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Colin D. Jones, Narciss Okhravi, Peter Adamson, Sharron Tasker, Susan Lightman; Comparison of PCR Detection Methods for B1, P30, and 18S rDNA Genes of T. Gondii in Aqueous Humor. Invest. Ophthalmol. Vis. Sci. 2000;41(3):634-644.
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© ARVO (1962-2015); The Authors (2016-present)
purpose. Comparison of polymerase chain reaction (PCR) amplification of three Toxoplasma gondii genes in aqueous humor.
methods. Nested PCRs carried out using published methods were optimized for
maximum sensitivity and specificity. Five pairs of oligonucleotide
primers, directed against the B1, P30, and ribosomal genes, were used
and compared to determine which sequences were most effective in
detecting T. gondii DNA. Methods were developed with DNA
templates in water and were subsequently applied to both normal and
results. After one round of PCR amplification, P30 and ribosomal primers were
able to detect 1 pg genomic T. gondii DNA. However,
those directed against the B1 gene were able to detect 50 fg
(approximately single tachyzoite). This level of sensitivity was also
achieved using the P30 primers after a second round of PCR; however,
only primers based on the B1 gene maintained this level of sensitivity
in both normal and inflamed aqueous. B1-specific primers did not
amplify sequences from fungal, bacterial, or human lymphocyte DNA. The
sensitivity of T. gondii detection using B1
gene–specific primers was not compromised when large amounts of human
lymphocyte DNA were present, and application to an ocular sample or
retinal section from patients with toxoplasma chorioretinitis was
successful in confirming the presence of T. gondii DNA.
conclusions. The B1 PCR protocol appears to be the most sensitive protocol
in the detection of T. gondii DNA and has been
successful in identification of T. gondii DNA in ocular
fluids and retinal sections. This provides direct evidence of the
presence of T. gondii within the eye and may therefore
help in the management of toxoplasma
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