Application of long-range and binding reverse transcription-quantitative PCR to indicate the viral integrities of noroviruses

• Published in: Applied and Environmental Microbiology 2014-10, Vol.80 (20), p.6473-6479
• Main Authors: Li, Dan, De Keuckelaere, Ann, Uyttendaele, Mieke
• Format: Article
• Language: English
• Subjects: Caco-2 Cells - virology; Capsid; Food Analysis - methods; Food Contamination; Food Microbiology; Genomics; Humans; Norovirus; Norovirus - genetics; Norovirus - isolation & purification; Norovirus - radiation effects; Polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; Rubus - virology; Shellfish - virology; Ultraviolet radiation; Ultraviolet Rays; Virology - methods; Virus Inactivation; Viruses
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/aem.02092-14

This study intends to establish and apply methods evaluating both viral capsid and genome integrities of human noroviruses (NoVs), which thus far remain nonculturable. Murine norovirus 1 (MNV-1) and human NoV GII.4 in phosphate-buffered saline suspensions were treated with heat, UV light, or ethanol and detected by reverse transcription-quantitative PCR (RT-qPCR), long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR. For MNV-1 heated at 60°C for 2 and 30 min, limited reductions of genomic copies (1.89-log reduction after 60°C for 30 min by binding long-range RT-qPCR). The human NoV GII.4 was found to be more heat resistant than MNV-1. For both MNV-1 and human NoV GII.4 after UV treatments of 20 and 200 mJ/cm(2), no significant difference (P > 0.05) was observed between the dose-dependent reductions obtained by the four detection methodologies. Treatment of 70% ethanol for 1 min was shown to be more effective for inactivation of both MNV-1 and human NoV GII.4 than the heat and UV treatments used in this study. Subsequently, eight raspberry and four shellfish samples previously shown to be naturally contaminated with human NoVs by RT-qPCR (GI and GII; thus, 24 RT-qPCR signals) were subjected to comparison by this method. RT-qPCR, long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR detected 20/24, 14/24, 24/24, and 23/24 positive signals, respectively, indicating the abundant presence of intact NoV particles.