Duplex real-time qRT-PCR for the detection of hepatitis A virus in water and raspberries using the MS2 bacteriophage as a process control.
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 10PFU for 1.5L of bottled water, 100PFU for 1.5L of tap water, 50PFU for 25g of fresh raspberries and 100PFU for 25g 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.
Blaise-Boisseau
Sandra
S
Unité VAE (Virologie des Aliments et de l'Eau), Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Laboratoire d'Etudes et de Recherches sur la Qualité des Aliments et sur les Procédés Agroalimentaires (LERQAP), 23 Avenue du Général de Gaulle, 94706 Maisons-Alfort Cedex, France.
Hennechart-Collette
Catherine
C
Guillier
Laurent
L
Perelle
Sylvie
S
D000818:Animals; D002460:Cell Line; D005638:Fruit / Q000382:microbiology*; D030041:Hepatitis A virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017909:Levivirus / Q000235:genetics*; D008253:Macaca mulatta; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000592:standards; D014871:Water Microbiology
D012367:RNA, Viral