Development of a differential multiplex PCR assay for equine herpesvirus 1 and 4 as a diagnostic tool.

The research article outlines the development of a new diagnostic tool for differentiating between strains of Equine Herpesvirus 1 and 4 using a method known as multiplex polymerase chain reaction (PCR).

Multiplex PCR Development

• The researchers developed a multiplex PCR assay specifically for differentiating the strains of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4).
• Multiplex PCR is a deviation of PCR technique used to amplify multiple target DNA sequences in a single PCR experiment. This technique uses multiple distinct sets of primers that can anneal to specific DNA sequences and amplify them simultaneously.
• The primer is a short piece of DNA that is complementary to a section of the template DNA. It can bind to the template DNA and start the DNA formation in PCR.

Methodology of Experiment

• In this particular study, the primers were designed to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4.
• Since the TK gene region in EHV-1 and EHV-4 sequences differs, the differences resulted in fragments of different lengths after PCR amplification. Thus, this provided a means to differentiate these two types of viruses.

Validation of Results

• The researchers also validated their methodology by confirming the specificity of the largest PCR amplicon for EHV-4 via restriction digestion with HindIII, an enzyme known for cutting DNA at specific places.

Practical Implications of the Assay

• This developed assay proved to be a quick and sensitive method for typing EHV-1 and EHV-4 isolates and for detection and differentiation of both viruses in field samples in which infectious virus may no longer be available.
• The sensitivity of the assay was also improved by optimizing the process of PCR cycling and by visualizing the PCR products in ethidium bromide and silver-stained acrylamide gels. Ethidium bromide and silver staining are common techniques used in molecular biology for visualization of nucleic acids after being separated by gel electrophoresis.