PCR amplification of the non-transcribed spacer region of the mini-exon gene was performed employing five primers in a multiplex reaction to discriminate between zymodemes I, II, III and T. rangeli, as previously described [47]. In the current nomenclature zymodeme I is equivalent to DTU TcI, zymodeme II comprises DTUs II, V and VI, while zymodeme III corresponds to DTUs III and IV. The amplified products were electrophoresed in 2% agarose gels and visualized with SYBR green under UV light. Samples co-infected with T. cruzi and T. rangeli displayed a two-band pattern. In such cases, gel excision and purification were performed for each fragment. Subsequently, amplicons were sequenced by NGS technology as detailed below.
Copyright and License information: The Author(s) ©2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this
article to respond.
Ask a
question
Post a Question
