Home › Biochemistry › High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR
Biochemistry JoVE (Open Access) Citable · DOI

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

DOI: 10.3791/54781-v
What you'll learn
  • ✓ Perform reverse transcription and pre-amplification of respiratory pathogen nucleic acids
  • ✓ Execute automated liquid handling for high-throughput PCR sample transfer
  • ✓ Analyze nanoscale PCR results using syndromic respiratory panels
  • ✓ Detect DNA and RNA pathogens in canine and equine specimens
Protocol

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Difficulty
advanced
Total time
~4–6 hours per batch (including RT, pre-amplification, liquid handling, and PCR run)
Biosafety
BSL-2

Steps

1
Perform reverse transcription and pre-amplification

Convert RNA pathogens to cDNA and amplify target sequences from extracted nucleic acids using syndromic respiratory PCR panels. This step prepares specimens for downstream nanoscale PCR detection.

▶ 01:00
2
Transfer amplified samples using liquid handler

Automate distribution of pre-amplified products to nanoscale PCR plates via automated liquid handling to enable high-throughput processing while minimizing contamination and user error.

▶ 03:47
3
Analyze nanoscale PCR amplification results

Review and interpret PCR output data, including threshold cycles and amplification curves, to determine pathogen detection status for respiratory pathogens.

▶ 07:58
4
Evaluate representative results from assays

Examine example outcomes from RNA assays (influenza virus A) and DNA assays (equine herpesvirus type 1) to validate assay performance and interpretation.

▶ 09:15
💬 Comments coming soon