Single-step duplex kDNA-PCR for detection of Leishmania donovani complex in human peripheral blood samples.
A duplex polymerase chain reaction (PCR) assay was performed using a sense Leishmania genus-specific oligonucleotides to amplify the conserved region of 120 bp of minicircle kDNA and an additional antisense Leishmania donovani oligonucleotide that amplifies a complex-specific fragment of 90 bp. All 12 tested reference isolates of the L. donovani complex yielded a complex-specific amplification product of 90 bp concurrently with a genus-specific 120-bp product. All 17 tested reference isolates pertaining to other complexes presented only the genus-specific 120-bp product. Peripheral blood samples of twenty patients with visceral leishmaniasis positive by the genus-specific PCR presented both fragments with the duplex assay. This duplex PCR can be applied as a 1-step diagnostic tool where discrimination of L. donovani complex species is relevant for clinical and epidemiologic purposes.
Disch
Jolande
J
Laboratório de Pesquisas Clínicas, Centro de Pesquisas René Rachou-FIOCRUZ, Belo Horizonte-MG, Brazil.
Caligiorne
Rachel B
RB
Maciel
Fabiana
F
Oliveira
Maria C
MC
Orsini
Marcela
M
Dias-Neto
Emmanuel
E
Rabello
Ana
A
D000293:Adolescent; D000328:Adult; D000818:Animals; D002648:Child; D002675:Child, Preschool; D018105:DNA, Kinetoplast / Q000235:genetics; D005260:Female; D006801:Humans; D007223:Infant; D007893:Leishmania donovani / Q000235:genetics / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis*; D008297:Male; D008875:Middle Aged; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013873:Thionucleotides
D018105:DNA, Kinetoplast; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides