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

Ruiqin Wu; Baozhong Meng; Milena Corredig; Mansel W Griffiths

doi:10.1007/s12560-022-09548-7

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

Food Environ Virol. 2023 Jun;15(2):144-157. doi: 10.1007/s12560-022-09548-7. Epub 2023 Jan 14.

Authors

Ruiqin Wu^{1

2}, Baozhong Meng³, Milena Corredig⁴, Mansel W Griffiths^{4

5}

Affiliations

¹ Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada. wuruiqin@yahoo.com.
² Canadian Research Institute for Food Safety, 43 McGilvray Street, Guelph, ON, N1G 2W1, Canada. wuruiqin@yahoo.com.
³ Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada.
⁴ Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada.
⁵ Canadian Research Institute for Food Safety, 43 McGilvray Street, Guelph, ON, N1G 2W1, Canada.

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 Mg²⁺ concentration (P < 0.05), in addition to primer quality. The optimal Mg²⁺ 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 × 10⁰ PFU/15 g, 8.3 × 10¹ PFU/50 g, 8.3 × 10⁰ PFU/5 g, and 8.3 × 10⁰ PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.

Keywords: Detection; Food; Hepatitis A virus; RT-LAMP-BART.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Hepatitis A virus*
Luminescent Measurements / methods
Nucleic Acid Amplification Techniques
Reverse Transcription*
Sensitivity and Specificity
Technology

Supplementary concepts

LAMP assay