Development and evaluation of PCR test for detection of Taylorella equigenitalis

Abstract

A PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis, was developed and evaluated. A genus-specific primer-probe set was derived from the 16S ribosomal DNA sequences. The PCR was specific and amplified a 585-bp product from all 64 available T. equigenitalis isolates. This PCR product hybridized with a specific probe in a dot spot assay. A variety of microorganisms from the genital tracts of horses or with a close phylogenetic relationship to T. equigenitalis did not yield a visible PCR product and were all negative in the dot spot hybridization assay. The results of the PCR assay were compared with those of culture by using 191 genital swabs from horses of several breeds. We demonstrate that the sensitivity of the PCR assay is superior to that of culture. The assay is most sensitive when DNA from culture plates incubated for at least 2 days is used. Of the tested samples, 1.5% were positive in the culture assay, whereas 35% were positive in the culture PCR assay. PCR-positive samples were obtained from all breeds tested. This means that many T. equigenitalis-carrying horses go unidentified by the current culturing technique. This affects current views about the spread and control of T. equigenitalis.

Prevalence of Taylorella equigenitalis infection in stallions in Slovenia: bacteriology compared with PCR examination

“…The simplicity, clarity and rapidity of the PCR assay should be noted when considering the PCR as a good supplementary procedure for detection of T. equigenitalis. Bleumink-Pluym et al (1994) reported the development and application of a PCR capable of detecting 10-15 bacteria based on the sequence of the 16S rRNA gene which is present in multiple copies in the bacterial genome. Anzai et al (1999) developed and evaluated a PCR with species-specific primer sets derived from the DNA sequence of a cloned DNA fragment of T. equigenitalis that did not hybridise with the genome of taxonomically related species.…”

“…The suspensions were heated at 100°C for 15 mins and then centrifuged at 15,000 g for 2 mins. Five µl supernatant were used for PCR, which was performed according to 2 different protocols with different primer sets as described previously (Bleumink-Pluym et al 1994;Anzai et al 1999). The PCR products were electrophoresed in 2% agarose gel and visualised with ethidium bromide.…”

Differences between Taylorella equigenitalis strains in their invasion of and replication in cultured cells

“…3,1996 INVASION AND REPLICATION OF T. EQUIGENITALIS traits, probably encoded by separate genes. A clinical evaluation of a PCR demonstrates that T. equigenitalis is present in 35% of tested healthy horses (2). This means that a considerable number of horses carry Taylorella spp.…”

Development of loop-mediated isothermal amplification methods for detecting <i>Taylorella equigenitalis</i> and <i>Taylorella asinigenitalis</i>

“…A few polymerase chain reaction (PCR)-based methods for detecting T . equigenitalis have been described [ 1 , 2 ]. However, it is difficult to use PCR-based methods in less well-equipped laboratories, because special equipment such as a thermal cycler is needed.…”

“…Two PCR-based methods—namely PCR [ 2 ] and semi-nested PCR [ 1 ], were used to compare the analytical sensitivity for experimentally spiked samples. The PCR assay detects both T .…”