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Applied and Environmental Microbiology, November 2005, p. 7113-7116, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7113-7116.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

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, University of California, Riverside, California 92521,2 Department of Chemical and Environmental Engineering, University of California, Riverside, California 925213

Received 20 July 2005/ Accepted 22 July 2005

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA 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, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

* Corresponding author. Mailing address: Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521. Phone: (951) 827-2473. Fax: (951) 827-5696. E-mail for W. Chen: wilfred{at}engr.ucr.edu. E-mail for A. Mulchandani: adani{at}engr.ucr.edu.

Applied and Environmental Microbiology, November 2005, p. 7113-7116, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7113-7116.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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