scholarly journals Characterization of Ozone Disinfection of Murine Norovirus

2009 ◽  
Vol 76 (4) ◽  
pp. 1120-1124 ◽  
Author(s):  
Mi Young Lim ◽  
Ju-Mi Kim ◽  
Jung Eun Lee ◽  
GwangPyo Ko
Keyword(s):  
Public Health ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Plaque Assay ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Human Noroviruses ◽  
Pcr Assays ◽  
Ozone Disinfection

ABSTRACT Despite the importance of human noroviruses (NoVs) in public health, little information concerning the effectiveness of ozone against NoVs is available. We determined the efficacy of ozone disinfection using murine norovirus (MNV) as a surrogate of human NoV. MNV in ozone demand-free buffer was exposed to a predetermined dose of ozone at two different pHs and temperatures. The virus remaining in the solution was analyzed by plaque assay, real-time TaqMan reverse transcriptase PCR (RT-PCR) (short template), and long-template conventional RT-PCR. Under all conditions, more than 99% of the MNV was inactivated by ozone at 1 mg/liter within 2 min. Both RT-PCR assays significantly underestimated the inactivation of MNV, compared with that measured by plaque assay. Our results indicate that NoV may be more resistant to ozone than has been previously reported. Nevertheless, proper ozone disinfection practices can be used to easily control its transmission in water.

scholarly journals Simultaneous Detection of North American and European Porcine Reproductive and Respiratory Syndrome Virus Using Real-Time Quantitative Reverse Transcriptase–PCR

2005 ◽  
Vol 17 (2) ◽  
pp. 165-170 ◽  
Author(s):  
Steven B. Kleiboeker ◽  
Susan K. Schommer ◽  
Sang-Myeong Lee ◽  
Sandy Watkins ◽  
Wayne Chittick ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
North American ◽  
Simultaneous Detection ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Field Isolates ◽  
Pcr Assays

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time ( TaqMan) reverse transcriptase (RT)–PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5–10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.

scholarly journals Inactivation and UV Disinfection of Murine Norovirus with TiO2 under Various Environmental Conditions

2008 ◽  
Vol 74 (7) ◽  
pp. 2111-2117 ◽  
Author(s):  
JungEun Lee ◽  
KyungDuk Zoh ◽  
GwangPyo Ko
Keyword(s):  
Real Time ◽  
Salt Concentration ◽  
Environmental Conditions ◽  
Plaque Assay ◽  
Surface Characteristics ◽  
Uv Disinfection ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Stool Suspension

ABSTRACT We studied inactivation and UV disinfection of murine norovirus (MNV) as a surrogate for human norovirus. We investigated the effects of different surface characteristics, temperatures, and NaCl concentrations on MNV survival using both a plaque assay and a real-time TaqMan reverse transcription (RT)-PCR assay. MNV survived more than 40 days on diaper material, on gauze, and in a stool suspension. Compared to inactivation at lower temperatures (−20 and 4°C), inactivation of MNV was greater at higher temperatures (18 and 30°C). On the surface of both gauze and diaper material, there was a <2-log10 reduction in the amount of infectious MNV in 40 days after incubation at both −20 and 4°C, compared to a >5-log10 reduction after incubation at 30°C in 24 days. MNV survived better in a stool suspension than on the surface of gauze or diaper material. A higher salt concentration increased the rate of inactivation of MNV. In 72 h, <0.3-, 1.5-, and 2.5-log10 reductions in the amount of infectious MNV occurred in distilled water and 0.5 and 1 M NaCl, respectively. We observed only minor reductions in the numbers of viral RNA copies as quantified by real-time TaqMan RT-PCR regardless of the temperature, the salt concentration, or the suspending medium. We also evaluated UV disinfection of infectious MNV with and without TiO2. The amount of MNV was significantly reduced by 254-nm UV with and without TiO2. When 25 mJ/cm2 UV was used, 3.3- and 3.6-log10 reductions in the amounts of infectious MNV occurred with and without TiO2, respectively. Our results demonstrate that MNV can persist in various environmental conditions and can be efficiently controlled by UV disinfection.

scholarly journals Internalization and Dissemination of Human Norovirus and Animal Caliciviruses in Hydroponically Grown Romaine Lettuce

