TRYPANOSOMA-CRUZI - SPECIFIC DETECTION OF PARASITES BY PCR IN INFECTED HUMANS AND VECTORS USING A SET OF PRIMERS (BP1 BP2) TARGETED TO A NUCLEAR-DNA SEQUENCE/

In the present work we evaluate Trypanosoma cruzi DNA detection by PCR using the nuclear oligonucleotides BP1/BP2 as primers. These primers are targeted to the 5' and 3' ends of the coding region for the flagel lar protein F29. An amplification product of BP1/BP2 is a DNA band 692 bp long. Titration assays were performed to evaluate the minimum amou nt of parasite DNA that can be detected by this assay, resulting in 10 fg (equivalent to about 1/20 of the genome). The assay was also perfo rmed using T. cruzi DNA from different strains, clones, and human-deri ved isolates obtaining, in all cases, amplification products. No DNA a mplification was observed when the PCR was performed using DNA from Le ishmania braziliensis, but when T. rangeli DNA was used, a 615-bp-long fragment was amplified. Under appropriate gel conditions T. cruzi and T. rangeli DNA amplicons could be differentiated. When both conventio nal xenodiagnosis and PCR detection of parasite DNA in the feces of in sect vectors fed with blood from infected patients were compared, 10 o f 20 samples were positive by both techniques. However, 2 other sample s with positive serology were also positive by PCR. When PCR was perfo rmed on blood samples from infected and uninfected individuals, 62 of 65 serologically positive human samples amplified the BP1/BP2 692-bp T . cruzi DNA fragment (sensitivity > 95%). The 3 negative samples were positive when Southern blot hybridization was performed using the radi olabeled PCR amplification product as probe (sensitivity 100%). (C) 19 97 Academic Press.