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A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses.

Buckley TC, Millar BC, Egan CL, Gibson P, Cosgrove H, Stanbridge S, Matsuda M, Moore JE - Ir Vet J (2005)

Bottom Line: Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis.Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions.Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Irish Equine Centre, Johnstown, Naas, Co Kildare, Republic of Ireland. iec@equine-centre.ie.

ABSTRACT
: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.

No MeSH data available.


Sensitivity of the two-step PCR assay; PCR amplification of a 706 bp region of the 16S rDNA of Taylorella equigenitalis.
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Figure 2: Sensitivity of the two-step PCR assay; PCR amplification of a 706 bp region of the 16S rDNA of Taylorella equigenitalis.

Mentions: Two primers [TE16SrRNABCHforward and TE16SrRNABCHreverse] were selected from the 16S rRNA gene of T. equigenitalis as outlined in Figure 1. We tested the specificity of the primers both by BLAST searching at http://www.blast.genome.ad.jp, as well as by challenging the primers to DNA from non-T. equigenitalis organisms. BLAST searching demonstrated a score of 50 [E value 6 × 10-5] and 49 [E value 2 × 10-4] for the forward and reverse primers, respectively. There was no shared homology with any other bacterial or eukaryotic DNA with either primer, nor with the other species member within the genus Taylorella (i.e., T. asinigenitalis) where there were eight and four nonaligning base pairs in the forward and reverse primers, respectively, between these two species. An amplified product of approximately 706 bp was generated with all tested T. equigenitalis isolates (Figure 2) and no other bacterial genomic DNA was amplified, demonstrating the specificity of the primers for T. equigenitalis. Of the 250 animals screened for T. equigenitalis, none was positive for this organism by either culture or PCR methods, where the molecular sensitivity of the assay was shown to be no lower than 36 cells, which compares favourably to the three-step PCR assay, previously described by Bleumink-Pluym et al. [2], who calculated the sensitivity of their assay as 15 bacteria.


A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses.

Buckley TC, Millar BC, Egan CL, Gibson P, Cosgrove H, Stanbridge S, Matsuda M, Moore JE - Ir Vet J (2005)

Sensitivity of the two-step PCR assay; PCR amplification of a 706 bp region of the 16S rDNA of Taylorella equigenitalis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113911&req=5

Figure 2: Sensitivity of the two-step PCR assay; PCR amplification of a 706 bp region of the 16S rDNA of Taylorella equigenitalis.
Mentions: Two primers [TE16SrRNABCHforward and TE16SrRNABCHreverse] were selected from the 16S rRNA gene of T. equigenitalis as outlined in Figure 1. We tested the specificity of the primers both by BLAST searching at http://www.blast.genome.ad.jp, as well as by challenging the primers to DNA from non-T. equigenitalis organisms. BLAST searching demonstrated a score of 50 [E value 6 × 10-5] and 49 [E value 2 × 10-4] for the forward and reverse primers, respectively. There was no shared homology with any other bacterial or eukaryotic DNA with either primer, nor with the other species member within the genus Taylorella (i.e., T. asinigenitalis) where there were eight and four nonaligning base pairs in the forward and reverse primers, respectively, between these two species. An amplified product of approximately 706 bp was generated with all tested T. equigenitalis isolates (Figure 2) and no other bacterial genomic DNA was amplified, demonstrating the specificity of the primers for T. equigenitalis. Of the 250 animals screened for T. equigenitalis, none was positive for this organism by either culture or PCR methods, where the molecular sensitivity of the assay was shown to be no lower than 36 cells, which compares favourably to the three-step PCR assay, previously described by Bleumink-Pluym et al. [2], who calculated the sensitivity of their assay as 15 bacteria.

Bottom Line: Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis.Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions.Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Irish Equine Centre, Johnstown, Naas, Co Kildare, Republic of Ireland. iec@equine-centre.ie.

ABSTRACT
: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.

No MeSH data available.