title: Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water.
abstract: A quadruplex Real-Time RT-PCR assay for the simultaneous quantitative detection of hepatitis A virus (HAV), norovirus (NoV) GI and GII, and mengovirus (used as process control for determination of the virus/nucleic acid extraction efficiency) has been developed. This multiplex assay has been comparatively evaluated with the individual monoplex assays and showed to be slightly less sensitive, with average ΔCq values of 0.90, 0.28 and 0.44 for HAV, NoV GI and NoV GII, respectively, in standard curves of viral RNA, or 0.32, 0.37 and 0.51 for the same viruses respectively, in naturally-contaminated samples. These ΔCq values were mostly negligible since it represented, in the worst case scenario, a loss of 0.43 log in genome copy numbers. The quadruplex assay shows similar theoretical detection limits than the monoplex assay for NoV GII, and 10 times higher for HAV and NoV GI. However, when naturally-contaminated food and water samples were tested, these theoretical detection thresholds were often exceeded and very low genome copy numbers (below the limit of detection) could be quantified. The quadruplex assay fulfills the requirements of the method developed by the European Committee on Standardization (CEN) for virus detection in selected foodstuffs with significant advantages in labor and reagent costs.
authors.affiliation: Enteric Virus Laboratory of the Department of Microbiology, University of Barcelona, Barcelona, Spain; Institute of Nutrition and Food Safety, University of Barcelona, Barcelona, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Fuentes
authors.affiliation: Enteric Virus Laboratory of the Department of Microbiology, University of Barcelona, Barcelona, Spain; Institute of Nutrition and Food Safety, University of Barcelona, Barcelona, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Guix
authors.affiliation: Enteric Virus Laboratory of the Department of Microbiology, University of Barcelona, Barcelona, Spain; Institute of Nutrition and Food Safety, University of Barcelona, Barcelona, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Pérez-Rodriguez
authors.affiliation: Enteric Virus Laboratory of the Department of Microbiology, University of Barcelona, Barcelona, Spain; Institute of Nutrition and Food Safety, University of Barcelona, Barcelona, Spain.
authors.forename: Noemí
authors.initials: N
authors.lastname: Fuster
authors.affiliation: Public Health Agency of Catalonia, Generalitat de Catalunya, Manresa, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Carol
authors.affiliation: Enteric Virus Laboratory of the Department of Microbiology, University of Barcelona, Barcelona, Spain; Institute of Nutrition and Food Safety, University of Barcelona, Barcelona, Spain. Electronic address: rpinto@ub.edu.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Pintó
authors.affiliation: Enteric Virus Laboratory of the Department of Microbiology, University of Barcelona, Barcelona, Spain; Institute of Nutrition and Food Safety, University of Barcelona, Barcelona, Spain. Electronic address: abosch@ub.edu.
authors.forename: Albert
authors.initials: A
authors.lastname: Bosch
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000821:virology*; D005506:Food Contamination / Q000032:analysis; D005618:Fresh Water / Q000821:virology*; D030041:Hepatitis A virus / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D029322:Norovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012758:Shellfish / Q000821:virology*
keywords: Foodborne viruses; Multiplex; Virus assay; Waterborne viruses