{"title":"开发并验证基于水解探针的多重定量 PCR 分析法,用于检测螯虾中的四种病原体。","authors":"Maris J. Daleo , Matthew C. Allender","doi":"10.1016/j.jviromet.2024.115077","DOIUrl":null,"url":null,"abstract":"<div><div>Many wildlife conservation efforts focus on the effects of one pathogen, but for many conservation efforts to be successful, researchers require an understanding of ecological processes that may include multiple co-occurring pathogens. We developed a multiplex quantitative PCR (qPCR) assay to detect four pathogens in eastern box turtles (<em>Terrapene carolina carolina</em>), including frog virus 3 (FV3), <em>Terrapene</em> herpesvirus 1 (TerHV1), box turtle <em>Mycoplasma</em> sp. (BTMyco), and <em>Terrapene</em> adenovirus (TerAdv). TaqMan™ primer probes were designed using previously published assays with four different fluorophores. Multiplex Cq values plotted against singleplex Cq values demonstrated slopes of 0.967, 1.00, 0.980, and 0.973 for TerHV1, TerAdv, FV3, and BTMyco, respectively, and R<sup>2</sup> values of 0.999 for all four pathogens. The assay was highly consistent with the intra-assay variation of all four pathogen targets, ranging from 0.05–1.826 % across all concentrations, while inter-assay variation ranged from 0.031–4.569 % among all four targets at all concentrations. Clinical samples were tested using previously collected samples from eastern box turtles and red-eared sliders (<em>Trachemys scripta elegans</em>) and performed similarly to singleplex assays. This multiplex assay is an effective, time-efficient diagnostic tool to quickly monitor chelonian pathogens by detecting FV3, TerHV1, BTMyco, and TerAdv within a single reaction. A validated and clinically utilized multiplex assay will be beneficial to characterizing a more complex pathogen profile for future chelonian epidemiological studies to better describe pathogen dynamics and their impacts on individual and population health.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115077"},"PeriodicalIF":2.2000,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Developing and validating a multiplex hydrolysis probe-based quantitative PCR assay for the detection of four pathogens in chelonians\",\"authors\":\"Maris J. Daleo , Matthew C. Allender\",\"doi\":\"10.1016/j.jviromet.2024.115077\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Many wildlife conservation efforts focus on the effects of one pathogen, but for many conservation efforts to be successful, researchers require an understanding of ecological processes that may include multiple co-occurring pathogens. We developed a multiplex quantitative PCR (qPCR) assay to detect four pathogens in eastern box turtles (<em>Terrapene carolina carolina</em>), including frog virus 3 (FV3), <em>Terrapene</em> herpesvirus 1 (TerHV1), box turtle <em>Mycoplasma</em> sp. (BTMyco), and <em>Terrapene</em> adenovirus (TerAdv). TaqMan™ primer probes were designed using previously published assays with four different fluorophores. Multiplex Cq values plotted against singleplex Cq values demonstrated slopes of 0.967, 1.00, 0.980, and 0.973 for TerHV1, TerAdv, FV3, and BTMyco, respectively, and R<sup>2</sup> values of 0.999 for all four pathogens. The assay was highly consistent with the intra-assay variation of all four pathogen targets, ranging from 0.05–1.826 % across all concentrations, while inter-assay variation ranged from 0.031–4.569 % among all four targets at all concentrations. Clinical samples were tested using previously collected samples from eastern box turtles and red-eared sliders (<em>Trachemys scripta elegans</em>) and performed similarly to singleplex assays. This multiplex assay is an effective, time-efficient diagnostic tool to quickly monitor chelonian pathogens by detecting FV3, TerHV1, BTMyco, and TerAdv within a single reaction. A validated and clinically utilized multiplex assay will be beneficial to characterizing a more complex pathogen profile for future chelonian epidemiological studies to better describe pathogen dynamics and their impacts on individual and population health.</div></div>\",\"PeriodicalId\":17663,\"journal\":{\"name\":\"Journal of virological methods\",\"volume\":\"332 \",\"pages\":\"Article 115077\"},\"PeriodicalIF\":2.2000,\"publicationDate\":\"2024-11-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of virological methods\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0166093424002027\",\"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/S0166093424002027","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Developing and validating a multiplex hydrolysis probe-based quantitative PCR assay for the detection of four pathogens in chelonians
Many wildlife conservation efforts focus on the effects of one pathogen, but for many conservation efforts to be successful, researchers require an understanding of ecological processes that may include multiple co-occurring pathogens. We developed a multiplex quantitative PCR (qPCR) assay to detect four pathogens in eastern box turtles (Terrapene carolina carolina), including frog virus 3 (FV3), Terrapene herpesvirus 1 (TerHV1), box turtle Mycoplasma sp. (BTMyco), and Terrapene adenovirus (TerAdv). TaqMan™ primer probes were designed using previously published assays with four different fluorophores. Multiplex Cq values plotted against singleplex Cq values demonstrated slopes of 0.967, 1.00, 0.980, and 0.973 for TerHV1, TerAdv, FV3, and BTMyco, respectively, and R2 values of 0.999 for all four pathogens. The assay was highly consistent with the intra-assay variation of all four pathogen targets, ranging from 0.05–1.826 % across all concentrations, while inter-assay variation ranged from 0.031–4.569 % among all four targets at all concentrations. Clinical samples were tested using previously collected samples from eastern box turtles and red-eared sliders (Trachemys scripta elegans) and performed similarly to singleplex assays. This multiplex assay is an effective, time-efficient diagnostic tool to quickly monitor chelonian pathogens by detecting FV3, TerHV1, BTMyco, and TerAdv within a single reaction. A validated and clinically utilized multiplex assay will be beneficial to characterizing a more complex pathogen profile for future chelonian epidemiological studies to better describe pathogen dynamics and their impacts on individual and population health.
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.