RESUMEN
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.
Asunto(s)
Ancylostomatoidea/clasificación , Ancylostomatoidea/genética , Animales , ADN Ribosómico , Electroforesis en Gel de Agar , Variación Genética , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , ARN de Helminto/genética , ARN Ribosómico/genética , Especificidad de la EspecieRESUMEN
A random amplified polymorphic DNA (RAPD) technique using 16 decamer oligonucleotide primers was employed to characterize isolates of Schistosoma japonicum from seven geographical locations (Sj1: Zhejiang; Sj2: Anhui; Sj3: Jiangxi; Sj4: Hunan; Sj5: Hubei; Sj6: Sichuan; Sj7: Yunnan) of the People's Republic of China. Distinct differences between some isolates were reproducibly detected in RAPD patterns produced using five of the primers. The analyses showed that both Sj6 and Sj7 were quite distinct genetically from Sj1-Sj5 based on the presence/absence of particular bands (A10-200 bp, A9-220 bp, B17-520 bp, P205-680 bp and P235-930 bp). These findings are in line with previous reports on the biological, biochemical, immunological and chemotherapeutic differences of S. japonicum from Sichuan and Yunnan compared with other geographical regions. The present study showed, based on RAPD profiles, that genetic differences exist within S. japonicum from mainland China. This finding may have important implications for studying the population biology, epidemiology and clinical forms of the disease in China, as well as for developing vaccines and diagnostic test systems.
Asunto(s)
ADN de Helmintos/genética , Variación Genética , Técnica del ADN Polimorfo Amplificado Aleatorio , Schistosoma japonicum/genética , Animales , China , Femenino , HumanosRESUMEN
Schistosoma japonicum from the People's Republic of China is considered to represent a single species comprising either 1 or 4 'strains'. We conducted an allozyme electrophoretic study to examine the extent of genetic variation in S. japonicum from mainland China. The allelic profiles of S. japonicum from 7 provinces were established at 16 enzyme loci. S. japonicum from Sichuan had 3-5 (19-31%) fixed differences compared with those from Zhejiang, Anhui, Jiangxi, Hunan, Hubei and Yunnan, suggesting that S. japonicum in mainland China represents a species complex. In addition, genetic markers were also established for different laboratory-maintained populations of S. japonicum which has significant implications for studying the biology of these organisms in human and animal hosts, and for the control and surveillance of human schistosomiasis in China.