2012 ◽  
Vol 78 (17) ◽  
pp. 6143-6152 ◽  
Author(s):  
Erin DiCaprio ◽  
Yuanmei Ma ◽  
Anastasia Purgianto ◽  
John Hughes ◽  
Jianrong Li
Keyword(s):  
Real Time ◽  
Fresh Produce ◽  
Plaque Assay ◽  
Feed Water ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Romaine Lettuce ◽  
Human Norovirus ◽  
Genotype 4 ◽  
Tulane Virus

ABSTRACTFresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both pre- and postharvest stages. However, the ecology of human NoV in fresh produce is poorly understood. In this study, we determined whether human NoV and its surrogates can be internalized via roots and disseminated to edible portions of the plant. The roots of romaine lettuce growing in hydroponic feed water were inoculated with 1 × 106RNA copies/ml of a human NoV genogroup II genotype 4 (GII.4) strain or 1 × 106to 2 × 106PFU/ml of animal caliciviruses (Tulane virus [TV] and murine norovirus [MNV-1]), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were homogenized, and viral titers and/or RNA copies were determined by plaque assay and/or real-time reverse transcription (RT)-PCR. For human NoV, high levels of viral-genome RNA (105to 106RNA copies/g) were detected in leaves, shoots, and roots at day 1 postinoculation and remained stable over the 14-day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (101to 103PFU/g) were detected in leaves and shoots at days 1 and 2 postinoculation, but virus reached a peak titer (105to 106PFU/g) at day 3 or 7 postinoculation. In addition, human NoV had a rate of internalization comparable with that of TV as determined by real-time RT-PCR, whereas TV was more efficiently internalized than MNV-1 as determined by plaque assay. Taken together, these results demonstrated that human NoV and animal caliciviruses became internalized via roots and efficiently disseminated to the shoots and leaves of the lettuce.

scholarly journals Immunomagnetic Separation Combined with Real-Time Reverse Transcriptase PCR Assays for Detection of Norovirus in Contaminated Food

2008 ◽  
Vol 74 (13) ◽  
pp. 4226-4230 ◽  
Author(s):  
YoungBin Park ◽  
You-Hee Cho ◽  
YoungMee Jee ◽  
GwangPyo Ko
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Food Borne ◽  
Contaminated Food ◽  
Microbial Contaminants ◽  
Pcr Assays ◽  
Sensitive Method

ABSTRACT We developed an immunomagnetic separation (IMS) technique combined with real-time TaqMan reverse transcriptase PCR (RT-PCR), which allowed detection of norovirus at a level as low as 3 to 7 RT-PCR units from artificially contaminated strawberries. The inoculum recovery rate ranged from 14 to 30%. The data demonstrate that IMS combined with real-time RT-PCR will be useful as a rapid and sensitive method for detecting food-borne microbial contaminants.

Tenacity of Human Norovirus and the Surrogates Feline Calicivirus and Murine Norovirus during Long-Term Storage on Common Nonporous Food Contact Surfaces

2015 ◽  
Vol 78 (1) ◽  
pp. 224-229 ◽  
Author(s):  
SASCHA MORMANN ◽  
CATHRIN HEIßENBERG ◽  
JENS PFANNEBECKER ◽  
BARBARA BECKER
Keyword(s):  
Stainless Steel ◽  
Real Time ◽  
Room Temperature ◽  
Plaque Assay ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Human Norovirus ◽  
Food Contact Surfaces ◽  
Contact Surfaces

The transfer of human norovirus (hNV) to food via contaminated surfaces is highly probable during food production, processing, and preparation. In this study, the tenacity of hNV and its cultivable surrogates feline calicivirus (FCV) and murine norovirus (MNV) on two common nonporous surface materials at two storage temperatures was directly compared. Virus titer reduction on artificially inoculated stainless steel and plastic carriers was monitored for 70 days at room temperature and at 7°C. Viruses were recovered at various time points by elution. Genomes from intact capsids (hNV, FCV, and MNV) were quantified with real-time reverse transcription (RT) PCR, and infectivity (FCV and MNV) was assessed with plaque assay. RNase treatment before RNA extraction was used to eliminate exposed RNA and to assess capsid integrity. No significant differences in titer reduction were found between materials (stainless steel or plastic) with the plaque assay or the real-time quantitative RT-PCR. At room temperature, infectious FCV and MNV were detected for 7 days. Titers of intact hNV, FCV, and MNV capsids dropped gradually and were still detectable after 70 days with a loss of 3 to 4 log units. At 7°C, the viruses were considerably more stable than they were at room temperature. Although only MNV infectivity was unchanged after 70 days, the numbers of intact capsids (hNV, FCV, and MNV) were stable with less than a 1-log reduction. The results indicate that hNV persists on food contact surfaces and seems to remain infective for weeks. MNV appears to be more stable than FCV at 7°C, and thus is the most suitable surrogate for hNV under dry conditions. Although a perfect quantitative correlation between intact capsids and infective particles was not obtained, real-time quantitative RT-PCR provided qualitative data about hNV inactivation characteristics. The results of this comparative study might support future efforts in assessment of foodborne virus risk and food safety.

