Comparison of PCR detection methods for B1, P30, and 18S rDNA genes of T-gondii in aqueous humor

PURPOSE. Comparison of polymerase chain reaction (PCR) amplification of thr ee 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 BI, P30, and ribosomal genes, were used and compared to determine which sequences were most effective in detecting T. gondii DN A. Methods were developed with DNA templates in water and were subsequently applied to both normal and inflamed aqueous. RESULTS. After one round of PCR amplification, P30 and ribosomal primers we re able to detect I pg genomic T. gondii DNA. However, those directed again st the B1 gene were able to detect 50 fg (approximately single tachpzoite). 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 lymphocy te DNA. The sensitivity of T gondii detection using B1 gene-specific primer s was not compromised when large amounts of human lymphocyte DNA were prese nt, and application to an ocular sample or retinal section from patients wi th 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 dire ct evidence of the presence of T. gondii within the eye and mag therefore h elp in the management of toxoplasma retinochoroiditis.