The use of biotin-labelled, synthetic DNA oligomers for the detection and identification of Plasmodium falciparum

Abstract

An oligonucleotide mixture based on the 21 base pair repeat sequence of Plasmodium falciparum was covalently coupled to biotin and used as a probe to detect P. falciparum DNA. The limit of detection was 10 ng. This method was further developed as a fingerprint assay for parasite strain typing. After restriction enzyme digestion, blotting and hybridization, distinct banding patterns were obtained for the strains tested and these were reproducible. In addition, discrete differences were found between PLF-3 S+/S-strains which may implicate genetic reorganization in the switching mechanism which occurs when parasites are passed from an intact to a splenectomized animal.

Antibodies and DNA probes used to analyze variant populations of the Indochina-1 strain of Plasmodium falciparum

“…5). However, the Indochina-1 pattern was quite different from the previously described pattern of the Palo Alto/PLF3 strain of P. falciparum (16) (comparative data not shown). The pattern of the Indochina-1 strain established in culture from the original isolate from an Aotus monkey had a fingerprint pattern that was substantially different from those of all 10 in vivo-maintained populations of Indochina-1 and that of the PLF3/Palo Alto strain.…”

“…Because these P. falciparum-specific 21-bp repeat sequences occur in blocks near telomeres (they may represent up to 1% of the total genome), restriction enzyme DNA fingerprinting can be used with accuracy to provide evidence for major changes in the overall genome organization (e.g., point mutation, deletion, or internal rearrangement of the DNA by movement of transposable genetic elements or by exchange of chromosome ends [6]). It has already been established that the fingerprinting pattern is very different between different isolates (16,20,22) but is stable in a given cloned isolate (PLF3/Palo Alto) maintained in vitro or in vivo by serial transfer over a number of years (3).…”

“…Variant-specific antibodies raised in hyperimmune squirrel monkeys were used to demonstrate diversity of erythrocyte-associated antigens, and DNA fingerprinting was used to examine the overall genetic background of the 10 populations studied. The rep-20 sequence, which had previously been shown to be of value in the identification of different P. falciparum isolates (16), was used for DNA fingerprinting after Southern blotting. The objective of the study was to establish whether the serological differences observed between variant populations were accompanied by detectable differences in gene organization.…”

Detection of variable DNA repeats in diverse eukaryotic microorganisms by a single set of polymerase chain reaction primers

“…4a) (8,31,43). An alternative approach is to use electrophoresis followed by Southern blotting and radioactive probe hybridization to detect variable-length restriction fragments (16,18,31,32,42). Both of these methods have been referred to as "RFLP analysis."…”

“…Both of these methods have been referred to as "RFLP analysis." For example, Hughes et al (16) accomplished parasite strain typing using an oligonucleotide probe mixture based on repeat DNA sequences in Plasmodium falciparum (16). Giardia duodenalis was typed by using an M13-based probe (42).…”