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Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 10/28/2003 Publication Date: 1/1/2004 Citation: RICHARDS, G.P., WATSON, M.A., KINGSLEY, D.H. A SYBR GREEN REAL-TIME RT-PCR METHOD TO DETECT AND QUANTITATE NORWALK VIRUS IN STOOLS. JOURNAL OF VIROLOGICAL METHODS. 2004. vol. 116. pg. 67-70. Interpretive Summary: Norwalk virus is a common cause of illness in the United States and tests to detect this virus in human stools have been complicated. We developed a new test, known as real-time reverse transcription-polymerase chain reaction, to detect Norwalk virus in stools. We also evaluated a simple dilution and heating process in place of more complicated procedures to extract the viruses from stools. These rapid and simple procedures were applied to 68 human stool isolates from a volunteer study and were successful in detecting Norwalk viruses in stools suspected to contain viruses. The levels of virus present were determined using a new procedure, referred to as the dilution end-point standard curve. One stool contained 60 billion Norwalk viruses per gram, which is far more than previously detected in human stools. Such high counts provide a clue to why Norwalk-like viruses are the leading cause of non-bacterial, gastrointestinal illness in the United States. Two of the volunteers shed Norwalk virus in their stools, even though they did not show any overt signs of illness. This suggests the possibility that health care workers and food handlers could unknowingly spread Norwalk virus in the workplace. Technical Abstract: This study was performed with three objectives: a) to develop a real-time reverse transcription-PCR (rt RT-PCR) procedure for a commonly used laboratory strain of Norwalk virus (NV), b) to evaluate the potential of sample dilution and heat release of viral RNA as an alternative to more complex procedures to extract viruses from stool specimens, and c) to identify the presence and relative quantity of NV in stools obtained from a highly controlled volunteer study. We describe the development of a simple, single tube, hot start, rt RT-PCR technique using SYBR green fluorescence for the detection of the 8FIIa strain of NV in diluted stools. Real-time RT-PCR was applied to 68 stool isolates from patients participating in a NV volunteer study. Ten of the 20 volunteers (50%) exhibited clinical signs of NV illness (diarrhea or vomiting) and seroconverted. The equivalent of 1 µl of a 1:1000 dilution of their stools was amplified by rt RT-PCR and consistently tested positive for NV amplicon without the need for virus extraction. Based on the development of a dilution end-point standard curve to semi-quantitate minimum virus levels, the number of RT-PCR amplifiable viruses in stool specimens is as high as 61.6 billion NV per gram of stool. Such high counts provide a clue to why Norwalk-like viruses are the number one cause of non-bacterial gastroenteritis in the United States. Two patients without clinical symptoms, but who seroconverted within 4 weeks of the challenge, shed NV in their stools, demonstrating a carrier state among asymptomatic individuals. This has major public health implications, particularly with health care workers and food handlers who could be at risk of unknowingly spreading NV contamination. |
