Sarah M. Stoppel , Bjørn Tore Lunestad , Mette Myrmel
{"title":"酶切和活力染料处理结合长程 PCR 对评估杜兰病毒感染性的影响","authors":"Sarah M. Stoppel , Bjørn Tore Lunestad , Mette Myrmel","doi":"10.1016/j.jviromet.2024.114919","DOIUrl":null,"url":null,"abstract":"<div><p>Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID<sub>50</sub>). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID<sub>50</sub> reductions of 0.3, 4.4 and 5.9 log<sub>10</sub> were observed. UV exposure (40/100/1000 mJ/cm<sup>2</sup>) resulted in 1.1, 2.5 and 5.9 log<sub>10</sub> reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log<sub>10</sub>. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID<sub>50</sub>. On average, RT-qPCR with pre-treatments deviated by 1.1–1.3 log<sub>10</sub> from TCID<sub>50</sub>. For UV light, long-range PCR was closest to TCID<sub>50</sub> results. Long-range reductions deviated from TCID<sub>50</sub> by ≤0.1 log<sub>10</sub> for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log<sub>10</sub> from TCID<sub>50</sub>. After chlorination the molecular methods repeatedly deviated from TCID<sub>50</sub> by >1.0 log<sub>10</sub>, Overall, each method needs to be further optimized for the individual types of inactivation treatment.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":2.2000,"publicationDate":"2024-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0166093424000430/pdfft?md5=cdc919b5c592e3bca10d7ce4c7e22793&pid=1-s2.0-S0166093424000430-main.pdf","citationCount":"0","resultStr":"{\"title\":\"The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity\",\"authors\":\"Sarah M. Stoppel , Bjørn Tore Lunestad , Mette Myrmel\",\"doi\":\"10.1016/j.jviromet.2024.114919\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID<sub>50</sub>). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID<sub>50</sub> reductions of 0.3, 4.4 and 5.9 log<sub>10</sub> were observed. UV exposure (40/100/1000 mJ/cm<sup>2</sup>) resulted in 1.1, 2.5 and 5.9 log<sub>10</sub> reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log<sub>10</sub>. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID<sub>50</sub>. On average, RT-qPCR with pre-treatments deviated by 1.1–1.3 log<sub>10</sub> from TCID<sub>50</sub>. For UV light, long-range PCR was closest to TCID<sub>50</sub> results. Long-range reductions deviated from TCID<sub>50</sub> by ≤0.1 log<sub>10</sub> for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log<sub>10</sub> from TCID<sub>50</sub>. After chlorination the molecular methods repeatedly deviated from TCID<sub>50</sub> by >1.0 log<sub>10</sub>, Overall, each method needs to be further optimized for the individual types of inactivation treatment.</p></div>\",\"PeriodicalId\":17663,\"journal\":{\"name\":\"Journal of virological methods\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.2000,\"publicationDate\":\"2024-03-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S0166093424000430/pdfft?md5=cdc919b5c592e3bca10d7ce4c7e22793&pid=1-s2.0-S0166093424000430-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of virological methods\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0166093424000430\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of virological methods","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0166093424000430","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity
Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID50). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID50 reductions of 0.3, 4.4 and 5.9 log10 were observed. UV exposure (40/100/1000 mJ/cm2) resulted in 1.1, 2.5 and 5.9 log10 reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log10. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID50. On average, RT-qPCR with pre-treatments deviated by 1.1–1.3 log10 from TCID50. For UV light, long-range PCR was closest to TCID50 results. Long-range reductions deviated from TCID50 by ≤0.1 log10 for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log10 from TCID50. After chlorination the molecular methods repeatedly deviated from TCID50 by >1.0 log10, Overall, each method needs to be further optimized for the individual types of inactivation treatment.
期刊介绍:
The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery.
The methods may include, but not limited to, the study of:
Viral components and morphology-
Virus isolation, propagation and development of viral vectors-
Viral pathogenesis, oncogenesis, vaccines and antivirals-
Virus replication, host-pathogen interactions and responses-
Virus transmission, prevention, control and treatment-
Viral metagenomics and virome-
Virus ecology, adaption and evolution-
Applied virology such as nanotechnology-
Viral diagnosis with novelty and comprehensive evaluation.
We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.