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. 2004 Jul;70(7):4371-4.
doi: 10.1128/AEM.70.7.4371-4374.2004.

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, S M Kheira, Andre M Salazar, Marylynn V Yates, Wilfred Chen, Ashok Mulchandani
Affiliations

Combined immunomagnetic separation-molecular beacon-reverse transcription-PCR assay for detection of hepatitis A virus from environmental samples

Khaled H Abd El Galil et al. Appl Environ Microbiol. 2004 Jul.

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.

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Figures

FIG. 1.
FIG. 1.
Sensitivity of the real-time RT-PCR assay. (A) Detection of serial dilutions of viral RNA at 1,000 (⧫), 100 (▪), 10 (▴), and 1 (•) PFU per reaction was done. A negative control (−) containing only water was used for comparison. (B) Standard curve generated by plotting the Ct value versus the number of PFU. The data represent the results of five independent experiments.
FIG. 2.
FIG. 2.
Detection of HAV in seeded groundwater samples by a combined IMS-MB-RT-PCR assay. Results are shown for 2,000 (▴), 200 (▪), and 20 (⧫) PFU and distilled water (◊) as a control.
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