Detection of hepatitis a virus from oyster by nested PCR using efficient extraction and concentration method

Area of Science:

• Food safety and microbiology
• Molecular virology
• Public health

Background:

• Polymerase chain reaction (PCR) is vital for detecting viral pathogens in food and water.
• Current PCR methods struggle with low sensitivity due to inhibitors and inefficient virus concentration/purification.
• Hepatitis A virus (HAV) detection in shellfish requires sensitive and reliable methods.

Purpose of the Study:

• To develop an improved protocol for detecting hepatitis A virus (HAV) in oyster digestive glands.
• To enhance the sensitivity of PCR-based viral detection in complex food matrices.
• To establish a more efficient method for concentrating and purifying viral nucleic acids.

Main Methods:

• Development of the TPTT (tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead) protocol.
• Comparison of HAV precipitation methods using zirconium hydroxide versus PEG/NaCl.
• Nested PCR for sensitive detection of HAV in oyster homogenates.

Main Results:

• The TPTT protocol demonstrated significantly higher sensitivity compared to zirconium hydroxide precipitation.
• Detection limit achieved was 0.0148 PFU/g of oyster homogenate using PEG/NaCl precipitation.
• The TPTT method proved 10(5) fold more sensitive than traditional methods.

Conclusions:

• The TPTT protocol offers an efficient and highly sensitive method for detecting HAV in oysters.
• This method is suitable for routine detection of HAV in shellfish, improving food safety surveillance.
• The developed protocol surpasses the sensitivity of most existing published tests for HAV detection.