**4.2 Variability of the segment of ribosomal RNA gene (24Sα)**

PCR amplification of ribosomal RNA gene (24Sα) using primers D71 and D72 resulted in fragments 125pb characteristic of lineage TcII (not shown).

#### **4.3 MSH2 gene**

The products of digestion with *Hha*I restriction enzyme resulted in fragments of 173pb, 207pb and 294pb for all isolates, which indicate a characteristic pattern for the TcII, so demonstrating that there is not possibly hybrids between our isolates (**Figure 2**).

## **4.4 DTU genotyping**

DTU was determined according to D´Ávila *et al*. [30] that propose a three-step assay: polymorphism of the mitochondrial cytochrome oxidase subunit 2 (COII) after digestion with restriction enzyme *Alu*I (**Figure 3**), amplification of the D7 divergent domain of the 24Sα rRNA gene (**Figure 4**) and amplification of the spliced leader intergenic region (SL-IRac) (**Figure 5**).

According to the results, it was possible to determine DTU. Most of them showed the presence of two mixed *T. cruzi* populations (**Table 1**).

#### **Figure 1.**

*Molecular characterization of Trypanosoma cruzi isolates by segment analysis of the non-transcribed spacer of the mini-exon gene obtained by electrophoresis in agarose gel stained with ethidium bromide. M—molecular marker (100bp), lanes 1 and 12—control sample of Tc I (Dm 28c), 2 and 13—control sample of Tc II (CL Brener), 3 and 14—ZIII (3663), 4 and 15—control sample of T. rangeli (R1625), 5—SMM1, 6—SMM9, 7—SMM10, 8—SMM30, 9—SMM34, 10—SMM36, 11—SMM39. 16—SMM51, 17—SMM57, 18—SMM82, 19—SMM88, 20—SMM89, 21—SMM98, 22—SMM106, B—negative control.*

## **Figure 2.**

*Electrophoresis in polyacrylamide Gel 7.5% of MSH2 gene after the restriction digestion enzyme HhaI. M—molecular marker (100 bp), 1—SMM1, 2—SMM9, 3—SMM10, 4—SMM30, 5—SMM34, 6—SMM36, 7—SMM39, 8—SMM51, 9—SMM57, 10—SMM82, 11—SMM88, 12—SMM89, 13—SMM98, 14—SMM106.*

*Evaluation of Molecular Variability of Isolates of* Trypanosoma cruzi *in the State… DOI: http://dx.doi.org/10.5772/intechopen.104498*

#### **Figure 3.**

*Electrophoresis in acrylamide gel of COII gene after digestion with AluI restriction enzyme. M—molecular marker (50 bp), 1–6—TcI-TcVI controls (1—haplotype A, 2—haplotype C, 3,5,6—haplotype B, 4—uncutted), 7–15 samples.*

#### **Figure 4.**

*Electrophoresis in acrylamide gel of 24Sα gene. M1—molecular marker (50 bp), 1—TcI, 2—TcII, 3–9—samples, M2 molecular marker (100 bp).*

#### **Figure 5.**

*Electrophoresis in acrylamide gel of SL-IRac gene. M1—molecular marker (100 bp), 1–7—samples, 8 and 9—controls, B—negative control.*


*Compatible results with TcII. b Compatible results with TcIV.*

#### **Table 1.**

*General results obtained in this work by the molecular characterization of isolates of* Trypanosoma cruzi *by different markers.*
