Publication Abstract

The development of polymerase chain reaction assays for detection of small round structured and other human enteric viruses in molluscan shellfish

The development of polymerase chain reaction assays for detection of small round structured and other human enteric viruses in molluscan shellfish

K. Henshilwood, J. Green, C.I. Gallimore, D.W.G. Brown and D.N. Lees

Public health controls for molluscan shellfish are hampered by the absence of methods for the detection in shellfish of the viral pathogens causing illness. The polymerase chain reaction (PCR) offers a potential way forward; however, its application to such complex samples as shellfish is hindered by the presence of potent amplification inhibitors. We describe the development of a procedure employing a combination of virus purification and nucleic acid extraction for the removal of the majority of such PCR inhibitors from shellfish. Initial developmental work used seeded poliovirus as a model and showed that PCR sample tolerance ranged from 2 to 5 g shellfish for highly polluted samples with over-all sensitivity limits of <10 plaque-forming units (PFCn poliovirus. These methods have recently been applied to the detection of hepatitis A virus and Norwalk and related small round structured viruses (SRSVs) in shellfish. Initial seeding experiments have shown that the method for removal of amplification inhibitors is equally applicable to the detection of these imses by PCR in shellfish. However a single round reverse transcriptase-polymerase chain reaction (RT-PCR) proved insufficiently sensitive to reliably detect SRSVs in shellfish samples associated with incidents of human infection. We describe the further development of a nested RT-PCR procedure for the detection of SRSVs in shellfish and the application of this assay for the detection of SRSV in commercially produced shellfish and in shellfish implicated in outbreaks of gastroenteritis. These studies show that detection of human enteric viruses in molluscan shellfish by PCR is feasible and may ultimately contribute to the further development of public health controls.

Reference:

K. Henshilwood, J. Green, C.I. Gallimore, D.W.G. Brown and D.N. Lees, 1998. The development of polymerase chain reaction assays for detection of small round structured and other human enteric viruses in molluscan shellfish. Journal of Shellfish Research, 17(5):1675-1678

K. Henshilwood*, J. Green, C.I. Gallimore, D.W.G. Brown and D.N. Lees*

Journal of Shellfish Research, 17(5):1675-1678