Parasitology Research, ( ISI ), Year (2007-4)

Title : ( Innovative restriction site created PCR-RFLP for detection )

Authors: پرویز شایان , علی اسلامی , Hassan Borji ,

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Abstract Benzimidazole compounds, especially albend a- zole, are the most commonly used anthelmintics for deworming of small ruminants in Iran. It is believed that the therapeutic effects of the benzimidazoles (BZs) come through their binding capacity to the -tubulin isotype 1. Substitution of phenylalanine to tyrosine at position 200 of this polypeptide confers resistance to BZs. Several investigators developed different biological- and molecu- lar-based techniques to demonstrate the occurrence of resistance in helminthes against BZs. To address the determination of resistance at position 200 of -tubulin isotype 1, we developed an innovative restriction site created polymerase chain reaction-restriction fragment length polymorphism, in which nucleotide A at the position of 637 upstream flanked by the first two coding sequences of the phenylalanine (TT) triplet was substitut- ed through the nucleotide G. The introduced modification in forward primer (UTvet MF-primer) leads to the creation of restriction site (AACGTT) for PSP1. Therefore, in the case of normal allele only, PSP1 can cut the corresponding PCR product. In the first step, the genomic DNA was isolated from each single Teladorsagia circumcincta col- lected either from the abomasa of untreated (n =35) or of 5 mg/kg BW 2.5% albendazole suspension-treated (n= 40) sheep. It was amplified with the primer pair, creating PCR product of 403 bp in length. In the second step, the PCR product was extracted from agarose gel and amplified with the modified forward primer (UTvet MF-primer) and the same reverse primer as in step 1, creating a PCR product of 222 bp. The PCR product was then cut with PSP1 to obtain in the case of normal allele two DNA products (183 and 39 bp). Eight of the 35 worms collected from the untreated sheep were BZSS homozygotes, and the rest (27) were BZR S heterozygotes. In our preliminary experiment, we could not find a BZRR homozygote form within the examined samples. Five out of 40 worms collected from the albendazole-treated sheep were BZR R homozygotes, whereas the rest (35) were BZR S heterozygotes. No BZSS homozygote form was detected within this group.


, benzimidazole resitance, Teladorsagia circumcincta, PCR-RFLP
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author = {پرویز شایان and علی اسلامی and Borji, Hassan},
title = {Innovative restriction site created PCR-RFLP for detection},
journal = {Parasitology Research},
year = {2007},
month = {April},
issn = {0932-0113},
keywords = {benzimidazole resitance;Teladorsagia circumcincta;PCR-RFLP},


%0 Journal Article
%T Innovative restriction site created PCR-RFLP for detection
%A پرویز شایان
%A علی اسلامی
%A Borji, Hassan
%J Parasitology Research
%@ 0932-0113
%D 2007