Detection of five viruses commonly implicated with Bovine Respiratory Disease using loop-mediated isothermal...

Area of Science:

• Veterinary Diagnostics
• Molecular Biology
• Virology

Background:

• Bovine respiratory disease (BRD) is a significant concern in cattle production.
• Accurate and rapid detection of causative agents is crucial for effective management.
• Existing diagnostic methods can be time-consuming and require extensive sample processing.

Purpose of the Study:

• To develop novel quantitative loop-mediated isothermal amplification (qLAMP) and reverse-transcription qLAMP (RT-qLAMP) assays.
• To detect five major viruses associated with BRD: BHV-1, BAV-3, BRSV, BVDV-1, and BPIV-3.
• To establish a streamlined pipeline for rapid assay development.

Main Methods:

• Development and validation of extraction-free qLAMP and RT-qLAMP assays.
• Testing with contrived samples spiked with whole viruses.
• Determination of limits of detection (LOD), analytical sensitivity, and analytical specificity.
• Implementation of a streamlined pipeline for assay design and characterization.

Main Results:

• Assays demonstrated limits of detection between 30 and 1,057 copies per reaction.
• LODs per swab ranged from 2 × 10^5 to 8.22 × 10^6 copies, depending on the virus.
• Analytical sensitivities ranged from 73% to 100%, and specificities ranged from 90% to 100%.
• A streamlined pipeline was established to accelerate assay development.

Conclusions:

• Novel qLAMP and RT-qLAMP assays provide sensitive and specific detection of key BRD viruses.
• The extraction-free nature and minimal sample processing enable rapid, chute-side diagnostics.
• The developed assays and pipeline facilitate timely diagnosis and treatment of BRD.
• This approach supports rapid development of diagnostic tools for emerging pathogens.