Pseudorabies virus latency: a quantitative approach by polymerase chain reaction

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Quantitative PCR (qPCR) is crucial for detecting and quantifying nucleic acids.
• Accurate quantification of viral DNA, especially in latent infections, remains challenging.

Purpose of the Study:

• To establish a highly specific and sensitive quantitative PCR assay for detecting low amounts of viral DNA.
• To validate the use of an internal standard (mimic) for accurate co-amplification and quantification of target DNA.

Main Methods:

• Development of a quantitative PCR assay utilizing an internal standard (mimic) differing by a small deletion.
• Co-amplification of target DNA and mimic, followed by fluorescent labeling.
• Separation and detection of PCR products using an automated sequencer.

Main Results:

• A highly specific and sensitive qPCR assay was developed for the envelope glycoprotein gp50 gene.
• The assay demonstrated high reproducibility with a detection limit of one copy of PRV DNA.
• The chosen strategy confirmed adequacy for quantifying low amounts of virus DNA in latently infected swine.

Conclusions:

• The developed quantitative PCR assay with an internal standard is effective for precise viral DNA quantification.
• This method offers high sensitivity and reproducibility, suitable for detecting low viral loads in latent infections.
• The strategy is validated for applications in swine virology and disease monitoring.