Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid.

The researchers compared two specific types of genetic detection methods for finding a virus called Equine arteritis virus (EAV) in horse semen and lab-grown cells. They found that one method was more sensitive for detecting the virus in horse semen, but it was less sensitive than the standard cell culture technique. Both methods were highly effective for detecting the virus in lab-grown cells. The researchers suggest confirmation tests are still needed for certain cases, and stress the importance of comparing and validating testing methods.

Comparison of Two RT-PCR Assays

• The researchers compared two 1-tube real-time reverse transcription polymerase chain reaction (RT-PCR) assays, called T1 and T2, for detecting Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF).
• Competing against the traditional method of virus isolation (VI) in cell culture, the assays were tested for their specificity (how well they detect only the intended target), and sensitivity (how well they can detect low amounts of the target).

Sensitivity and Specificity of the Assays

• The T1 was more sensitive than the T2 with 93.4% versus 42.6% sensitivity for detection of EAV RNA in semen.
• However, the T1 assay was less sensitive than the gold standard VI test as prescribed by the World Organization for Animal Health (OIE).
• Both PCR assays showed high sensitivity levels when detecting EAV RNA in TCF, with T1 having a full 100% sensitivity and T2 following close behind with a 95.2% sensitivity.

Implications of the Findings

• The study points out discrepancies in sensitivity between these RT-PCR assays and the VI test, particularly noting that horse semen samples from EAV-seropositive stallions that test negative for EAV by the RT-PCR assays should be further confirmed free of virus by the VI method.
• Similarly, TCF samples that are VI-positive but RT-PCR negative should also be confirmed using a one-way neutralization test or fluorescent antibody test specifically targeting EAV.
• If these further tests confirm the absence of EAV, such samples should then be tested for the presence of other equine viral pathogens to ascertain what other viruses may be present.
• This study highlights the importance of making comparative evaluations and validations of real-time RT-PCR assays before their use in diagnostic settings for the detection and identification of specific infectious agents.