Application of real-time PCR for evaluation of distribution of equine herpesvirus type 1 in tissues of aborted fetuses.

This research paper details the use of real-time PCR (Polymerase Chain Reaction), a highly sensitive and precise method, for detecting and quantifying the distribution of equine herpesvirus type 1 (EHV-1) in the different internal organs of aborted horse fetuses.

Methodology

• Twenty-three tissue samples were taken from aborted horse fetuses and submitted to the National Veterinary Research Institute in Pulawy for testing. These samples were collected over the period of 2012 to 2013.
• Total DNA was extracted from these samples using a standard protocol involving phenol-chloroform-isoamyl alcohol.
• The researchers utilised a real-time PCR assay. This method is a modern variation on the polymerase chain reaction, which amplifies DNA to create millions of copies of a particular DNA sequence. This allows for the detection and quantification of specific DNA sequences, in this case, EHV-1.
• For the real-time PCR assay, researchers used specific forward and reverse primers, which are short pieces of DNA that complement and bind to specific sequences in the EHV-1 DNA. These primers were targeted at a highly conserved region that encodes for viral glycoprotein B, a significant component of EHV-1.

Findings

• The real-time PCR assay was shown to be highly sensitive, with a detection limit of 6.0 x 10⁰ of EHV-1 DNA copies. This means that the assay can detect as few as 6 viral DNA copies, demonstrating its high level of sensitivity.
• The assay also showed a linear range from 10⁰ to 10⁷ molecules, indicating its capacity to accurately measure a wide range of viral loads.
• A substantial 69.5% (16 out of 23) of tested fetuses were found to have EHV-1. This reinforces the notion that EHV-1 is a significant cause of equine abortion.
• The EHV-1 DNA load varied among the different tissues tested. The highest DNA loads were found in the liver and lungs, while the lowest were in the thymus and placenta. This suggests that the virus may preferentially target certain tissues.

This study presents a highly sensitive and specific method for detecting EHV-1 in aborted horse fetuses, providing valuable insights into the distribution of the virus in fetal tissues.