Norwalk-like viruses, referred to as Noroviruses (NV), are a group of small nonenveloped RNA viruses that belong to the Caliciviridae family. NVs cause epidemic acute gastrointestinal illness in humans and are thought to be the most common cause of viral gastroenteritis worldwide (Fankhauser et al., 1998; Lopman et al., 2003). NV strains causing human infections can be subdivided into at least two genogroups (GI and GII) and as many as 20 subgroups. GII strains are responsible for most (73% of outbreaks from 1997-2000) NV cases in the US (Fankhauser et al., 2002) and 90% in Florida (L. Stark, personal communication). With an incubation period of only 24-48 hours and as many as 30% of the infections being asymptomatic, this highly contagious virus often results in rapid disease transmission that affects large numbers of exposed individuals (Greene et al., 2002). For this reason, NV outbreaks are common in closed environments like daycare centers, nursing homes (Thornton et al., 2004) and cruise ships (Widdowson et al., 2004).
Acute gastroenteritis has also been identified as a significant cause of substantial morbidity for US military personnel during deployment (Oyofo et al., 1999). The most common disability amongst soldiers in Operation Desert Storm and Operation Desert Shield was gastroenteritis resulting from NV infection (Brown et al., 2002; McCarthy et al., 2000). Outbreaks of NV have been commonly identified on US Naval vessels. From March to May in 1996, 49 of 721 personnel aboard the USS Germantown reported to sick call with acute gastroenteritis, and 45% of the stool samples taken were positive for NV. From 1992 to 1997, four large outbreaks on Navy aircraft carriers were attributed to NV's (McCarthy et al., 2000). NV also caused an acute outbreak of gastroenteritis amongst British soldiers deployed in Afghanistan in May of 2002 (Ahmad, 2002; Brown et al., 2002). Although tainted food tends to be the primary mode of transmission, contact with contaminated surfaces and water play a significant role in the spread of this disease (Barker et al., 2004; Green et al., 1998; Green, 1998). NV is a significant worldwide public health concern and there is a need for rapid field detection and diagnosis such that patterns of transmission can be interrupted (Brown et al., 2000). This challenge is complicated by two main issues: [1.] the inability to grow NV in culture and [2.] the high genetic diversity that exists among NVs. We have developed a method to detect NV based on nucleic-acid sequence based amplification (NASBA) coupled with detection using a molecular beacon. Although other NASBA assays have been developed for detection of NV (Greene et al., 2003; Moore et al., 2004), these assays rely on end-point detection and therefore require more time for analysis. Furthermore, a real time NASBA approach provides a semi-quantitative method to estimate viral load.
The NASBA assay can routinely detect 1,000 or more copies of the target RNA. Furthermore, 100 copies are detected in most runs (>80%) and as few as 10 copies are occasionally amplified. These results are similar to the detection limits obtained using a real time (Taqman) RT-PCR assay for NV. However, NASBA amplification occurs in as little as 22 minutes for 105 copies and all positive samples (>100 copies of target RNA) amplify within 50 minutes, versus a minimum time requirement of 2.5 hours with Taqman RT-PCR.
Figure 1. Amplification curves for NV transcript over four orders of magnitude. Reactions were done in triplicate. Click here to view Figure 1.
Figure 2. Standard curve of NV transcript (100 -100,000 copies of target) using time to positivity (TTP) measurements from the amplification curve data. Click here to view Figure 2.
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