Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes

T K Walsh, A A Donnan, F Jackson, P Skuce, A J Wolstenholme

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Benzimidazole resistance is a common problem in parasitic nematodes of ruminants and early detection is vital if its spread is to be monitored and controlled. Real time PCR offers a fast and reliable method for rapid detection and measurement of resistance allele frequencies. In Haemonchus contortus a single nucleotide polymorphism at codon 200 of the P-tubulin gene (TTC to TAC), causing a phenylalanine to tyrosine amino acid substitution, has been shown to be involved in many cases of resistance. Locked nucleic acid (LNA) Taqman probes have been used in this work to detect and measure the frequency of resistance alleles in individual and multiple H. contortus. Detection of resistant genotypes using LNA Taqman probes in individual H. contortus is simpler and more reliable than with previously described assays. Measurement of the frequency of resistant alleles in populations of H. contortus was achieved by using the cycle threshold (Q values and a standard curve derived from populations with known allele frequencies. Results using the LNA probes on individual and multiple worms gave similar results to the allele specific PCR. The sensitivity of the LNA assay on multiple nematodes allowed reliable detection of >= 10% resistance allele frequency. Using the final fluorescence method, it was possible to differentiate populations with similar to 0, 5 and 10% resistance allele frequencies. (c) 2006 Elsevier B.V. All rights reserved.
Original languageEnglish
Pages (from-to)304-312
Number of pages9
JournalVeterinary Parasitology
Volume144
Issue number3-4
DOIs
Publication statusPublished - 2007

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Nucleic Acid Probes
Haemonchus
benzimidazole
Haemonchus contortus
Gene Frequency
nucleic acids
gene frequency
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
Alleles
alleles
animal parasitic nematodes
amino acid substitution
assays
tubulin
phenylalanine
codons
rapid methods
single nucleotide polymorphism
Population

Cite this

Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes. / Walsh, T K; Donnan, A A; Jackson, F; Skuce, P; Wolstenholme, A J.

In: Veterinary Parasitology, Vol. 144, No. 3-4, 2007, p. 304-312.

Research output: Contribution to journalArticle

Walsh, T K ; Donnan, A A ; Jackson, F ; Skuce, P ; Wolstenholme, A J. / Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes. In: Veterinary Parasitology. 2007 ; Vol. 144, No. 3-4. pp. 304-312.
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AB - Benzimidazole resistance is a common problem in parasitic nematodes of ruminants and early detection is vital if its spread is to be monitored and controlled. Real time PCR offers a fast and reliable method for rapid detection and measurement of resistance allele frequencies. In Haemonchus contortus a single nucleotide polymorphism at codon 200 of the P-tubulin gene (TTC to TAC), causing a phenylalanine to tyrosine amino acid substitution, has been shown to be involved in many cases of resistance. Locked nucleic acid (LNA) Taqman probes have been used in this work to detect and measure the frequency of resistance alleles in individual and multiple H. contortus. Detection of resistant genotypes using LNA Taqman probes in individual H. contortus is simpler and more reliable than with previously described assays. Measurement of the frequency of resistant alleles in populations of H. contortus was achieved by using the cycle threshold (Q values and a standard curve derived from populations with known allele frequencies. Results using the LNA probes on individual and multiple worms gave similar results to the allele specific PCR. The sensitivity of the LNA assay on multiple nematodes allowed reliable detection of >= 10% resistance allele frequency. Using the final fluorescence method, it was possible to differentiate populations with similar to 0, 5 and 10% resistance allele frequencies. (c) 2006 Elsevier B.V. All rights reserved.

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