Norovirus (NV) sometimes causes acute nonbacterial gastroenteritis through contaminated oysters in Japan. RT-PCR reaction or real-time RT-PCR reaction is generally used to detect NV from oysters; however, it is thought that some component in oyster frequently inhibits the PCR reaction. We examined the RNA extraction method for real-time RT-PCR reaction to detect NV: extraction with QIAamp Viral RNA MiniKit after extraction with an equal volume of phenol-chloroform-isoamyl alcohol (PQ method) using 10% homogenized oyster with added NV as a positive control. We tested both genogroups of NV, GI and GIIas positive control, using both domestic and imported oysters in this study. PQ method showed a higher sensitivity (97%) and collection rate compared with the standard method (extraction with QIAamp Viral RNA MiniKit, sensitivity: 57%) or CTAB compound method (extraction with QIAamp Viral RNA MiniKit after processing by the Cetyl trimethyl ammonium bromide method, sensitivity: 88%) irrespective of the genogroup or growing area of the oysters. Our results suggest that the PQ method may effectively remove some PCR inhibiter contained in oysters and is useful in a general laboratory as the extraction method for detection of Norovirus from oyster specimens.

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Arabinogalactan (AG) is a kind of water-soluble polysaccharide, which is long, densely branched and consists of galactose and arabinose. Type II AG composed of β-(1→3)-linked galactan main chain with frequent arabinose and galactose residues containing side chains. Furthermore, Type II AG is frequently distributed in coffee beans. AG from coffee beans was investigated for its effect on the growth of established enterobacterial strains. Thirty-four species of enterobacteria were cultivated in AG-containing media. After 72 hours cultivation, the cell number of Bifidobacterium longum and Bifidobacterium pseudocatenulatum especially increased, pH of the media significantly decreased and both DL-lactic acid and acetic acid accumulated. The enzyme produced by B. longum in the supernatant of its culture showed hydrolyzing activity toward AG from coffee bean and AG from larch wood. This enzyme generated arabinose, galactose, and unknown oligosaccharide when it was incubated with AGs. These observations indicate that B. longum and B. pseudocatenulatum selectively utilize AG as carbon-source. The digestion of AG by artificial digestive juices was investigated in vitro. AG artificial pancreatic juice, and only slightly digested by small intestinal enzymes. Therefore it could be expected that AG from coffee beans had potential as a prebiotic to promote the improvement of healthful human intestinal microflora.

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Salmonella infections of egg contents can be related to external contamination of the shell. In this study, the efficiency of recovery methods using swab-wipe (SW) and crush (CR) was evaluated for bactericidal activity of Salmonella inoculated onto the eggshell surface (10⁷ CFU/egg). When 200 ppm of sodium hypochlorite was used to sanitize the eggshell, the SW method could not detect bacteria after treatment for 2 min. However, the CR method could still detect Salmonella from the sample. There were 10⁵ CFU/swab-area reductions when the egg surface was sanitized with 200 ppm of sodium hypochlorite for 10 min.
However, when the egg contents (organic matter) were mixed with hypochlorite solutions, the reduction rate was decreased significantly. When the liquid egg was mixed above 0.5% in 200 ppm of sodium hypochlorite, the available chlorine concentration decreased immediately. There were 10³ CFU/swab-area reductions when hypoclorite solutions were mixed with 0.1% liquid egg and recovered by the CR method. However, the SW method yielded higher estimates of bactericidal activity against Salmonella than CR method. We tested 4,000 of commercial eggs to estimate bacterial contamination levels by both methods. The CR method with 37°C of PBS showed the best recovery condition, and showed 10-times higher recovery than the SW method. Therefore, the CR method could be a better alternative than the conventional SW method for detecting bacteria from eggshells.

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In our previous report, we evaluated and reported some VIDAS protocols for the detection of Salmonella. In this report, we evaluated the new additional protocol “Easy SLM” with “SX broth”, a new unique enrichment broth. This method needs about 2 days to complete the total protocol, 1-2 days less than the conventional culture method. We tested 77 food samples with and without artificial inoculation with Salmonella strains. The positive rate for each method of testing food samples with and without artificial inoculation of Salmonella strains were almost the same, and as a result, the sensitivity and specificity of the Easy SLM protocol and the conventional culture method were equal. Therefore, the Easy SLM protocol is useful for routine quality control for the detection of Salmonella because of its rapidity, ease of handling and automation for standardization.

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In 2003, the Ministry of Health, Labour and Welfare in Japan presented the official assay using ABI PRISM 7000^® for the detection of Norovirus (NV) genogroups I (GI) and II (GII). We have recently modified the official assay and, using LightCycler^® PCR equipment, have developed the “LC cDNA” and “LC One-Step” detection assays. These two assays enabled detection and quantitation of the NV genome within 6 hrs using the LC cDNA assay, and within 3 hr using the LC One-Step assay. The primers and probes used in both assays were identical to those used in the official assay, because there are no idiosyncrasies between them. The detection limits of our assays for the GI and GII genomes were 10 copies/5 μl. Results of field samples using both the LC cDNA and One-Step assays were the same. In correlation analysis between log transformed genome copy numbers of the LC cDNA and One-Step assays, fecal samples from food poisoning cases (n = 27) were y = 0.9627x-0.1034 (R² = 0.9888), and patient samples from a hospital (n = 22) were y = 1.0064x-0.3473 (R² = 0.9610). Utilizing LightCycler^® PCR equipment, our assays detected NV GI and GII. In particular, the One-Step assay could detect within 3 hrs; hence, it is a very useful laboratory technique for the detection of NV from field samples.

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[in Japanese]

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