Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients.
Following sequence analysis of a Leishmania donovani kinetoplast DNA (kDNA) minicircle, we have developed synthetic oligonucleotides for use in the polymerase chain reaction (PCR). With these primers, we have amplified L. donovani kDNA from splenic aspirates and blood samples taken from kala-azar patients. Treatment of the samples for PCR requires only limited DNA purification by lysis in SDS, digestion with proteinase K, phenol extraction and ethanol precipitation of the resulting nucleic acid. We have obtained amplified product routinely with DNA prepared from the equivalent of 2.5-25 microliters of splenic aspirate or of 50-500 microliters of blood from infected patients. In dilution experiments a visible product has been obtained on amplification of DNA from the equivalent of 2.5 x 10(-7) microliters of splenic material. We therefore propose the amplification of L. donovani kDNA by PCR as a rapid and highly sensitive method for the diagnosis of kala-azar.
Smyth
A J
AJ
Department of Pathology, University of Cambridge, UK.
Ghosh
A
A
Hassan
M Q
MQ
Basu
D
D
De Bruijn
M H
MH
Adhya
S
S
Mallik
K K
KK
Barker
D C
DC
D000818:Animals; D001483:Base Sequence; D004270:DNA, Circular / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D004311:Double-Blind Method; D005784:Gene Amplification; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics* / Q000302:isolation & purification; D007898:Leishmaniasis, Visceral / Q000175:diagnosis* / Q000188:drug therapy; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013154:Spleen / Q000469:parasitology*
D004270:DNA, Circular; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan; D009838:Oligodeoxyribonucleotides