Effect of Temperature, pH, and NaCl on the Inactivation Kinetics of Murine Norovirus

2012 ◽  
Vol 75 (3) ◽  
pp. 533-540 ◽  
Author(s):  
KYEONGJIN SEO ◽  
JUNG EUN LEE ◽  
MI YOUNG LIM ◽  
GWANGPYO KO
Keyword(s):  
Real Time ◽  
Environmental Conditions ◽  
Low Ph ◽  
Effect Of Temperature ◽  
Inactivation Kinetics ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Human Norovirus ◽  
Pcr Assays ◽  
Kinetics Of

We investigated the resistance of murine norovirus (MNV) and coliphage MS2, a culturable human norovirus surrogate, to temperature, salt, and pH. Virus inactivation was measured by plaque, real-time TaqMan reverse transcription (RT) PCR, and long-template RT-PCR assays. Both MNV and MS2 were rapidly inactivated at temperatures above 60°C. Similarly, MNV tolerated low salt concentrations (0.3% NaCl) to a greater degree than high salt concentrations (3.3 to 6.3% NaCl). MNV was relatively resistant to strong acidic conditions (pH 2) and was more tolerant of slightly acidic (pH 4) or neutral (pH 7) conditions. In contrast, MS2 was resistant to high salinity. Overall, temperature had a greater effect on infectivity than salt or low pH. Additionally, temperature and low pH had a synergistic effect on MNV infectivity. Both real-time and long-template RT-PCR assays significantly underestimated the inactivation by temperature, salt, and pH. The inactivation kinetics of both MNV and MS2 under various environmental conditions gave a good fit by the Weibull model (R2 &gt; 0.9). This study suggests both the capacity of infectious human norovirus to persist in the face of various environmental conditions and its sensitivity to high temperatures, which may provide a mechanism of protection against this virus.

Critical Issues in Detecting Viable Listeria monocytogenes Cells by Real-Time Reverse Transcriptase PCR

2012 ◽  
Vol 75 (3) ◽  
pp. 512-517 ◽  
Author(s):  
LINLIN XIAO ◽  
LU ZHANG ◽  
HUA H. WANG
Keyword(s):  
Listeria Monocytogenes ◽  
16S Rrna ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Pcr Primers ◽  
Detection Methods ◽  
Rt Pcr ◽  
Heat Treated ◽  
Pcr Assays ◽  
The Impact

Rapid and specific detection of viable Listeria monocytogenes cells, particularly in processed foods, is a major challenge in the food industry. To assess the suitability of using RNA-based detection methods to detect viable cells, several sets of PCR primers and florescent probes were designed targeting the 16S rRNA, internalin A, and ribosomal protein L4 genes. One-step real-time reverse transcriptase (RT) PCR assays were conducted using RNAs extracted from control and heat-treated L. monocytogenes samples. The cycle threshold values were significantly higher in heat-treated cells than in controls. However, real-time RT-PCR amplification signals were still detected even in samples stored at room temperature for 24 h after lethal treatments, and the intensity of the signals was correlated with the cell population. The 16S rRNA molecules were the most stable of the three targets evaluated, and the impact on detection efficacy of the relative positions of the PCR primers within the target genes was limited under the experimental conditions. These results suggest that real-time RT-PCR assays have advantages over conventional PCR assays for assessing viable L. monocytogenes cells, but the results are affected by the stability of the RNA molecules targeted. These findings could have a major impact on interpretation of RNA-based detection data and gene expression studies.

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