Rapid Detection of Hepatitis A Virus in Foods Using a Bioluminescent Assay in Real-Time (BART) and Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Technology

Abstract

Foodborne hepatitis A infections have been considered as a major threat for public health worldwide. Increased incidences of hepatitis A virus (HAV) infection has been associated with growing global trade of food products. Rapid and sensitive detection of HAV in foods is very essential for investigating the outbreaks. Real-time RT-PCR has been most widely used for the detection of HAV by far. However, the technology relies on fluorescence determination of the amplicon and requires sophisticated, high-cost instruments and trained personnel, limiting its use in low resource settings. In this study, a robust, affordable, and simple assay, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in combination with a bioluminescence-based determination of amplification in real-time (BART), was developed for the detection of HAV in different food matrices, including green onion, strawberry, mussel, and milk. The efficiencies of a one-step RT-LAMP-BART and a two-step RT-LAMP-BART were investigated for the detection of HAV in different food matrices and was compared with that of real-time RT-PCR. The sensitivity of the RT-LAMP-BART assay was significantly affected by Mg 2+ concentration ( P < 0.05), in addition to primer quality. The optimal Mg 2+ concentration was 2 mM for one-step RT-LAMP-BART and 4 mM for two-step RT-LAMP-BART. Compared with cartridge-purified primers, HPLC-purified primers could greatly improve the sensitivity of the RT-LAMP-BART assay ( P < 0.05). For detecting HAV in different food matrices, the performance of two-step RT-LAMP-BART was comparable with that of real-time RT-PCR and was better than that of one-step RT-LAMP-BART. The detection limit of the two-step RT-LAMP-BART for HAV in green onion, strawberry, mussel, and milk was 8.3 × 10 0 PFU/15 g, 8.3 × 10 1 PFU/50 g, 8.3 × 10 0 PFU/5 g, and 8.3 × 10 0 PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.

Hepatitis A Virus and Hepatitis E Virus as Food- and Waterborne Pathogens—Transmission Routes and Methods for Detection in Food

“…In the case of LAMP, the nucleic acid amplification is conducted at a constant temperature (between 60 and 65 °C), so a thermal cycler is not needed. Wu and colleges have developed an RT-LAMP assay for the detection of HAV in different food matrices, including green onion, strawberry, mussel, and milk [ 236 ]. Their assay showed comparable sensitivity with RT-qPCR in certain food matrices.…”

Point-of-Care Testing for Hepatitis Viruses: A Growing Need

“…Simplifying food and water testing with POC-compatible formats would move testing outside of the laboratory and potentially allow for routine screening of items such as shellfish, leafy produce, and frozen vegetables that are commonly associated with outbreaks. An assay that uses RT-LAMP in combination with real-time bioluminescent detection was developed and shown to be capable of detecting low-level HAV contamination of green onions, strawberries, mussels, and milk [73]. However, the laboratory methods used for the processing of the samples and the length of time needed to perform the assay (>100 min) were not necessarily attractive for POC use.…”

Development of a cell-free toehold switch for hepatitis A virus type I on-site detection

“…Despite the potential effectiveness of this strategy in addressing common issues related to RT-PCR, the prevalence of false-positive results has hindered its widespread adoption in ordinary practice. 21 HAV antigen and antibody testing can quickly provide immunological evidence for the clinical diagnosis but have relatively poor sensitivity among all virus detection methods. 22 The emergence of toehold switches has provided novel ideas for detecting viral RNA in recent years.…”