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 flagellar protein F29. An amplification product of BP1/BP2 is a DNA band 692 bp long. Titration assays were performed to evaluate the minimum amount 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 performed using T. cruzi DNA from different strains, clones, and human-derived isolates obtaining, in all cases, amplification products. No DNA amplification was observed when the PCR was performed using DNA from Leishmania 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 conventional xenodiagnosis and PCR detection of parasite DNA in the feces of insect vectors fed with blood from infected patients were compared, 10 of 20 samples were positive by both techniques. However, 2 other samples with positive serology were also positive by PCR. When PCR was performed 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 radiolabeled PCR amplification product as probe (sensitivity 100%).
Silber
A M
AM
Instituto Nacional de Chagas Dr. Mario Fatala Chaben, Ministerio de Salud y Accion Social, Buenos Aires, Argentina.
Búa
J
J
Porcel
B M
BM
Segura
E L
EL
Ruiz
A M
AM
D000818:Animals; D015139:Blotting, Southern; D014355:Chagas Disease / Q000097:blood / Q000175:diagnosis / Q000469:parasitology*; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000032:analysis* / Q000097:blood / Q000235:genetics; D005243:Feces / Q000469:parasitology; D006801:Humans; D007303:Insect Vectors / Q000469:parasitology*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014225:Triatominae / Q000469:parasitology*; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D016054:DNA, Protozoan