{"title":"开发并验证用于检测普通鲍曼不动杆菌的多靶点 TaqMan qPCR 方法。","authors":"Sébastien Masséglia , Magalie René-Martellet , Maxime Rates , Cecilia Hizo-Teufel , Volker Fingerle , Gabriele Margos , Xavier Bailly","doi":"10.1016/j.mimet.2024.106941","DOIUrl":null,"url":null,"abstract":"<div><p>Reliable detection of bacteria belonging to the <em>Borrelia burgdorferi</em> sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of <em>B. burgdorferi</em> s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of <em>B. burgdorferi</em> s. l.</p><p>Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of <em>B. afzelii</em>, <em>B. burgdorferi</em> sensu stricto or <em>B. bavariensis</em>. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays.</p><p>The developed qPCR assays allow exclusive detection of <em>B. burgdorferi</em> s. l. with the exception of the M16 marker which also detect relapsing fever <em>Borreliae</em>. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the <em>B. burgdorferi</em> genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays.</p><p>Within the defined limits, this multi-target qPCR method allows a versatile detection of <em>B. burgdorferi</em> s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106941"},"PeriodicalIF":1.7000,"publicationDate":"2024-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224000538/pdfft?md5=ad306daec5af9aebe13a7a368dae4ebd&pid=1-s2.0-S0167701224000538-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato\",\"authors\":\"Sébastien Masséglia , Magalie René-Martellet , Maxime Rates , Cecilia Hizo-Teufel , Volker Fingerle , Gabriele Margos , Xavier Bailly\",\"doi\":\"10.1016/j.mimet.2024.106941\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Reliable detection of bacteria belonging to the <em>Borrelia burgdorferi</em> sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of <em>B. burgdorferi</em> s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of <em>B. burgdorferi</em> s. l.</p><p>Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of <em>B. afzelii</em>, <em>B. burgdorferi</em> sensu stricto or <em>B. bavariensis</em>. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays.</p><p>The developed qPCR assays allow exclusive detection of <em>B. burgdorferi</em> s. l. with the exception of the M16 marker which also detect relapsing fever <em>Borreliae</em>. 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引用次数: 0
摘要
可靠地检测脊椎动物蓄水池、蜱虫媒介和患者体内属于广义鲍曼不动杆菌的细菌是回答莱姆病流行病学问题的关键。然而，对用于检测枸椽酸杆菌的 qPCRs 特性的描述往往很有限。本研究涵盖了针对 B. burgdorferi s. l 基因种染色体上四个标记的两种双联 taqman qPCR 检测方法的开发和验证。最后，使用从蜱虫和哺乳动物活体组织中提取的 DNA 对检测结果的有效性进行了评估，这些哺乳动物的感染状况已由其他检测方法确定。所开发的 qPCR 检测方法只能检测 B. burgdorferi s.l.，但 M16 标记除外，它还能检测复发性热鲍里氏菌。每个 qPCR 反应的检测限在 10 到 40 个拷贝之间，具体取决于样品类型、布氏杆菌基因种和目标标记。对各种样品进行的检测测试表明，我们的 qPCR 检测方法准确可靠。在规定的限度内，这种多靶标 qPCR 方法可以对 B. burgdorferi s. l.进行多种检测，而不受基因种属和所分析样本材料的影响，其灵敏度符合大多数应用的要求，在检测限度的测量条件下，重现性达到 100%，从而限制了结果的不确定性。
Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato
Reliable detection of bacteria belonging to the Borrelia burgdorferi sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of B. burgdorferi s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of B. burgdorferi s. l.
Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of B. afzelii, B. burgdorferi sensu stricto or B. bavariensis. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays.
The developed qPCR assays allow exclusive detection of B. burgdorferi s. l. with the exception of the M16 marker which also detect relapsing fever Borreliae. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the B. burgdorferi genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays.
Within the defined limits, this multi-target qPCR method allows a versatile detection of B. burgdorferi s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.
期刊介绍：
The Journal of Microbiological Methods publishes scholarly and original articles, notes and review articles. These articles must include novel and/or state-of-the-art methods, or significant improvements to existing methods. Novel and innovative applications of current methods that are validated and useful will also be published. JMM strives for scholarship, innovation and excellence. This demands scientific rigour, the best available methods and technologies, correctly replicated experiments/tests, the inclusion of proper controls, calibrations, and the correct statistical analysis. The presentation of the data must support the interpretation of the method/approach.
All aspects of microbiology are covered, except virology. These include agricultural microbiology, applied and environmental microbiology, bioassays, bioinformatics, biotechnology, biochemical microbiology, clinical microbiology, diagnostics, food monitoring and quality control microbiology, microbial genetics and genomics, geomicrobiology, microbiome methods regardless of habitat, high through-put sequencing methods and analysis, microbial pathogenesis and host responses, metabolomics, metagenomics, metaproteomics, microbial ecology and diversity, microbial physiology, microbial ultra-structure, microscopic and imaging methods, molecular microbiology, mycology, novel mathematical microbiology and modelling, parasitology, plant-microbe interactions, protein markers/profiles, proteomics, pyrosequencing, public health microbiology, radioisotopes applied to microbiology, robotics applied to microbiological methods,rumen microbiology, microbiological methods for space missions and extreme environments, sampling methods and samplers, soil and sediment microbiology, transcriptomics, veterinary microbiology, sero-diagnostics and typing/identification.
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