Efficacy of cold atmospheric plasma for inactivation of viruses on raspberries

In this study, the effectiveness of cold atmospheric plasma (CAP) in inactivating murine norovirus (MNV/human norovirus surrogate) and hepatitis A virus (HAV) on aerosol-inoculated dark red Willamette raspberries was explored. Pulsed positive corona discharge system fed by synthetic air was used for the production of CAP. Raspberries were treated for 1, 3, 5, 7, and 10 min at 25 W. Application of CAP enabled a 4 log₁₀ infectivity reduction in <5 min for MNV and approximately 10 min for HAV (from starting level of 6.91 and 7.84 log₁₀ PFU/mL, respectively). Viral genome copies reduction of 3.18 log₁₀ for MNV and 4.32 for HAV were found from starting level of 5.76 and 6.47 log₁₀ gc/μL, respectively. CAP treatment did not result in significant degradation of fruit color, an important quality attribute. The study demonstrated CAP as an efficient post-harvest decontamination method to reduce viral load in raspberries without significantly affecting its quality parameters.Industrial relevanceDue to the fast-processing paces required in the raspberry industry, it is difficult to assure the complete microbiological safety of this fruit. Cold atmospheric plasma is a practical, environmentally-friendly, non-thermal tool for the effective reduction of microbial pathogens. The model developed in this study demonstrated that CAP treatment of fresh raspberries not only inactivated hazardous enteric viruses in a short time (10 min) but also unaffected fruit color stability. The simplicity of described CAP design and low-cost inputs (air and electricity) enable the commercial application of inexpensive plasma chambers for continuous surface decontamination of large volumes of raspberries without bringing processing to a standstill.     

Rapid virus detection procedure for molecular tracing of shellfish associated with disease outbreaks

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III-spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.     

An ultracentrifugation-based approach to the detection of hepatitis A virus in soft fruits

A method was developed for detection of hepatitis A virus (HAV) in soft fruits (raspberries and strawberries). After washing the sample in 1 M sodium bicarbonate with added soya protein, fruits were removed by slow speed centrifugation, then particulate material and residual pectin were removed from the supernatant by flocculation and pectinase treatment during another slow speed centrifugation. Virus particles were then sedimented by ultracentrifugation. RNA was extracted from the virus particles, and nested RTPCR was performed on the nucleic acid extract. Nested RTPCR comprised an RTPCR, followed by PCR to amplify sequences within the amplicon. Internal amplification controls (IACs) were constructed for both the RTPCR and the PCR. The sensitivity of the nested RTPCR was approximately 10 RTPCRU. The overall method was shown to be able to detect 10(4) RTPCRU HAV in 90 g fresh strawberries, and 10(3) RTPCRU HAV in 60 g fresh raspberries. It is estimated that the lowest possible limit of detection of the method should be between 40 and 400 RTPCRU HAV per fruit sample. The method can be performed within one day, in suitably equipped microbiological laboratories, and is suitable for routine screening of food samples, and for analysis of suspected samples, e.g. during outbreak investigations.     

Differences in the binding of human norovirus to and from romaine lettuce and raspberries by water and electrolyzed waters

Food contamination by human norovirus (hNoV) is a major cause of gastrointestinal disease. We evaluated the effectiveness of removing inoculated hNoV from the surfaces of raspberries and romaine lettuce by a simple wash in tap water and in different forms of electrolyzed water (EW), including acidic EW (AEW), neutral EW (NEW), and basic EW (BEW). A simple rinsing or soaking in water was able to remove >95% of hNoV from surface-inoculated raspberries. In contrast, only 75% of hNoV was removed from surface-inoculated romaine lettuce by rinsing in tap water. An AEW wash enhanced the binding of hNoV to raspberries and lettuce. Only 7.5% (±10%) and 4% (±3.1%) of hNoV were removed by AEW wash from surface-inoculated raspberries and lettuce, respectively. When raspberries and lettuce were prewashed with NEW or BEW prior to surface inoculation, an AEW wash likewise resulted in significantly less removal of hNoV compared with untreated samples. A prewash with AEW significantly decreased the removal of hNoV from raspberries and lettuce when they were washed with NEW, from 90.6 to 51% and from 76 to 51.3% , respectively. There are minimal or no improvements gained by use of any of the EWs instead of a regular tap water wash in removal of hNoV from produce. However, use of AEW shows a significant decrease in the removal of hNoV from contaminated produce compared with other water rinses. The ability to remove hNoV from different types of produce varies, possibly due to differences among types of ligand-like molecules that bind hNoV. The distribution of hNoV on raspberries and lettuce was studied using recombinant Norwalk-like particles (rNVLP). By immunofluorescence microscopy, we were able to observe binding of rNVLP only to vein areas of romaine lettuce, suggesting that the virus was binding to specific molecules in these areas. Random binding of rNVLP occurred only with raspberries prewashed with AEW or washed with AEW.     

微滴式数字PCR检测冷冻草莓中GⅠ、GⅡ型诺如病毒

目的:建立一种检测冷冻草莓中诺如病毒(GⅠ和GⅡ)的逆转录微滴数字PCR (RT-ddPCR)方法。方法:根据ISO标准选定检测引物,优化反应体系,退火温度,进行了方法学实验,建立了一种快速检测冷冻草莓中GⅠ和GⅡ亚型诺如病毒的新方法。结果:确定了数字PCR检测GⅠ型诺如病毒退火温度为56.5℃,GⅡ型诺如病毒退火温度为58.1℃。RT-ddPCR检测GⅠ质粒标准品标准曲线的R2=0.9947,RT-ddPCR检测GⅡ质粒标准品标准曲线的R2=0.9950,说明该方法具有良好的线性关系。与RT-qPCR灵敏度对比,RT-ddPCR法的灵敏度比RT-qPCR法高一个数量级。在检测范围内,最低检测限低至个位拷贝数。RSD最小为3.8%,表明该实验重复性良好。浓度较低100 copies/μL左右时,RT-ddPCR的重复性不佳。结论:本研究建立的诺如病毒数字PCR法具有特异性强、灵敏度高、检测限低等优点,可用于冷冻草莓中诺如病毒的定量检测。