Hydroxymethylbilane synthase (HMBS) gene-based endogenous internal control for avian species

Area of Science:

• Veterinary diagnostics
• Molecular biology
• Avian pathology

Background:

• Polymerase Chain Reaction (PCR) is crucial for diagnosing poultry infectious diseases.
• A need exists for an endogenous internal control (EIC) to ensure PCR assay reliability in poultry.
• Existing controls may not adequately address variations in sample quality and quantity.

Purpose of the Study:

• To develop and validate a SYBR-quantitative PCR (qPCR) assay for an avian-specific EIC.
• To target the poultry homolog of the hydroxymethylbilane synthase (HMBS) gene for this EIC.
• To assess the EIC's effectiveness in diverse poultry samples and diagnostic scenarios.

Main Methods:

• Developed a SYBR-qPCR assay targeting the avian HMBS gene.
• Tested the assay's sensitivity and specificity using avian DNA.
• Evaluated the EIC's performance across 11 different chicken organs and tissues.
• Applied the EIC to clinically and experimentally infected poultry samples.

Main Results:

• The avian HMBS-based qPCR demonstrated high sensitivity, detecting a single gene copy.
• The assay showed high specificity for avian species.
• Amplification was successful from 11 diverse chicken organs and tissues.
• The EIC application revealed that false negatives and variations stem from sampling and storage methods.

Conclusions:

• The avian HMBS-based qPCR is a sensitive and specific EIC ideal for poultry DNA and RNA diagnostics.
• This EIC effectively quantifies DNA from various tissues and poultry species.
• The study underscores the critical importance of proper sample collection and storage for accurate molecular diagnostics.