Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR

Lee, Shin-Young; Kim, Mi-Ju; Kim, Hyun‐Joong; Jeong, Kwangcheol C.; Kim, Hae‐Yeong

doi:10.4014/jmb.1710.10008

Cited by 9 publications

(4 citation statements)

References 31 publications

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“…The lack of a multiplex, sensitive, and field-based tool is the main factor that limits routine food safety testing. RT-PCR and RT-qPCR are widely used sensitive techniques for multiplex detection of foodborne pathogens in food and shellfish (Lee et al, 2018). However, their uses in a field setting are restricted because of the need for expensive reagents and equipment (a thermal cycler or a real-time PCR machine), long processing times, and skilled, trained personnel.…”

Section: Discussionmentioning

confidence: 99%

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Development of a Reverse Transcription-Recombinase Polymerase Amplification Assay for Duplex Detection of Foodborne Viruses in Oysters

Chutoam¹,

Jinda²,

Lethochavalit³

et al. 2023

NLSC

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Viral contamination may occur at any stage of food processing. The study aim was to develop a two-step reverse transcription (RT)-recombinase polymerase amplification (RPA) assay and evaluate for in-field duplex detection of hepatitis A virus (HAV) and norovirus in oysters. The RNA expression plasmids were generated by amplifying a fragment of the VP1 gene of HAV and norovirus through PCR and cloning it into an expression vector. The RNAs were transcribed in vitro from the plasmids and further used for reverse transcription. The resulting single-stranded cDNAs were used as the purified or spiking templates to determine the sensitivities of simplex and duplex RT-RPA compared with RT-PCR, and RT-qPCR assays. The reproducibility and application of duplex RT-RPA in the field were also evaluated. Our results showed that simplex RT-RPA was at least 100 times more sensitive than RT-PCR and RT-qPCR and even more than duplex detection using purified targets. Unlike RT-PCR, the RT-RPA reaction was unaffected by inhibitors found in food, allowing simple sample preparation methods for detection within a fraction of the time. The duplex assay detected HAV, norovirus, or both in 12/30 (40%) oyster samples tested. Duplex RT-RPA proved to be a rapid, accurate, and reproducible method in a field test for detecting HAV and norovirus. Thus, duplex RT-RPA should be suitable for use in minimally equipped laboratories and field settings. If in-field RT-RPA services are provided to oyster farmers, the technique can minimize the risk of infection to consumers, thereby improving food safety. Keywords: Food safety, Hepatitis A virus, Direct extraction, Norovirus, Nucleic acid amplification

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Section: Discussionmentioning

confidence: 99%

“…Multiplex PCR has been widely used to detect foodborne pathogens (Lee et al, 2018;Sheng et al, 2018). Compared with the simplex reaction, multiplex technology can minimizing the number of operational steps and reagents required to produce simultaneous multiple detections in a single reaction.…”

Section: Discussionmentioning

confidence: 99%

Development of a Reverse Transcription-Recombinase Polymerase Amplification Assay for Duplex Detection of Foodborne Viruses in Oysters

Chutoam¹,

Jinda²,

Lethochavalit³

et al. 2023

NLSC

View full text Add to dashboard Cite

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“…It can amplify a low concentration of target DNA in a few hours [13]. In addition, multiplex PCR has also been used in the detection of real food samples [14,15]. Nevertheless, methods based on PCR are not suitable for wide application in the field due to the dependence on thermal cycling instruments.…”

Section: Introductionmentioning

confidence: 99%

Multiplex Recombinase Polymerase Amplification Assay for the Simultaneous Detection of Three Foodborne Pathogens in Seafood

Chen

et al. 2020

Foods

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Foodborne pathogens can cause foodborne illness. In reality, one food sample may carry more than one pathogen. A rapid, sensitive, and multiple target method for bacteria detection is crucial in food safety. For the simultaneous detection of Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella Enteritidis, multi-objective recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD) was developed in this study. The whole process, including amplification and reading, can be completed in 15 min at 37 °C. The detection limits were 2.6 × 101 CFU/mL for Staphylococcus aureus, 7.6 × 101 CFU/mL for Vibrio parahaemolyticus, and 1.29 × 101 CFU/mL for Salmonella Enteritidis. Moreover, colored signal intensities on test lines were measured by a test strip reader to achieve quantitative detection for Staphylococcus aureus (R2 = 0.9903), Vibrio parahaemolyticus (R2 = 0.9928), and Salmonella Enteritidis (R2 = 0.9945). In addition, the method demonstrated good recoveries (92.00%–107.95%) in the testing of spiked food samples. Therefore, the multiplex LFD-RPA assay is a feasible method for the rapid, sensitive, and quantitative detection of bacterial pathogens in seafood.

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“…The following supporting information can be downloaded at: , Figure S1: specificity of detection for the multiplex qPCR method. Table S1: table of six shellfish collection samples; Table S2: shellfish intake and frequency in the population; Table S3: the virus infection risk classification matrix; Table S4: detection limit results of the single- and multiplex qPCR; Table S5: repeatability test of the multiplex qPCR; Table S6: number of shellfish samples with multiple viruses detected [ 23 , 35 , 51 ].…”

mentioning

confidence: 99%

Quantitative Risk Assessment of Five Foodborne Viruses in Shellfish Based on Multiplex qPCR

Yu,

Xu,

Chen

et al. 2023

Foods

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Foodborne diseases are currently the most critical food safety issue in the world. There are not many hazard identification and exposure assessments for foodborne viruses (Norovirus GI, GII, Hepatitis A Virus, Rotavirus, Adenovirus) in shellfish. Multiplex qPCR for the simultaneous detection of five foodborne viruses was established and used to assess infection risk based on a 1-year pathogenesis study. The sensitivity, specificity and reproducibility of the multiplex qPCR method are consistent with that of conventional qPCR, which saves more time and effort. Overall, 37.86% of shellfish samples had one or more foodborne viruses. Risk assessment formulae and matrices were used to develop risk assessments for different age groups, different seasons and different shellfish. The annual probability of contracting a foodborne virus infection from shellfish is greater than 1.6 × 10−1 for all populations, and even for infants aged 0–4 years, it is greater than 1.5 × 10−2, which is much higher than the risk thresholds recommended by WHO (10−6) and the US EPA (10−4). High risk (level IV) is associated with springtime, and medium risk (level III) is associated with Mussel consumption. This study provides a basis for the risk of foodborne viral infections in people of different ages, in different seasons, and by consuming different shellfish.

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Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR

Cited by 9 publications

References 31 publications

Development of a Reverse Transcription-Recombinase Polymerase Amplification Assay for Duplex Detection of Foodborne Viruses in Oysters

Development of a Reverse Transcription-Recombinase Polymerase Amplification Assay for Duplex Detection of Foodborne Viruses in Oysters

Multiplex Recombinase Polymerase Amplification Assay for the Simultaneous Detection of Three Foodborne Pathogens in Seafood

Quantitative Risk Assessment of Five Foodborne Viruses in Shellfish Based on Multiplex qPCR

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