Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food
Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantifi...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-03-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/16/3/413 |
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| author | Gianfranco La Bella Maria Grazia Basanisi Gaia Nobili Anna Mattea D’Antuono Elisabetta Suffredini Giovanna La Salandra |
| author_facet | Gianfranco La Bella Maria Grazia Basanisi Gaia Nobili Anna Mattea D’Antuono Elisabetta Suffredini Giovanna La Salandra |
| author_sort | Gianfranco La Bella |
| collection | DOAJ |
| description | Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control. |
| first_indexed | 2024-04-24T17:45:57Z |
| format | Article |
| id | doaj.art-48b72afb9b964a6597c45dd1691169e9 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-04-24T17:45:57Z |
| publishDate | 2024-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-48b72afb9b964a6597c45dd1691169e92024-03-27T14:07:48ZengMDPI AGViruses1999-49152024-03-0116341310.3390/v16030413Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in FoodGianfranco La Bella0Maria Grazia Basanisi1Gaia Nobili2Anna Mattea D’Antuono3Elisabetta Suffredini4Giovanna La Salandra5Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyIstituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyIstituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyIstituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyDepartment of Food Safety Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, 00161 Rome, ItalyIstituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyHepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.https://www.mdpi.com/1999-4915/16/3/413ddPCRhepatitis Efood safetyquantificationduplexinternal control |
| spellingShingle | Gianfranco La Bella Maria Grazia Basanisi Gaia Nobili Anna Mattea D’Antuono Elisabetta Suffredini Giovanna La Salandra Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food Viruses ddPCR hepatitis E food safety quantification duplex internal control |
| title | Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food |
| title_full | Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food |
| title_fullStr | Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food |
| title_full_unstemmed | Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food |
| title_short | Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food |
| title_sort | duplex droplet digital pcr assay for quantification of hepatitis e virus in food |
| topic | ddPCR hepatitis E food safety quantification duplex internal control |
| url | https://www.mdpi.com/1999-4915/16/3/413 |
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