Ruiqin Wu, Baozhong Meng, Milena Corredig, Mansel W. Griffiths
{"title":"利用实时生物发光法(BART)和逆转录环介导等温扩增(RT-LAMP)技术快速检测食品中甲型肝炎病毒","authors":"Ruiqin Wu, Baozhong Meng, Milena Corredig, Mansel W. Griffiths","doi":"10.1007/s12560-022-09548-7","DOIUrl":null,"url":null,"abstract":"<div><p>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<sup>2+</sup> concentration (<i>P</i> < 0.05), in addition to primer quality. The optimal Mg<sup>2+</sup> 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 (<i>P</i> < 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<sup>0</sup> PFU/15 g, 8.3 × 10<sup>1</sup> PFU/50 g, 8.3 × 10<sup>0</sup> PFU/5 g, and 8.3 × 10<sup>0</sup> PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.</p></div>","PeriodicalId":563,"journal":{"name":"Food and Environmental Virology","volume":"15 2","pages":"144 - 157"},"PeriodicalIF":4.1000,"publicationDate":"2023-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s12560-022-09548-7.pdf","citationCount":"2","resultStr":"{\"title\":\"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\",\"authors\":\"Ruiqin Wu, Baozhong Meng, Milena Corredig, Mansel W. Griffiths\",\"doi\":\"10.1007/s12560-022-09548-7\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>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<sup>2+</sup> concentration (<i>P</i> < 0.05), in addition to primer quality. The optimal Mg<sup>2+</sup> 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 (<i>P</i> < 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<sup>0</sup> PFU/15 g, 8.3 × 10<sup>1</sup> PFU/50 g, 8.3 × 10<sup>0</sup> PFU/5 g, and 8.3 × 10<sup>0</sup> PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.</p></div>\",\"PeriodicalId\":563,\"journal\":{\"name\":\"Food and Environmental Virology\",\"volume\":\"15 2\",\"pages\":\"144 - 157\"},\"PeriodicalIF\":4.1000,\"publicationDate\":\"2023-01-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://link.springer.com/content/pdf/10.1007/s12560-022-09548-7.pdf\",\"citationCount\":\"2\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Food and Environmental Virology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s12560-022-09548-7\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"ENVIRONMENTAL SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Food and Environmental Virology","FirstCategoryId":"97","ListUrlMain":"https://link.springer.com/article/10.1007/s12560-022-09548-7","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ENVIRONMENTAL SCIENCES","Score":null,"Total":0}
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
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.
期刊介绍：
Food and Environmental Virology publishes original articles, notes and review articles on any aspect relating to the transmission of pathogenic viruses via the environment (water, air, soil etc.) and foods. This includes epidemiological studies, identification of novel or emerging pathogens, methods of analysis or characterisation, studies on survival and elimination, and development of procedural controls for industrial processes, e.g. HACCP plans. The journal will cover all aspects of this important area, and encompass studies on any human, animal, and plant pathogenic virus which is capable of transmission via the environment or food.
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