Use of Restriction Fragment Length Polymorphism of 18S rRNA Gene for Identification of Trypanosome and Other Protozoan Species

Abstract

Oligonucleotide primers based on conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of Trypanosoma brucei, Leishmania donovani, Triponema aequale and Legenidium gigantum were designed, synthesized and used to generate PCR products from different trypanosome species, subspecies and Leishmania donovani. The PCR products were digested with restriction enzyme Sau3A1, electrophoresed, transferred to nylon membrane and hybridized to a DNA probe (18pNS-1), which is specific to Trypanosoma congolense 18S rRNA gene. The restriction fragment length polymorphism (RFLP) pattern of PCR products obtained was the same for T. brucei subspecies: T.b. brucei and T.b. gambiense but different for other trypanosome species and L. donovani. RFLP analysis was also done with genomic DNA from different trypanosome species, subspecies and other protozoan species digested with Sau3A1 and other restriction enzymes. The genomic DNA RFLP pattern was the same for trypanosomes of the subgenus Trypanozoon: T. evansi, T.b. gambiense and T.b. brucei but different for L. donovani, Theilera parva and T. simiae. The genomic DNA RFLP pattern for T. Congolese genotypes: Savanna-type, Kilifi-type and West African riverine/forest type was also different. The results indicate intra- and inter-species genetic heterogeneity of 18S rRNA gene among the protozoans tested. The results further indicate that 18S rRNA gene of protozoans can potentially be used to identify different protozoan species and subspecies.