Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/26/2009
Publication Date: 10/28/2009
Citation: Kingsley,D.,2009.Challenges for Novovirus detection within shellfish [abstract].Purdue/ARS Meeting.West Lafayette, Indiana.p.1.
Technical Abstract: The GPTT method was developed for rapid extraction of viral RNA that contains a polyA tail from contaminated shellfish to facilitate RT-PCR-based detection methods. This is a four-step method that involves blending shellfish tissue in pH 9.5 glycine buffer, and concentrating the extracted virus by polyetheylene glycol precipitation, followed by separation of the virion from its RNA using Tri-reagent, a mixture of guandinium isothiocyanate, phenol and chloroform. Once the RNA is extracted, poly-A containing RNA is separated from impurities by annealing the 3’ polyA tail to a poly dT oligo attached to a magnetic bead followed by extensive washing. This method permits RT-PCR detection of as low as 0.015 plaque-forming units of hepatitis A virus and 22.4 RT-PCR units of norovirus. The procedure was used to identify both norovirus and HAV within imported clams implicated in an outbreak of viral food poisoning in the US and used by Canadian health officials to identify norovirus in shellfish associated with an outbreak in British Columbia. The GPTT method detects other viruses that are potential threats to shellfish consumers and research surrogate viruses. These include Aichi virus, human parechovirus, coxsackieviruses A9 and B5, as well as murine norovirus and feline calicivirus. The GPTT method only requires commercially available reagents and has been successfully used worldwide. Users of this method include the NY State Health Department, Health Canada, the Office of Inspections and Quarantine in Shanghai China, and the University of Bari in Bari, Italy. Goals for improvement of this extraction and detection method include increasing sensitivity, particularly for norovirus and improving the expediency of the method, which currently only permits extraction of a handful of samples per day.