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

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.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 Molecular methods such as reverse transcription (RT)-PCR and integrated cell culture-PCR (6, 11, 15) have largely superseded the cell culture methods, offering improved sensitivity and specificity.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...

Diagnosis of Hepatitis A Virus Infection: a Molecular Approach

“…Small amplicons are recommended, and this may also play a role in the speed; however, it has been shown that decreasing the size of the PCR fragment does not necessarily improve PCR performance (174). Molecular beacons (1,155,240) and TaqManbased assays (53) are a few of the chemistries that have been used for real-time PCR detection of HAV. Real-time PCR chemistries use primers and probes with separate fluorogenic labels; the generation of a fluorescent signal depends on the interaction between PCR products and/or probes (24,53,155,240).…”

Immunomagnetic Separation Combined with Real-Time Reverse Transcriptase PCR Assays for Detection of Norovirus in Contaminated Food

“…from various foods (5,16,23). Antibodies specific for a particular microorganism are used, which gives the assay a higher specificity for concentrating and purifying microorganisms in both food and other environmental samples (1,6). The aim of this study was to evaluate IMS combined with real-time TaqMan RT-PCR for rapid and quantitative detection of NoV from artificially contaminated strawberries (11,20).…”

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

“…A molecular beacon (HAV MB: 6-FAM-5Ј-CTTGCGGGATAGGGTAAC AGCGGCGGCGCAAG-3Ј-DABCYL [the stem sequence of the MB is underlined, and the target sequence of HAV is in bold]) (Midland Certified Reagent Co., Midland, TX), which is perfectly complementary to a 20-bp region of the amplicon, was designed based on aligning the sequences of 15 different HAV strains obtained from GenBank as described previously (1). The molecular beacon was labeled at the 5Ј-end with the fluorophore FAM (6-carboxyfluorescein), and DABCYL [4-(4Ј-dimethylaminophenylazo) benzoic acid] was used as the quencher at the 3Ј end.…”