Evaluation of PCR methods to detect and characterise flagelate-protozoans in fecal specimens from triatomines

MARCET PL; BURGOS JM; CARDINAL MV; BISIO M; LAURICELLA M; DUFFY T; LEVIN MJ; GÜRTLER RE; SCHIJMAN AG

Rosario - Santa Fe - Argentina.

Congreso; XX Reunión de la Sociedad Argentina de Protozoología; 2004

Sociedad Argentina de Protozoología (SAP)

PCR methods were evaluated in order to 1) improve the sensitivity and specificity of detection of T.cruzi DNA in fecal samples of triatomines, which were negative by direct microscopical observation and II) to discriminate between T. cruzi and other flagelates in faeces from T. infestans (Ti), T. guasayana (Tg) and T. garciabesi (Tgb) with positive MO findings, collected in October 2002, Amamá, Sgo del Estero. 1) MO negative samples: A) DNA from 32 faecal samples of T.i was isolated with DNAzol (Invitrogen, USA) or boiled during 10 minutes after incubation with Chelex-100 (Sigma, USA). A test of PCR inhibitors was carried out spiking 10 pg of a cloned internal standard to each DNA lysate. B) We compared the PCR concordance between 121-122 ( amplicon 330 bp) and 34-67 (amplicon 120 bp) based PCR procedures in 57 DNAzol lysates; C) 93 T.i samples lysed using DNAzol were tested by PCR, and 41 of them, with negative PCR findings, were re-tested by a Hot Start approach using a Taq polymerase bound to Antibody (Taq Platinum, Invitrogen, USA). RESULTS: A) An inhibition of 98% and 20% was detected from lysates prepared by boiling or DNAzol, respectively. B) PCR concordance between 121-122 and 34-67 was 100% (3 + /57). C) PCR detected 3 positive cases out of 93, whereas the Hot start-PCRallowed detection of 5 new cases out of the 41 tested. Moreover, 22 samples of T.g and 19 of Tgb, prepared with DNAzol were studied. None of them was positive for T. cruzi DNA. II) MO positive specimens: 43 T.i, 2 T.g y one T.gb samples were analysed. All T.i were PCR positive confirming their infection with T. cruzi. Tg and Tgb samples were PCR negative. In 21 T.i, the lineage was characterised directly from the lysates using miniexon spacer-PCR and D7 rDNA PCR. 19 were T. cruzi II, 1 was T. cruzi I and the other one was a mixed T.cruzi I and II infection. Tg and Tgb samples were negative by the miniexon-PCR assay. However, they gave rise to a 255 bp amplicon when amplified by D7 rDNA-PCR. This product had the same molecular weightthan one obtained from a Blastochrithidia strain. T. cruzi I generated a 270 bp band, T. cruzi II a 300 bp amplicon, showing the usefullness of this approach to identify among flagelated protozoans in triatomine vectors.