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Applied and Environmental Microbiology, September 2001, p. 4152-4157, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.4152-4157.2001

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

David H. Kingsley* and Gary P. Richards

Microbial Food Safety Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Delaware State University, Dover, Delaware 19901

Received 14 March 2001/Accepted 21 June 2001

As part of an effort to develop a broadly applicable test for Norwalk-like viruses and hepatitis A virus (HAV) in shellfish, a rapid extraction method that is suitable for use with one-step reverse transcription (RT)-PCR-based detection methods was developed. The method involves virus extraction using a pH 9.5 glycine buffer, polyethylene glycol (PEG) precipitation, Tri-reagent, and purification of viral poly(A) RNA by using magnetic poly(dT) beads. This glycine-PEG-Tri-reagent-poly(dT) method can be performed in less than 8 h on hard-shell clams (Mercenaria mercenaria) and Eastern oysters (Crassostrea virginica) and, when coupled with RT-PCR-based detection, can yield results within 24 h. Observed sensitivities for seeded shellfish extracts are as low as 0.015 PFU of HAV and 22.4 RT-PCR50 units for Norwalk virus. Detection of HAV in live oysters experimentally exposed to contaminated seawater is also demonstrated. An adaptation of this method was used to identify HAV in imported clams (tentatively identified as Ruditapes philippinarum) implicated in an outbreak of food-borne viral illness. All of the required reagents are commercially available. This method should facilitate the implementation of RT-PCR testing of commercial shellfish.

* Corresponding author. Mailing address: U.S. Department of Agriculture; Agricultural Research Service, Microbial Food Safety Research Unit; W. W. Baker Center, 1200 N. Dupont Hwy., Delaware State University, Dover, DE19901. Phone: (302) 857-6406. Fax: (302) 857-6451. E-mail: dkingsle{at}dsc.edu.

Applied and Environmental Microbiology, September 2001, p. 4152-4157, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.4152-4157.2001


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