Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) through the implementation of a High-Resolution Melting (HRM) genotyping assay

Particle and Fibre Toxicology

Juan David Ramírez¹  Felipe Guhl¹  Sonia L Higuera¹ 

[1] Centro de Investigaciones en Microbiología y Parasitología Tropical, CIMPAT, Universidad de Los Andes, Cra 1ª No. 18ª-10, Bogotá, Colombia

关键词: Genotyping;    high-resolution melting;    DTU;    Trypanosoma cruzi;    Chagas disease;   

Others  :  1227990
DOI  :  10.1186/1756-3305-6-112

 received in 2013-03-05, accepted in 2013-04-11,  发布年份 2013

【 摘 要 】

Background

Chagas disease, caused by Trypanosoma cruzi, is a geographically widespread anthropozoonosis that is considered a major public health problem in Latin America. Because this parasite presents high genetic variability, a nomenclature has been adopted to classify the parasite into six discrete typing units (DTUs): TcI, TcII, TcIII, TcIV, TcV, and TcVI, which present different eco-epidemiological, clinical, and geographic associations. Currently, the available genotyping methods present a series of drawbacks that implies the need for developing new methods for characterizing T. cruzi DTU’s. The aim of this work was to genotype reference populations from T. cruzi by means of a High-Resolution Melting (HRM) genotyping assay.

To genotype the DTUs of 38 strains and 14 reference clones of T. cruzi from diverse sources, real-time PCR (qPCR) was coupled to high-resolution melting (HRM) based on the amplification of two molecular markers—the divergent domain of the 24 sα rRNA gene and the intergenic region of the mini-exon gene.

Findings

Amplification of the mini-exon gene allowed the genotyping of three distinct groups: TcI, TcII- TcIV- TcV, and TcIII-TcVI, while amplification of the 24sα gene generated non-overlapping melting temperature ranges for each DTU that were used to identify the groups in the six existing DTUs of Trypanosoma cruzi.

Conclusions

The proposed genotyping assay allowed discrimination of the six genetic groups by obtaining specific melting curves for each DTU. The application of this technique is proposed because of its specificity, sensitivity, high performance, and low cost compared with other previously described characterization methods.

