Real-Time PCR in faecal samples of Triatoma infestans obtained by xenodiagnosis: proposal for an exogenous internal control

Particle and Fibre Toxicology

Inés Zulantay¹  Werner Apt¹  Eduardo Araya¹  Gabriela Martínez¹  Miguel Saavedra¹  Nicolás Nazal²  Catalina Muñoz¹  Nicolás Bravo¹ 

[1] Faculty of Medicine, Institute of Biomedical Sciences, Cellular and Molecular Biology Program, Basic-Clinical Laboratory of Parasitology, University of Chile, PO 427, Santiago 3, Chile;Gene X-Press, Avenida Sucre 2418, Santiago, Ñuñoa, Chile

关键词: Trypanosoma cruzi;    Faecal samples;    Triatoma infestans;    Xenodiagnosis;    Real-time PCR;   

Others  :  1233017
DOI  :  10.1186/1756-3305-5-59

 received in 2012-01-10, accepted in 2012-03-26,  发布年份 2012

【 摘 要 】

Background

The polymerase chain reaction (PCR) has proved to be a sensitive technique to detect Trypanosoma cruzi in the chronic phase of Chagas disease, which is characterized by low and fluctuating parasitemia. Another technique proposed for parasitological diagnosis in this phase of infection combines a microscopic search for motile trypomastigote forms in faecal samples (FS) obtained by xenodiagnosis (XD) with conventional PCR (XD-PCR). In this study we evaluate the use of human blood DNA as an exogenous internal control (EIC) for real time PCR (qPCR) combined with XD (XD-qPCR) using chromosome 12 (X12) detection.

Findings

None of the FS-XD evaluated by qPCR amplified for X12. Nevertheless, all the EIC-FS-XD mixtures amplified for X12.

Conclusions

We determined that X12 is useful as an EIC for XD-qPCR because we showed that the FS-XD does not contain human DNA after 30 or more days of XD incubation. This information is relevant for research on T. cruzi by XD-qPCR since it allows ruling out inhibition and false negative results due to DNA loss during the process of extraction and purification.

【 授权许可】

   
2012 Bravo et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Virreira M, Serrano G, Maldonado L, Svoboda M: Trypanosoma cruzi: typing of genotype (sub) lineages in megacolon samples from Bolivian patients. Acta Trop 2006, 100:252-255.
• [2]Britto C: Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations. Mem Inst Oswaldo Cruz 2009, 104:122-135.
• [3]Botto-Mahan C, Campos R, Acuña-Retamar M, Coronado X, Cattán PE, Solari A: Temporal variation of Trypanosoma cruzi infection in native mammals in Chile. Vector Borne Zoonotic Dis 2010, 10:317-319.
• [4]Schijman A, Bisio M, Orellana L, Sued M, Duffy T, Mejia-Jaramillo AM, Cura C, Auter F, Veron V, Qvarnstrom Y, Deborggraeve S, Hijar G, Zulantay I, Lucero RH, Velázquez E, Tellez T, Sánchez-León Z, Galvão L: International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl Trop Dis 2011, 5:e931.
• [5]Zulantay I, Apt W, Valencia C, Torres A, Saavedra M, Rodríguez J, Sandoval L, Martínez G, Thieme P, Sepúlveda E: Detection of Trypanosoma cruzi in untreated chagasic patients is improved by using three parasitological methods simultaneously. J Antimicrob Chemother 2011, 66:2224-2226.
• [6]Duffy T, Bisio M, Altcheh J, Burgos JM, Diez M, Schijman AG: Accurate Real-Time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients. PLoS Negl Trop Dis 2009, 3:e419.
• [7]Cencig S, Coltel N, Truyens C, Carlier Y: Parasitic loads in tissues of mice infected with Trypanosoma cruzi and treated with Am Bisome. PLoS Negl Trop Dis 2011, 5:e1216.
• [8]Castro C, Santos MC, Silveira CA: Comparative study between artificial xenodiagnosis performed immediately and four hours after venous punch. Rev Soc Bras Med Trop 2004, 37:128-130.
• [9]Schenone H: Xenodiagnosis. Mem Inst Oswaldo Cruz 1999, 94:289-294.
• [10]Zulantay I, Apt W, Gil LC, Rocha C, Mundaca K: The PCR-based detection of Trypanosoma cruzi in the faeces of Triatoma infestans fed on patients with chronic American tripanosomiasis gives higher sensitivity and a quicker results than routine xenodiagnosis. Ann Trop Med Parasitol 2007, 101:673-679.
• [11]Pita-Pereira D, Lins R, Oliveira M, Lima R, Pereira B, Moreira O: SYBR Green-based Real-Time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniasis. Parasit Vectors 2012, 5:15. BioMed Central Full Text