title: A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System.
abstract: Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.
authors.affiliation: Université Paris-Est, ANSES, Food Safety Laboratory, Enteric viruses Unit, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.
authors.forename: Coralie
authors.initials: C
authors.lastname: Coudray-Meunier
authors.affiliation: Université Paris-Est, ANSES, Food Safety Laboratory, Enteric viruses Unit, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Fraisse
authors.affiliation: Université Paris-Est, ANSES, Food Safety Laboratory, Enteric viruses Unit, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Martin-Latil
authors.affiliation: Université Paris-Est, ANSES, Food Safety Laboratory, Identypath, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.
authors.forename: Sabine
authors.initials: S
authors.lastname: Delannoy
authors.affiliation: Université Paris-Est, ANSES, Food Safety Laboratory, Identypath, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Fach
authors.affiliation: Université Paris-Est, ANSES, Food Safety Laboratory, Enteric viruses Unit, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Perelle
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D052660:Bocavirus / Q000235:genetics; D017931:DNA Primers / Q000138:chemical synthesis*; D004770:Enterovirus / Q000235:genetics; D030041:Hepatitis A virus / Q000235:genetics; D016752:Hepatitis E virus / Q000235:genetics; D006801:Humans; D052699:Kobuvirus / Q000235:genetics; D056656:Lab-On-A-Chip Devices; D001257:Mamastrovirus / Q000235:genetics; D008574:Mengovirus / Q000235:genetics; D049329:Nanostructures; D029322:Norovirus / Q000235:genetics; D017992:Parvovirus / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012401:Rotavirus / Q000235:genetics; D029341:Sapovirus / Q000235:genetics; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted / Q000295:instrumentation; D014777:Virus Diseases / Q000175:diagnosis* / Q000821:virology
chemical_list: D017931:DNA Primers
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