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Human noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis worldwide withnumerous outbreaks being attributed to consumption of NoV-contaminated water. Based onepidemiological outbreak investigations and limited human volunteer challenge studies, it hasbeen reported that NoVs are resistant to environmental degradation and chemical inactivation.The absence of a cell culture infectivity model for human NoVs has required the use ofmolecular methods to monitor changes in NoV nucleic acid persistence in conjunction with theuse of viral surrogates amenable to cell culture. To evaluate the stability of human NoVs insource water used by water treatment plants, the prototype genogroup I NoV; Norwalk virus(NV) and the viral surrogates feline calicivirus (FCV), poliovirus (PV) and MS2 bacteriophagewere seeded into surface (n=3) and ground (n=2) waters used as source water for drinking watertreatment plants. Infectivity of the viral surrogates and reduction of viral nucleic acid for all theviruses was monitored using cell culture-based viral infectivity and quantitative reversetranscription-PCR (qRT-PCR), respectively. qRT-PCR measures the increase of PCR productduring each amplification cycle by continuously monitoring the intensity of a target-specificfluorescent reporter oligoprobe. The oligoprobe produces a signal proportional to the amount ofgenerated PCR product. Template concentration is determined based on the threshold positivecycle value. The threshold cycle (i.e. Ct) is the cycle at which the fluorescent reporter signal isfirst observed over background. PCR product detection occurs in real-time during theexponential phase of the reaction rather than following completion of the total reaction (30-40cycles) at the plateau stage. Therefore, qRT-PCR is less affected by amplification limitingcomponents during amplification and better represents initial template concentration. Moreover,the amplicon detection and confirmation are conducted at the same time by incorporating afluorescent reporter probe saving time and effort during amplicon confirmation.

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Edition: Vol. - No. Published: 11/01/2005 Number of Pages: 2File Size: 1 file , 87 KB