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Applied and Environmental Microbiology, July 2004, p. 4371-4374, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4371-4374.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

Khaled H. Abd El Galil,1 M. A. El Sokkary,1 S. M. Kheira,1 Andre M. Salazar,2 Marylynn V. Yates,2 Wilfred Chen,3* and Ashok Mulchandani3*

Department of Microbiology, Mansoura University, Mansoura, Egypt,1 Department of Environmental Sciences,2 Department of Chemical and Environmental Engineering, University of California, Riverside, California 925213

Received 10 November 2003/ Accepted 16 March 2004

ABSTRACT

In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

* Corresponding author. Mailing address: Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521. Phone: (909) 787-2473. Fax: (909) 787-5696. E-mail for Wilfred Chen: wilfred{at}engr.ucr.edu. E-mail for Ashok Mulchandani: adani{at}engr.ucr.edu.

Applied and Environmental Microbiology, July 2004, p. 4371-4374, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4371-4374.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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