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. 2005 Nov;71(11):7113-6.
doi: 10.1128/AEM.71.11.7113-7116.2005.

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus

Khaled H Abd el-Galil  1 , M A el-Sokkary, S M Kheira, Andre M Salazar, Marylynn V Yates, Wilfred Chen, Ashok Mulchandani
Affiliations

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus

Khaled H Abd el-Galil et al. Appl Environ Microbiol. 2005 Nov.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Sensitivity of the real-time NASBA assay. (A) Detection of serial dilutions of HAV viral RNA at 1,000, 100, 0, and 1 PFU per reaction. A negative control containing only water was used for comparison. (B) Standard curve generated by plotting the time required for signal detection versus PFU. Fluorescence intensity data were recorded every minute of the NASBA reaction. The threshold cycle of each amplification reaction was calculated based on the first cycle at which the fluorescence was 10-fold higher than the standard deviation of the mean baseline emission. The data represent the results from four independent experiments.
FIG. 2.
FIG. 2.
Detection of HAV in seeded lake water samples by a combined IMS-MB NASBA assay. Different dilutions of hepatitis A virus were added to surface water samples obtained from Lake Elsinore in southern California. An IMS technique was used to concentrate the virus particles from the water sample. Results for 1 to 1,000 PFU are shown. A control using lake water alone was also used.
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