-fold repetitive gene, with 2214 nucleotides in each repeat, and is highly conserved among Toxoplasma strains [8,9]. Primers Tg1 (5′-AAAAATGTGGGAATGAAAGAG-3′) and Tg2 (5′-ACGAATCAACGGAACTGTAAT-3′) were designed and tested for specificity in the BLASTN data base (http://www.ncbi.nlm.nih.gov/BLAST) and were predicted to be 100% specific for the B1 gene. PCR with these primers amplified a 469-bp DNA fragment of the B1 gene. The PCR control primers used were β1 (5′-ACCACCAACTTCATCCACGTTCACC-5′) and β2 (5-CTTCTGACACAACTGTGTTCACTAGC-3′); these amplified a 140-bp DNA fragment of the human β-globin gene [10]. Both reactions were run simultaneously with the same temperature protocol in parallel tubes in the same PCR machine.

Twenty clinical samples, consisting of eight amniotic fluid samples, two cerebrospinal fluid samples, four vitreous fluid samples and six tissue samples, were received from the Department of Virology, Helsinki University Central Hospital, Helsinki, Finland, and the Department of Microbiology, Ullevaal University Hospital, Oslo, Norway. All the samples were coded upon receipt. The tissue samples were collected in physiological saline in sterile tubes and stored at − 70°C immediately upon arrival until analysed. The liquid samples were transferred to 2-mL sterile microcentrifuge tubes and centrifuged at 10 000 g for 10 min, after which the supernatant was discarded. The microcentrifuge tubes with pellets were stored at − 70°C until analysed. The samples stored at − 70°C were thawed once before DNA extraction. Total genomic DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). The supplier's protocol was modified, as described below, for maximum recovery of DNA.

In order to test the sensitivity of the method, a suspension of T. gondii tachizoites was made and the cells were counted by microscopy. Ten-fold serial dilutions (106−101) of this suspension were then made in 2-mL aliquots of amniotic fluid and/or fresh human blood to yield concentrations between 500 000 and 5 parasites/mL.

The amniotic fluid suspension was centrifuged at 10 000 g for 10 min, and the supernatant was discarded. The pellet was resuspended in 180 µL ATL buffer (supplied in the QIAamp DNA Mini Kit), and the protocol recommended for tissue samples was followed.

Blood samples were collected in Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA) and then aliquoted (1 mL) into two 2-mL microcentrifuge tubes. Blood cells were lysed by the addition of an equal volume of lysis buffer A (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.32 mM saccharose and Triton X-100 1% v/v) [11]. The lysates were vortexed briefly, incubated on ice for 5 min, and centrifuged at 8000 g for 3 min. The supernatants were discarded and the pellet resuspended in 0.1 mL lysis buffer A. Following brief centrifugation, the supernatants from the two tubes were pooled to give a total volume of 0.2 mL. The QIAamp tissue protocol was then followed.

Tissue samples were cut into small pieces or homogenised mechanically before suspension in 180 µL ATL lysis buffer (Qiagen). The QIAamp tissue protocol was then followed. DNA was eluted twice in elution buffer in a total volume of 100 µL. DNA from vitreous fluid and cerebrospinal fluid was extracted using the same protocol as described above for amniotic fluid.

A PCR master mix was prepared with 1 × PCR Gold buffer, 200 µM each dNTP, 0.4 pmol of each primer (Tg1 and Tg2), 1.5 mM MgCl2, and 1.25 U of AmpliTaq Gold enzyme for each 50 µL reaction. Reactions were preheated in a PTC-200 DNA engine (MJ Research Inc., Waltham, MA, USA) for 10 min at 95°C, followed by 35 cycles of 94°C for 1 min, 52°C for 30 s and 72°C for 1 min, with a final extension step at 72°C for 7 min. Samples (15 µL) of each reaction were analysed on agarose 1.8% w/v mini-gels by electrophoresis for 25 min at 120 V, followed by staining with ethidium bromide and visualisation under UV light. For each sample, five reaction tubes were interpreted, as indicated in Table 1.

Table 1.  Interpretation of the five PCR results obtained for each patient sample
1 Negative control2 Positive control3 β-globin4 DNA: 10 µ/reaction5 DNA: 5 µL/reactionInterpretation
  • a

    In the case of PCR inhibition and/or inhibition of the control PCR, the template DNA was repurified and/or diluted (1:10) before repeating the PCR.

++++Positive
++Negative
+Inhibitiona
++Inhibition of controla

The reproducible detection limit of the method was two parasites/sample (Fig. 1). The sample containing DNA equivalent to one parasite genome yielded negative results in one of three experiments.

image

Figure 1. Sensitivity of detection of Toxoplasma PCR product on agarose 1.8% w/v agarose gels. Lane 1: negative control. Lane 2: equivalent to 32 genomes. Lane 3: equivalent to 16 genomes. Lane 4: equivalent to eight genomes. Lane 5: equivalent to four genomes. Lane 6: equivalent to two genomes. Lane 7: equivalent to one genome.

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The results obtained with the 20 clinical samples using the described method were in good agreement with the results of analysis obtained by nested PCR or PCR-ELISA using internal probes in Oslo and Helsinki [6]. Nine of ten samples reported positive in Oslo were also positive by PCR. One sample, a piece of dark tissue (c. 5 mg), was negative for T. gondii DNA in the present study, but was described as an ‘uncertain’ positive in Oslo. This result could be caused by lack of sufficient sample material. Similarly, nine of ten samples from Helsinki were also positive by PCR. One tissue sample (2–3 mg) was toxoplasma DNA-positive in Helsinki, but negative in the present study, again probably because of lack of sufficient sample material. The sample volume is important for a reproducible result. A minimum of 2 mL of liquid sample or 10 mg of solid sample was found to yield a reproducible result.

The lengths of the T. gondii-specific PCR product (469 bp) and the β-globin inhibition control (PCR) product (140 bp) were different. The effect of this product-length difference on the PCR was tested in a series of experiments, but no influence on specificity or sensitivity was detected. In addition, it was observed that the β-globin primers were capable of amplifying a specific PCR product at annealing temperatures ranging from 48°C to 64°C.

It was concluded that the PCR method was a rapid and sensitive approach to detecting T. gondii DNA directly from clinical samples. Quantification of DNA with real-time PCR experiments is being used increasingly for the detection and follow-up of various infections, and it would be interesting to study the use of quantitative real-time PCR for detection of T. gondii.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References

We would like to thank M. Holberg-Petersen for her generous cooperation and provision of samples for this study, and A. Magnusson for his skilful microscopic assistance.

References

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References