A mutation scanning approach for the identification of hookworm species and analysis of population variation

• Published in: Molecular and biochemical parasitology Vol. 92; no. 2; pp. 303 - 312
• Main Authors: Gasser, Robin B, Monti, Jennifer R, Bao-Zhen, Qian, Polderman, Anton M, Nansen, Peter, Chilton, Neil B
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.1998
• Subjects: Ancylostomatidae; Ancylostomatoidea - classification; Ancylostomatoidea - genetics; animal parasitic nematodes; Animals; biochemical techniques; chemotaxonomy; DNA, Ribosomal; Electrophoresis, Agar Gel; genbank/aj001594; genetic markers; genetic polymorphism; Genetic Variation; Hookworms; identification; intergenic DNA; Internal transcribed spacer (ITS); internal transcribed spacers; molecular sequence data; Mutation; Mutation scanning; Polymerase Chain Reaction; Polymerase chain reaction-linked single strand conformation polymorphism (PCR–SSCP); Polymorphism, Single-Stranded Conformational; Ribosomal DNA; RNA, Helminth - genetics; RNA, Ribosomal - genetics; Sequence variation; single strand conformation polymorphism; Species identification; Species Specificity; Polymerase chain reaction-linked single strand conformation polymorphism (PCR–SSCP); Ribosomal DNA; ITS, internal transcribed spacer; MDE, mutation detection enhancement; ITS-2, second internal transcribed spacer; Hookworms; Internal transcribed spacer (ITS); RFLP, restriction fragment length polymorphism; SSCP, single strand conformation polymorphism; Sequence variation; rDNA, ribosomal DNA; Mutation scanning; PCR, polymerase chain reaction; ITS-1, first internal transcribed spacer; Species identification
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(98)00008-5

To overcome limitations in the morphological identification of different developmental stages of hookworms to species, we have established a polymerase chain reaction-linked single strand conformation polymorphism technique (PCR–SSCP) utilizing the internal transcribed spacers (ITS) of ribosomal (r)DNA. These spacers were specifically chosen because they provide reliable species markers for strongylid nematodes. ITS spacers were amplified by PCR from DNA derived from individual parasites of seven species of hookworm, then denatured and subjected to electrophoresis in a mutation detection enhancement (MDE) (non-denaturing) gel matrix. PCR–SSCP analysis showed that the single-strand ITS patterns produced allowed the unequivocal identification of all species. The method also allowed the direct display of sequence variation within some species where multiple individual worms were examined. These findings demonstrate the usefulness of the SSCP approach for hookworm identification, the detection of population variation and the direct display of sequence variation in rDNA.