【 授权许可】

   
2013 Higuera et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Rassi A Jr, Rassi A, de Rezende M: American Trypanosomiasis Chagas Disease. Infect Dis Clin North Am 2012, 262:275-291.
• [2]Zingales B, Miles MA, Campbell DA, Tibayrenc M, Macedo AM, Teixeira MM, Schijman AG, Llewellyn MS, Lages-Silva E, Machado CR, Andrade SG, Sturm NR: The revised Trypanosoma cruzi subspecific nomenclature: Rationale, epidemiological relevance and research applications. Infect Genet Evol 2012, 122:240-253.
• [3]Brisse S, Verhoef J, Tibayrenc M: Characterization of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 2001, 311:1218-1226.
• [4]Souto RP, Fernandes O, Macedo AM, Campbell DA, Zingales B: DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol Biochem Parasitol 1996, 83:141-152.
• [5]Burgos JM, Diez M, Vigliano C, Bisio M, Risso M, Duffy T, Cura C, Brusses B, Favaloro L, Leguizamon MS, Lucero RH, Laguens R, Levin MJ, Favaloro R, Schijman AG: Molecular Identification of Trypanosoma cruzi Discrete Typing Units in End-Stage Chronic Chagas Heart Disease and Reactivation after Heart Transplantation. Clin Inf Dis 2010, 515:485-495.
• [6]Rozas M, Doncker S, Adaui V, Coronado X, Barnabé C, Tibayrenc M, Solari A, Dujardin JC: Multilocus polymerase chain reaction restriction fragment-length polymorphism genotyping of Trypanosoma cruzi (Chagas disease): taxonomic and clinical applications. J Infect Dis 2007, 195:1381-8.
• [7]Lewis MD, Ma J, Yeo M, Carrasco HJ, Llewellyn MS, Miles MA: Genotyping of Trypanosoma cruzi: systematic selection of assays allowing rapid and accurate discrimination of all known lineages. AmJTrop Med Hyg 2009, 81:1041-1049.
• [8]Llewellyn MS, Miles MA, Carrasco HJ, Lewis MD, Yeo M, Vargas J, Torrico F, Diosque P, Valente SA, Gaunt MW: Genome-scale multilocus microsatellite typing of Trypanosoma cruzi discrete typing unit I reveals phylogeographic structure and specific genotypes linked to human infection. PLoS Pathog 2009, 5:e1000410.
• [9]Yeo M, Mauricio IL, Messenger LA, Lewis MD, Llewellyn MS, Acosta N, Bhattachaeyya T, Diosque P, Carrasco HJ, Miles MA: Multilocus Sequence Typing MLST for Lineage Assignment and High Resolution Diversity Studies in Trypanosoma cruzi. PLoS Negl Trop Dis 2011, 56:e1049.
• [10]Valadares HMS, Pimenta JR, Segatto M, Veloso VM, Gomes ML, Chiari E, Gollob KJ, Bahia M, de Lana M, Franco GR, Machado CR, Pena SD, Macedo AM: Unequivocal Identification of Subpopulations in Putative Multiclonal Trypanosoma cruzi Strains by FACs Single Cell Sorting and Genotyping. PLoS Negl Trop Dis 2012, 67:e1722.
• [11]Schütz E, von Ahsen N: Influencing factors of dsDNA dye high.resolution melting curves and improved genotype call based on thermodynamic considerations. Anal Biochem 2009, 3851:143-152.
• [12]Zhang P, Liu Y, Alsarakibi M, Li J, Liu T, Li Y, Li G: Application of HRM assays with EvaGreen dye for genotyping Giardia duodenalis zoonotic assemblages. Parasitol Res 2012, 111:2157-2163.
• [13]Al-Mohammed H: Genotypes of Giardia intestinalis clinical isolates of gastrointestinal symptomatic and asymptomatic Saudi children. Parasitol Res 2011, 108:1375-1381.
• [14]Ngui R, Lim YA, Chua KH: Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting HRM Analysis. PLoS One 2012, 77:e41996.
• [15]Costa JM, Cabaret O, Moukoury S, Bretagne S: Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene. J Microbiol Met 2011, 863:357-363.
• [16]Ramírez JD, Guhl F, Rendón LM, Rosas F, Marin-Neto JA, Morillo CA: Chagas Cardiomyopathy Manifestations and Trypanosoma cruzi Genotypes Circulating in Chronic Chagasic Patients. PLoS Neg Trop Dis 2010, 41:e899.
• [17]Moncayo A, Luquetti AO: Multicentre double blind study for evaluation of Trypanosoma cruzi defined antigens as diagnostic reagents. Mem Inst Oswaldo Cruz 1990, 85:489-495.
• [18]Peña VH, Fernández GJ, Gómez-Palacio AM, Mejía-Jaramillo AM, Cantillo O, Triana-Chávez O: High-Resolution Melting HRM of the Cytochrome B Gene: A Powerful Approach to Identify Blood-Meal Sources in Chagas Disease Vectors. PLoS Neg Trop Dis 2012, 62:e1530.
• [19]Cardinal MV, Lauricella MA, Ceballos LA, Lanati L, Marcet PL, Levin MJ, Kitron U, Gürtler RE, Schijman AG: Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina. Int J Parasitol 2008, 381:1533-1543.
• [20]Messenger LA, Llewellyn MS, Bhattacharyya T, Franzén O, Lewis MD, Ramírez JD, Carrasco HJ, Andersson B, Miles MA: Multiple Mitochondrial Introgression Events and Heteroplasmy in Trypanosoma cruzi Revealed by Maxicircle MLST and Next Generation Sequencing. PLoS Neg Trop Dis 2012, 64:e1584.
• [21]Minning TA, Weatherly DB, Flibotte S, Tarleton RL: Widespread, focal copy number variations CNV and whole chromosome aneuploidies in Trypanosoma cruzi strains revealed by array comparative genomic hybridization. BMC Genom 2011, 121:139-146.
• [22]Hong H, Xu L, Liu J, Jones WD, Su Z, Ning B, Perkins R, Ge W, Miclaus K, Zhang L, Park K, Green B, Han T, Fang H, Lambert CG, Vega SC, Lin SM, Jafari N, Czika W, Wolfinger RD, Goodsaid F, Tong W, Shi L: Technical Reproducibility of Genotyping SNP Arrays Used in Genome-Wide Association Studies. PLoS One 2012, 79:e44483.