Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes.
In situ hybridization on cultured promastigotes and sandfly smears were performed with nonradioactively labeled total DNA and recombinant DNA probes containing minicircle kinetoplast DNA (kDNA) or nuclear DNA inserts. Total DNA probes lack specificity whereas recombinant nuclear DNA probes work only if they contain repetitive sequences. Minicircle kDNAs of five Leishmania isolates, representative of five Leishmania taxa found in Kenya, were sequenced. Comparison of the sequences showed a 150-bp region with around 80% homology, whereas the rest of the minicircles had about 50% homology. Nevertheless, application of these probes in in situ hybridization assays as tested on Leishmania promastigotes in the vector gave good specificity and hybridization signal. Two types of labeling were tested: incorporation of biotin-labeled dUTP or directly horseradish peroxidase (HRP)-labeled nucleotides. Both techniques provided good sensitivity and signal-to-noise ratio on cultured promastigotes. Hybridization with HRP-labeled kDNA probes gave a superior signal-to-noise ratio if tested on sandfly preparations. This method provided a reliable and fast identification and facilitated the detection of promastigotes in sandflies. The technique presented here may be helpful in rapid identification of Leishmania promastigotes, and thus make epidemiological studies easier and less time consuming.
Schoone
G J
GJ
Laboratory of Tropical Hygiene, Royal Tropical Institute, Amsterdam, The Netherlands.
van Eys
G J
GJ
Ligthart
G S
GS
Taub
F E
FE
Zaal
J
J
Mebrahtu
Y
Y
Laywer
P
P
D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D004270:DNA, Circular / Q000032:analysis / Q000235:genetics*; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics*; D004274:DNA, Recombinant; D007891:Leishmania / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010691:Phlebotomus / Q000469:parasitology
D015342:DNA Probes; D004270:DNA, Circular; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan; D004274:DNA, Recombinant