PCR-ELISA for diagnosis of mucocutaneous leishmaniasis.
In this work we demonstrate that the PCR-ELISA technique is sufficiently sensitive and specific for use as a diagnostic test in cases of mucocutaneous leishmaniasis. DNA was extracted from cultures of Leishmania braziliensis, Leishmania infantum, Leishmania tropica, Leishmania mexicana, Trypanosoma cruzi, and blood samples from individuals who presented a clinical diagnosis of leishmaniasis as well as from healthy individuals. The DNA was PCR amplified and the product obtained was hybridised with a biotin-labelled probe, the sequence of which was designed in our laboratory. The result of the hybridisation was visualised by means of an ELISA technique using antifluorescein antibody labelled with alkaline phosphatase and p-nitrophenylphosphate (pNFF) as chromogen. The optical density of the products of the pNFF hydrolysis was quantified in a spectrophotometer at a wavelength of 405 nm. Using this technique the percentage of detection was 83.3% in blood samples from patients clinically diagnosed as having mucocutaneous leishmaniasis. No false positive results were obtained.
Piñero
J
J
Departamento de Parasitología, Ecología y Genética, Facultad de Farmacia, Universidad de la Laguna, Tenerife, Islas Canarias, Spain. jpinero@ull.es
Martínez
E
E
Pacheco
R
R
Aragón
Z
Z
De Armas
F
F
Del Castillo
A
A
Valladares
B
B
D000818:Animals; D003124:Colorimetry; D015342:DNA Probes; D018105:DNA, Kinetoplast / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007892:Leishmania braziliensis / Q000235:genetics / Q000302:isolation & purification*; D007897:Leishmaniasis, Mucocutaneous / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015342:DNA Probes; D018105:DNA, Kinetoplast