NDLI: Innovative restriction site created PCR-RFLP for detection of benzimidazole resistance in Teladorsagia circumcincta

Content Provider	Springer Nature Link
Author	Shayan, Parviz Eslami, Ali Borji, Hassan
Copyright Year	2006
Abstract	Benzimidazole compounds, especially albendazole, 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 β-tubulin isotype 1. Substitution of phenylalanine to tyrosine at position 200 of this polypeptide confers resistance to BZs. Several investigators developed different biological- and molecular-based techniques to demonstrate the occurrence of resistance in helminthes against BZs. To address the determination of resistance at position 200 of β-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 substituted 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 collected 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 BZRS 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 BZRR homozygotes, whereas the rest (35) were BZRS heterozygotes. No BZSS homozygote form was detected within this group.
Starting Page	1063
Ending Page	1068
Page Count	6
File Format	PDF
ISSN	09320113
Journal	Parasitology Research
Volume Number	100
Issue Number	5
e-ISSN	14321955
Language	English
Publisher	Springer-Verlag
Publisher Date	2006-11-30
Publisher Place	Berlin/Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Immunology Microbiology Medical Microbiology
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Veterinary Parasitology Insect Science

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