Yenisey Alfonso, Jorge Fraga, Zhaily Gonzalez, Narciso Jimenez, Yainais Borrero, Raymundo Cox, Carlos Fonseca, Francisco Bandera, Virginia Capo and Dora Ginorio
Polymerase Chain Reaction (PCR) has made a significant improvement in the diagnosis of toxoplasmosis. Nevertheless, a wide variety of targets and primers has been evaluated in different independent assays, and only few comparative studies have been performed. Even when the B1 gene and the 529 repetitive element have been the most studied markers, there no concluding remarks respect the best.
Objetive: The aim of the present study was to design a multiplex PCR assay to simultaneously detect these two markers in a single multiplex PCR to diagnose T. gondii infection.
Methods: Specific PCR primers targeting the B1 gene and 529 repetitive element were evaluated. After a careful optimization of multiplex PCR process, analytical sensitivity and specificity were evaluated. The usefulness of multiplex assay was evaluated using DNA from 8 T. gondii reference strains, corresponding to the three principal genotypes, and 70 clinical samples (blood) from AIDS patients with and without Toxoplasmic encephalitis (TE).
Results: Our preliminar results show that multiplex PCR assay is able to amplify both targets in using DNA from one parasite in the mix PCR reaction. Unspecific reactions for other microorganisms were not observed. The diagnostic sensitivity of multiplex assay in blood, according to the Centers for Disease Control and Prevention (CDC) criteria, was 86.6% while the diagnostic specificity was 100%. Only one sample was positive to one marker (B1 gene) in the multiplex reaction.
Conclusion: This multiplex PCR method is the first multiplex PCR evaluating the detection of T. gondii DNA in TE cases and it proved to be rapid, enough sensitive, highly specific and inexpensive respect to perform independent assays for each marker, real time PCR or nested PCR assays.
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