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.Hepatitis A virus (HAV) is an important cause of acute hepatitis worldwide that can lead to severe illness or even death. It is transmitted by the fecal-oral route through the consumption of contaminated foods or water. Traditional methods for HAV detection based on cell culture propagation are often difficult to perform and can take more than 1 week before any visual cytopathic effects are observed (2). Molecular methods such as reverse transcription (RT)-PCR and integrated cell culture-PCR (6, 11, 15) have largely superseded the cell culture methods, offering improved sensitivity and specificity. However, positive detection still relies on visual detection of an appropriately sized DNA band, followed by specific hybridization with a radiolabeled DNA probe, which can take up to 15 h and is not amenable to automation. Recent developments in the real-time RT-PCR technique have engendered wider acceptance of the PCR assay, as it eliminates the need for gel electrophoresis and improves the speed, sensitivity, and reproducibility of detection (13).Molecular beacons (MBs) are single-stranded fluorescence probes that form a stem-loop structure. In the presence of a target sequence, the MB undergoes a spontaneous conformational reorganization that forces the stem hybrid to dissociate and causes fluorescence to occur. Since unhybridized MBs do not have to be separated, they can be included in PCRs, permitting the progress of the reaction to be followed in real time (21).Pretreatments of environmental samples before RT-PCR are often necessary to reduce reaction volumes and to remove PCR inhibitors naturally present in the samples (1, 10, 18). Immunomagnetic separation (IMS) is a simple and powerful tool for quick and effective separation and isolation of bacteria and viruses from environmental water samples (4,5,7,12). IMS depends on isolation of the antigen from the sample with either monoclonal or polyclonal antibodies coupled to magnetic beads. In this paper, an MB-based real-time RT-PCR assay for HAV was developed. We demonstrate that the reported assay is highly sensitive and specific for HAV and, when combined with IMS, allows detection of HAV in seeded ground water samples.Selection of the target region...

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.Outbreaks of acute gastroenteritis due to hepatitis A virus (HAV) have been attributed to consumption of drinking water and foods considered safe on the basis of bacterial standards (23). These outbreaks indicate that meeting bacterial standards does not always ensure the absence of infectious viruses, and more reliable approaches are needed to detect HAV and other enteroviruses in environmental samples.Conventional methods for the detection of HAV are based on cell culture propagation, which is often difficult to perform and can take several weeks and continuous propagation in one or more cell lines before a sufficient amount of viral antigen or nucleic acid is produced to allow detection (5, 6). In addition, no single cell line is currently recommended for the detection of HAV (19,22).To date, molecular methods, such as the reverse transcription (RT)-PCR technique, are the most commonly studied, offering improved sensitivity, specificity, and the possibility of direct detection of HAV in environmental samples (11,16). In RT-PCR, the viral RNA is first converted to a single-stranded complementary DNA in a reverse transcription step, followed by PCR amplification of the target complementary DNA sequences to a detectable level. However, RT-PCR procedures have the disadvantages of requiring a two-step amplification process and relying on the use of expensive thermal-cycling equipment, which add to the complexity and the cost of their implementation for routine testing programs.Unlike RT-PCR, nucleic acid sequence-based amplification (NASBA) is a homogeneous, isothermal nucleic acid amplification method (17) that is particularly suited to RNA targets in a double-stranded DNA background (12). A cocktail of three enzymes (reverse transcriptase, T7 RNA polymerase, and RNase H) acting in concert allows the rapid amplification of target sequences by more than 10 8 -fold without the use of expensive thermal-cycling equipment. NASBA has proved successful in the detection of various mRNAs (3, 9) and in the detection of both viral (17) and bacterial (21) RNAs. Diagnostic procedures based on NASBA methodology have bee...