A rapid single-tube protocol for HAV detection by nested real-time PCR

Area of Science:

• Food safety
• Virology
• Molecular diagnostics

Background:

• Food-borne viral infections, such as hepatitis A virus (HAV), pose significant global public health risks.
• Accurate detection of food-borne viruses is challenging due to difficulties in direct isolation from contaminated food matrices.
• Highly sensitive molecular techniques are crucial for detecting viral genetic material in food samples.

Purpose of the Study:

• To develop and validate a novel single-tube nested real-time PCR (NRT-PCR) assay for the sensitive detection of HAV in food samples.
• To improve upon existing NRT-PCR methods by reducing sample manipulation and minimizing cross-contamination risks.
• To assess the performance of the single-tube NRT-PCR assay in terms of sensitivity, specificity, and efficiency.

Main Methods:

• Development of a single-tube NRT-PCR protocol integrating reverse transcriptase PCR, nested-PCR, and RT-PCR steps.
• Isolation of HAV RNA from both HAV-spiked food samples (green onions) and HAV-infected cell cultures.
• Validation of the assay by comparing results with conventional RT-PCR and nested-PCR methods.

Main Results:

• The single-tube NRT-PCR assay demonstrated comparable specificity and robustness to the original NRT-PCR method.
• All samples positive by conventional RT-PCR and nested-PCR were also detected by the single-tube NRT-PCR assay.
• The assay achieved detection limits of 0.1 PFU for HAV-infected cell cultures and 1 PFU for HAV-spiked green onions.

Conclusions:

• The single-tube NRT-PCR assay offers a streamlined and efficient approach for HAV detection in food.
• This method significantly reduces sample handling, turnaround time, and the risk of carry-over contamination.
• The developed assay is a promising tool for enhancing food safety and protecting public health from HAV contamination.