Abstract
The accuracy and sensitivity of PCR-based methods for detection of hepatitis A virus (HAV) are dependent on the methods used to separate and concentrate the HAV from the infected cells. The pH and ionic strength affect the binding affinity of the virus to cells. In this study, we initially investigated the effects of pH (4.0–10.0) and metal ions (Fe2+, Co2+, Cu2+, Mg2+, K+, and Ca2+) on the binding of HAV to oyster digestive cells. The lowest relative binding (RB) of HAV to the cells was found at pH 4.0 and in FeSO4 solution (64.6% and 68.1%, respectively). To develop an alternative to antibody-dependent immunomagnetic separation prior to detection of HAV using RT-PCR, the binding of HAV to five lectins, peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Ulex europaeus agglutinin (UEA-1) and soybean agglutinin (SBA), was evaluated using ELISAs. SBA showed significantly higher RB to HAV than the other lectins tested. In addition, HAV could be concentrated within 30 min using SBA-linked magnetic bead separation (SMS) prior to the RT-PCR assay. Our findings demonstrate the feasibility of using SMS combined with RT-PCR to detect HAV at dilutions ranging from 10−1–10−4 of a HAV stock (titer: 104 TCID50/mL).
Highlights
Viruses are a major cause of foodborne illness, due in large part to the contamination of shellfish by hepatitis A virus (HAV) [1]
(MgSO4), K+ (KCl), and Ca2+ (CaCl2)), we used ELISAs to assess the relative binding (RB) of HAV to the cells by comparing the optical densities of test solution to that of positive control, which was prepared by treating the cells with distilled water and HAV (Figure 1)
The precise reason for the lower RB at acidic pH remains uncertain, it may be associated with a pH-related conformational change within the HAV [42] or the cells
Summary
Viruses are a major cause of foodborne illness, due in large part to the contamination of shellfish by hepatitis A virus (HAV) [1]. To overcome these limitations, immunomagnetic separation (IMS) has been applied to concentrate viruses present in food or water samples prior to PCR [24,25,26,27,28,29]. Immunomagnetic separation (IMS) has been applied to concentrate viruses present in food or water samples prior to PCR [24,25,26,27,28,29] With this method, magnetic beads coated with specific antibodies are used to separate specific viral proteins and microorganisms under a magnetic field and extract them from water containing interfering and inhibiting substances. We assessed the feasibility of using the selected lectin in lectin-linked magnetic beads separation (LMS) prior to RT-PCR as a rapid and less expensive method of HAV detection
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