Virion Concentration Method for the Detection of Human Enteric Viruses in Extracts of Hard-Shelled Clams

Abstract

A method to extract and concentrate intact human enteric viruses from oyster extracts for detection using reverse transcription-polymerase chain reaction (RT-PCR) was applied to hard-shelled clams (mercenaria mercenaria). Fifty-gram clam samples were processed by an adsorption-elution-precipitation method and then seeded with 10(1) to 10(5) PFU to poliovirus 1 (PV1) and/or hepatitis A virus (HAV). Seeded viruses in extracts were purified by fluorocarbon (Freon) extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Efficiency of virion recovery from PEG precipitates was dependent upon PEG concentration and elution buffer volume, with optimized variable yielding recoveries as high as 99% for PV1 and 45% for HAV, as evaluated by cell culture infectivity assay. To further concentrate viruses, remove inhibitors, and reduce sample volumes, the protein-precipitating agent Pro-Cipitate was used in an adsorption-elution-precipitation scheme. The final concentrate was of low volume (< 1 ml) and directly compatible with viral genomic amplification using RT-PCR. When extracts from 50-g clam samples were seeded and processed by the combined concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 10(3) PFU for PV1 and HAV. Corresponding virus recoveries based on cell culture infectivity were 7 to 50% and 0.3 to 8% for pV1 and HAV, respectively, when extracts of clams were artificially contaminated with the Norwalk virus, direct detection of virion RNA using RT-PCR and subsequent oligoprobe hybridization was possible at levels as low as 450 RT-PCR amplifiable units of the Norwalk virus per extract of 50-g clam sample.

Rapid and Efficient Extraction Method for Reverse Transcription-PCR Detection of Hepatitis A and Norwalk-Like Viruses in Shellfish

“…(37), using a clam extraction procedure involving the use of guanidinium isothiocyanate extraction and immunomagnetic capture, reported detecting less than 10 PFU of HAV. Dix and Jaykus (8) reported the detection of 10 3 PFU for HAV and 450 RT-PCR units of NV from 50 g of clams. However, our method may not be as sensitive as that of Goswami et al (10), who detected 400 HAV RNA particles by using random primed RT-PCR and reported achieving a sensitivity of as little as 10 viral RNA molecules by using oligo(dT) primer for RT.…”

“…Some tests utilize extraction of dissected digestive diverticula and shellfish stomachs rather that whole shellfish (2,8,21). Ostensibly, this enhanced sensitivity is due to the presence of less RT-PCR inhibitors and an elevated virus concentration.…”

“…Although seemingly straightforward, successful RT-PCRs from samples derived from shellfish present formidable challenges, which prevent direct testing as a practical means of preventing shellfish-borne viral illness. These difficulties have been attributed to the presence of humic substances, large amounts of glycogen, and the properties of shellfish extracts (2,17,24,37).Current methods described for the extraction of enteroviruses from shellfish samples are cumbersome, often requiring several days to perform and involving many steps, including multiple polyethylene glycol 8000 (PEG) precipitations, pH changes, flocculant applications, and the use of Freon (trichlorotrifluoroethane) (2,3,8,11,12,20,21,23,36). Although the average infectious dose of Norwalk virus (NV) or HAV is not known, it may be less than 100 virions (6).…”

“…Current methods described for the extraction of enteroviruses from shellfish samples are cumbersome, often requiring several days to perform and involving many steps, including multiple polyethylene glycol 8000 (PEG) precipitations, pH changes, flocculant applications, and the use of Freon (trichlorotrifluoroethane) (2,3,8,11,12,20,21,23,36). Although the average infectious dose of Norwalk virus (NV) or HAV is not known, it may be less than 100 virions (6).…”

Detection of hepatitis a virus from oyster by nested PCR using efficient extraction and concentration method

“…However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification such as polyethylene glycol 8000 (PEG), zircodium chloride, and Freon (trichlorotrifluoroethane) due to the low sensitivity of PCR in the presence of PCR inhibitors such as polysaccharides and humic acids, particularly abundant in food samples (Lewis and Metcalf, 1988;Dix and Jaykus, 1998;D'Souza and Jaykus, 2002). In order to detect HAV inoculated in oyster digestive glands, we developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol modified from the GPTT procedure (Kingsley and Richards, 2001).…”