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The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.

The aims of the study were to determine the survival of Escherichia coliO157 on lettuce as a function of temperature and light intensity, and to use that Information in a screening-level quantitative microbial risk assessment (QMRA) in order to evaluate risk-reducing strategies including irrigation water quality guidelines, rinsing, and holding time between last irrigation and harvest. Iceberg lettuce was grown in a climate chamber and inoculated with E. coliO157. Bacterial numbers were determined with the standard plate count method after inoculation and 1, 2, 4, and 7 day(s) postinoculation. The experiments were carried out at 11, 18, and 25°C in light intensities of 0, 400, and 600 mmol (m2)_1s_1. There was a significant effect of temperature and light intensity on survival, with less bacteria isolated from lettuce incubated at 25 and 18°C compared with 11°C (P < 0.0001), and in light intensities of 400 and 600 mmol (m2)–1s–1compared with 0 mmol (m2)_1s_1(P < 0.001). The average log reductions after 1, 2, 4, and 7 day(s) were 1.14, 1.71, 2.04, and 3.0, respectively. The QMRA compared the relative risk with lettuce consumption from 20 scenarios. A stricter water quality guideline gave a mean fivefold risk reduction. Holding times of 1, 2, 4, and 7 day(s) reduced the risk 3, 8, 8, and 18 times, respectively, compared with harvest the same day as the last irrigation. Finally, rinsing lettuce for 15 s in cold tap water prior to consumption gave a sixfold risk reduction compared with eating unrinsed lettuce. Sensitivity analyses indicated that variation in bacterial inactivation had the most significant effect on the risk outcome. A QMRA determining the relative risks between scenarios reduces uncertainty and can provide risk managers with decision support.