Selection and validation of a real-time rt-PCR assay for the detection of equine arteritis virus and preliminary investigations into the prevalence of carrier stallions in the Netherlands

Abstract

use of a labeled probe, to enable detection of amplified products using a LFD. The assay to identify T. equigenitalis successfully amplified a 121bp fragment of the 23S rDNA genes. The assay detected as few as 46 gene copies in 12 minutes. The assay did not cross-hybridise with any of the 38 different bacterial organisms tested. The RPA assay was tested against a panel of 60 clinical equine genital swabs. All of the culture negative DNA extracts were correctly identified. Of the 30 culture positive DNA extracts, 24 were correctly identified. Culture positive, RPA negative DNA extracts had high real-time PCR CT values, indicating low levels of target DNA present. The introduction of a second probe into the assay enabled the simultaneous detection and differentiation of T. equigenitalis and T. asinigenitalis. The T. equigenitalis assaywas successfully adapted to enable detection of amplification products LFDs. We have successfully developed a rapid and sensitive isothermal assay to identify T. equigenitalis. Additionally, the inclusion of a second probe enables the detection of T. asinigenitalis. The amplification products can be detected in real time or via LFD. This test provides a fast (under 15 minutes) and accurate means of identification. The low energy requirements, lack of expensive equipment and LFD readout makes this test a suitable candidate for rapid diagnosis in low technology laboratories or near to the point of sampling.