Pandemic A(H1N1)2009 influenza virus detection by real time RT-PCR: is viral quantification useful?

Area of Science:

• Virology
• Molecular Biology
• Infectious Diseases

Background:

• The 2009 H1N1 influenza pandemic highlighted the need for sensitive and reliable influenza A virus detection methods.
• Real-time reverse transcription polymerase chain reaction (RT-PCR) offers viral quantification capabilities.
• Accurate viral load assessment is crucial for disease management and understanding influenza pathogenesis.

Purpose of the Study:

• To develop and evaluate a universal influenza A RT-PCR assay targeting the matrix (M) gene.
• To integrate RNaseP detection for real-time assessment of sample quality.
• To establish a reliable method for quantifying influenza A virus RNA in clinical samples.

Main Methods:

• A universal influenza A RT-PCR targeting the M gene was designed.
• An RNaseP RT-PCR was incorporated for internal control and sample quality assessment.
• The combined RT-PCR method was evaluated on 209 clinical samples from pediatric patients.
• Viral loads (Ct values) were corrected based on RNaseP Ct values to account for sample quality.

Main Results:

• The universal M gene RT-PCR detected all influenza A subtypes, including H1N1 2009.
• RNaseP detection provided real-time quality control for specimen integrity.
• Mean viral load was estimated at 6.84 log RNA copies/mL.
• Corrections for poor quality samples (RNaseP Ct > 27) adjusted M gene Ct values by +3 to +8.
• Corrected influenza Ct values were lower in samples collected later in the disease course.

Conclusions:

• Real-time RT-PCR targeting the matrix gene is a reliable tool for quantifying influenza A virus.
• Incorporating cellular gene quantification (RNaseP) is essential for accurate viral load estimation.
• This validated method can aid in disease management, particularly when serial specimens are collected.