| Popis: |
Molecular approaches have been shown to be valuable tools contributing to the diagnosis of congenital Trypanosoma cruzi infection and to the follow-up of infected patients having received trypanocidal drugs, as well as for identifying lineages and sublineages of T. cruzi. In the first part of this chapter molecular analyses are considered as diagnostic tools at the individual and/or epidemiological level; this is followed, in the second part, by detailed protocols for detecting T. cruzi DNA using qualitative PCR and quantitative PCR (qPCR) as well as identifying (sub)lineages by hybridization with specific DNA probes. PCR could help in the diagnosis of T. cruzi infection/Chagas’ disease or in the management of infected patients in three main cases: (i) the diagnosis of congenital infection, (ii) the early detection of a reactivation of Chagas’ disease in immunosuppressed patients, and (iii) the follow-up of infected patients treated with antiparasitic drugs. PCR, particularly qPCR, was also demonstrated to be useful for the early diagnosis of Chagas’ disease reactivation in immunosuppressed patients. The detection of DNA is made with the SYBR Green fluorophore, an intercalating dye, fluorescing only when bound to dsDNA. The fluorescence of each sample is measured after each cycle. The SYBR Green signal measured is proportional to the dsDNA synthesized de novo in the course of the PCR cycles. High specificity of amplification is mandatory and must be assessed at the end of the amplification by electrophoresis or by analysis of the melting curves of the amplicons. |
