Application of a simple method using minute particles of amorphous calcium phosphate for recovery of norovirus from cabbage, lettuce, and ham.

In this study, the amorphous calcium phosphate (ACP) method developed previously for calicivirus concentration from water was applied for norovirus detection from food. The viral recovery from cabbage, lettuce, or ham (10g of each) was firstly examined in seeding experiments with feline caliciviruses (FCVs). The viruses were concentrated by viral adsorption to ACP particles (0.3g) in the eluent solution (40ml) from foods, collection of the particles by centrifugation, followed by dissolution of the particles with 3.3M citric acid (3ml). In ham, FCV recovery was improved by addition of ascorbic acids into the eluent solution before ACP-particle adsorption. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed that FCV recoveries were 32-33%, 50-55%, and 37-46% from cabbage, lettuce, and ham, respectively, when seeded with 10(3)-10(4) viruses, and detection limits were estimated ∼10(3) genomic copies in all 3 foods. Subsequently, the ACP-concentration method was evaluated for norovirus (NoV) detection from these 3 foods. The recoveries and detection limit of NoVs determined by qRT-PCR were 12-41% and 10(3) (genomic copies) from cabbage, 30-57% and 10(3) from lettuce, and 20-26% and 10(4) from ham, when seeded with 10(3)-10(5) viruses. This simple method may be suitable for NoV detection from these foods.

Novel concentration method for the detection of norovirus and sapovirus from water using minute particles of amorphous calcium phosphate.

A novel concentration method using minute particles of amorphous calcium phosphate (ACP) was developed for the detection of caliciviruses including norovirus and sapovirus, agents of human gastroenteritis, from water. In seeding experiments with feline calicivirus (FCV), ACP particles were able to adsorb efficiently the viruses in water, and the FCV-concentrated solution was obtained by dissolution of the virus-adsorbing ACP particles with citric acid after centrifugation. By quantitative real-time RT-PCR, the recovery efficiencies from 300 ml ultrapure water seeded with 10³, 104 and >105 copies of FCV were 48, 68 and >100 %, respectively. A comparative study showed that in the addition of viruses at <105 copies, the recovery efficiency of our method was significantly higher (P<0.05) than that of the similar calcium flocculation-citrate dissolution method. Using our newly developed method, we successfully detected 2.1 x 104 copies l⁻¹ of norovirus (each of genogroups I and II) and 5.4 x 10³ copies l⁻¹ of sapovirus (genogroups I, II, IV and V) from river water. The data suggest that our new viral concentration is a rapid, simple, cost efficient and high virus recovery method, and it can be used for routine monitoring of norovirus and sapovirus in water, especially environmental water.

[An epidemiologic study on prevalence of allergen-specific IgE antibodies in school children living in rural area of Saitama Prefecture, Japan].

BACKGROUND: In order to estimate the sensitization to inhalant allergen in childhood, prevalence of allergen-specific IgE antibodies was retrospectively surveyed on sera from school children aged from 9 to 15 living in rural area of Saitama prefecture, Japan, in 2001 and five years ago from 2001 (1996). METHODS: Allergen-specific IgE antibodies against Japanese cedar pollen (JCP), ragweed, sweet vernal grass, Dermatophagoides pteronyssinus (Dp), house dust (HD) and cat dander were examined on sera from 79 school children aged from 9 to 11 (group A) and 119 school children aged from 12 to 15 (group B) collected in 2001, and sera from 117 school children aged from 9 to 11 and 56 school children aged from 12 to 15 collected in 1996. Furthermore, a questionnaire survey about diagnosis of any allergic diseases and symptoms associated with the diseases was conducted on the same subjects in 2001. RESULTS: On the survey from school children, group A and B, collected in 2001, percentage of positive cases for allergen-specific IgE antibodies against any of 6 allergens were 49 in both of the groups for JCP, 10 and 12 for ragweed, 18 and 19 for sweet vernal grass, 39 in both of the groups for Dp, 42 in both of the groups for HD, 23 and 14 for cat dander, respectively. On the survey from school children collected in 1996, there is no significant difference from the percentage of positive cases for allergen-specific IgE antibodies against each allergen in 2001. Among the subjects followed-up on the survey in both of the years, the percentage of positive cases for allergen-specific IgE antibodies against each of six allergens on the survey in 2001 increased as compared with those in 1996. As a result of questionnaire survey in 2001, percentage (70: group A, 89: group B) of positive cases for allergen-specific IgE antibodies against any 6 allergens in the subjects with any symptoms associated with allergic diseases, but not diagnosed, was significantly higher than the percentage (34: group A, 48: group B) of that in the subjects without any symptoms associated with the diseases. CONCLUSION: Further prospective study is required to clarify a cause of allergic diseases in childhood, and it should be taken precautionary measures during childhood against sensitization with inhalant allergen to prevent allergic diseases in surveyed area.