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TaqMan minor groove binder real-time PCR analysis of β-tubulin codon 200 polymorphism in small strongyles (Cyathostomin) indicates that the TAC allele is only moderately selected in benzimidazole-resistant populations

Published online by Cambridge University Press:  17 October 2003

G. VON SAMSON-HIMMELSTJERNA ,
S. BUSCHBAUM ,
N. WIRTHERLE ,
M. PAPE  and
T. SCHNIEDER
G. VON SAMSON-HIMMELSTJERNA
Affiliation:
Institute of Parasitology, Hannover School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover, Germany
S. BUSCHBAUM
Affiliation:
Institute of Parasitology, Hannover School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover, Germany
N. WIRTHERLE
Affiliation:
Institute of Parasitology, Hannover School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover, Germany
M. PAPE
Affiliation:
Institute of Parasitology, Hannover School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover, Germany
T. SCHNIEDER
Affiliation:
Institute of Parasitology, Hannover School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover, Germany
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Abstract

TaqMan minor groove binder probes were evaluated as to their suitability for the real-time allelic discrimination of the β-tubulin codon 200 TTC/TAC single nucleotide polymorphism in cyathostomin species. Amplification of titrated cloned full-length β-tubulin cDNA revealed that the TaqMan minor groove binder PCR is capable of specifically detecting as few as 10 copies. Testing of DNA from single adult and larval stages of several different species of cyathostomin allowed reproducible genotyping of individual worms. Using the real-time PCR approach, the throughput of samples was considerably increased compared with conventional post-PCR readout procedure. Only 7·8% homozygous TAC L3 were found among 102 L3 which were genotyped from phenotypically BZ-resistant small strongyle populations. The percentages of the homozygous TTC and heterozygous TTC/TAC were 41·3% and 50·9%, respectively. This resulted in a total TAC-allele percentage of only 33·3%. These findings correspond to data obtained by genotyping of an experimentally selected BZ-resistant cyathostomin population. It is concluded that the β-tubulin codon 200 polymorphism is not the sole mechanism involved in the process of BZ resistance in cyathostomins.

Keywords

TaqMan PCRminor groove binderBZ resistancecyathostominβ-tubulincodon 200 polymorphism
Type
Research Article
Information
Parasitology , Volume 127 , Issue 5 , November 2003 , pp. 489 - 496
Copyright
2003 Cambridge University Press

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