Duplex real-time qRT-PCR for the detection of hepatitis A virus in water and raspberries using the MS2 bacteriophage as a process control

Abstract

Hepatitis A virus (HAV) infection is the leading worldwide cause of acute viral hepatitis. An important aspect of viral control is rapid diagnosis. Epidemiological studies have linked hepatitis A outbreaks to the consumption of drinking water or soft fruits exposed to faecal contamination. Real-time reverse transcriptase PCR (qRT-PCR) is now widely used for detecting RNA viruses in food samples. Efficiency of viral concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction must be monitored to prevent false negative results. In this study, the MS2 bacteriophage used as a process control was detected simultaneously with HAV in a one-step duplex real-time qRT-PCR. The assay was developed for testing water and raspberries. Adding MS2 showed no loss of sensitivity for HAV detection in water and raspberry samples. The limit of detection of HAV with this new approach was 10 PFU for 1.5 L of bottled water, 100 PFU for 1.5 L of tap water, 50 PFU for 25 g of fresh raspberries and 100 PFU for 25 g of frozen raspberries. The data show that the MS2 offers a very reliable and simple way to monitor false-negative results, making it a valuable tool in the routine diagnostics laboratory.