Improved detection of Tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time PCR assay

McMillen, Lyle and Lew, Ala E. (2006) Improved detection of Tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time PCR assay. Veterinary Parasitology, 141 3-4: 204-215. doi:10.1016/j.vetpar.2006.06.012


Author McMillen, Lyle
Lew, Ala E.
Title Improved detection of Tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time PCR assay
Journal name Veterinary Parasitology   Check publisher's open access policy
ISSN 0304-4017
1873-2550
Publication date 2006-11
Sub-type Article (original research)
DOI 10.1016/j.vetpar.2006.06.012
Volume 141
Issue 3-4
Start page 204
End page 215
Total pages 12
Place of publication Amsterdam, Netherlands
Publisher Elsevier
Language eng
Formatted abstract
A Tritrichomonas foetus-specific 5' Taq nuclease assay using a 3' minor groove binder-DNA probe (TaqMan® MGB) targeting conserved regions of the internal transcribed spacer-1 (ITS-1) was developed and compared to established diagnostic procedures. Specificity of the assay was evaluated using bovine venereal microflora and a range of related trichomonad species. Assay sensitivity was evaluated with log10 dilutions of known numbers of cells, and compared to that for microscopy following culture (InPouchTM TF test kit) and the conventional TFR3-TFR4 PCR assay. The 5' Taq nuclease assay detected a single cell per assay from smegma or mucus which was 2500-fold or 250-fold more sensitive than microscopy following selective culture from smegma or mucus respectively, and 500-fold more sensitive than culture followed by conventional PCR assay. The sensitivity of the conventional PCR assay was comparable to the 5' Taq nuclease assay when testing purified DNA extracted from clinical specimens, whereas the 5' Taq nuclease assay sensitivity improved using crude cell lysates, which were not suitable as template for the conventional PCR assay. Urine was evaluated as a diagnostic specimen providing improved and equivalent levels of T. foetus detection in spiked urine by both microscopy following culture and direct 5' Taq nuclease detection, respectively, compared with smegma and mucus, however inconclusive results were obtained with urine samples from the field study. Diagnostic specimens (n = 159) were collected from herds with culture positive animals and of the 14 animals positive by 5' Taq nuclease assay, 3 were confirmed by selective culture/microscopy detection (Fisher’s exact test P < 0.001). The 5' Taq nuclease assay described here demonstrated superior sensitivity to traditional culture/microscopy and offers advantages over the application of conventional PCR for the detection of T. foetus in clinical samples.
Keyword Tritrichomonas foetus
Bovine venereal disease
5 ' Taq nuclease
Minor groove binder
TaqMan (R)
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: School of Agriculture and Food Sciences
Queensland Alliance for Agriculture and Food Innovation
 
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Created: Mon, 07 Mar 2011, 15:14:49 EST