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A novel procedure for the quantification of antifungal activity against filamentous fungi, mycelial invasion distance (MID) method 一种新的丝状真菌抗真菌活性定量程序--菌丝侵袭距离(MID)法。
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-21 DOI: 10.1016/j.mimet.2024.106958
Shinobu Oda , Sonomi Karasawa , Kurea Satoh

A novel method for the quantification of antifungal activity of fungicides and painted surfaces, mycelial invasion distance (MID) method, was developed and applied to the quantification of activities of parabens and an antifungal paint. In this method, the MID of aerial mycelia on a test paper or a panel placed on a nutrient agar plate was measured with a stereoscopic microscope and a micro-ruler. The antifungal activities of the parabens and painted surfaces were expressed as the MID. The higher the hydrophobicity of parabens, the longer the MID, that is the lower the antifungal activity, were observed. Conversely, relatively polar parabens, such as methyl and ethyl parabens, exhibited stronger antifungal activity, that is shorter MID. The most hydrophobic paraben, benzyl paraben, showed the weakest antifungal activity. Furthermore, it was confirmed that the MID method was effective for the evaluation of the painted surfaces.

研究人员开发了一种用于定量分析杀真菌剂和涂料表面抗真菌活性的新方法--菌丝侵袭距离(MID)法,并将其应用于对羟基苯甲酸酯和一种抗真菌涂料的活性定量分析。在该方法中,使用立体显微镜和微尺测量放置在营养琼脂平板上的试纸或面板上气生菌丝的 MID。对羟基苯甲酸酯和涂漆表面的抗真菌活性用 MID 表示。据观察,对羟基苯甲酸酯类的疏水性越高,MID 越长,即抗真菌活性越低。相反,对羟基苯甲酸甲酯和对羟基苯甲酸乙酯等相对极性的对羟基苯甲酸酯类则表现出更强的抗真菌活性,即更短的 MID。疏水性最强的对羟基苯甲酸甲酯和对羟基苯甲酸乙酯的抗真菌活性最弱。此外,还证实了 MID 法对油漆表面的评估是有效的。
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引用次数: 0
Cryptosporidium occultus in disguise 伪装的隐孢子虫
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-20 DOI: 10.1016/j.mimet.2024.106957
Christen Rune Stensvold , Alba Martí-Marco , Samantha Moratal , Marianne Lebbad , David Carmena

As data accumulate in GenBank, the difficulties of delineating species of Cryptosporidium based on nuclear small subunit ribosomal RNA (ssu rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several ssu rDNA sequences primarily referred to as Cryptosporidium suis (some of them from non-suid hosts) should be considered Cryptosporidium occultus.

随着 GenBank 中数据的积累,仅凭核小亚基核糖体 RNA(ssu rRNA)基因信息来划分隐孢子虫种类的困难日益明显。在此,我们总结了目前可用的证据,这些证据表明,一些主要被称为猪隐孢子虫(其中一些来自非猪宿主)的 ssu rDNA 序列应被视为隐孢子虫。
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引用次数: 0
A novel biosensing strategy for identification of three important bacteria causing meningitis 用于识别引起脑膜炎的三种重要细菌的新型生物传感策略
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-15 DOI: 10.1016/j.mimet.2024.106954
Azam Yaghoobi , Ramin Abiri , Amirhoushang Alvandi , Iraj Manouchehri , Elham Arkan , Ali R. Jalalvand

Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis (N. meningitidis), and Haemophilus influenzae (H. influenzae). The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify S. pneumoniae were 119 × 102 CFU/mL (0.27 ng/μL) and 380 CFU/mL (9 pg/μL), respectively. The LODs of homemade PCR and FBC for detection of N. meningitidis were 4.49 CFU/mL (1.1 pg/μL) and 13 × 103 CFU/mL (30 pg/μL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of H. influenzae were 15.1 CFU/mL (30 fg/μL) and 41 × 102 CFU/mL (90 pg/ μL), respectively. Both techniques had appropriate sensitivity and specificity in detection of S. pneumoniae, N. meningitidis and H. influenzae.

细菌性脑膜炎是一种急性传染病,死亡率高,后果严重,因此需要快速诊断和治疗。本研究的目的是设计一种自制的多重 PCR 和新型芯片荧光生物传感器(FBC),以检测三种重要的脑膜炎病原体,包括肺炎链球菌(S. pneumoniae)、脑膜炎奈瑟菌(N. meningitidis)和流感嗜血杆菌(H. influenzae)。自制的多重 PCR 可同时诊断三种细菌。FBC 的制作基于热蒸发法在石英载玻片上沉积纳米铅颗粒。然后,将 SH-Cap Probe/Target ssDNA /FAM-Rep 探针装载到铅膜上。探针与目标 ssDNA 结合时的荧光反应由 Cytation 5 细胞成像多模阅读器 Bio-Tek 进行评估。自制 PCR 和 FBC 鉴定肺炎双球菌的检测限(LOD)分别为 119 × 102 CFU/mL(0.27 ng/μL)和 380 CFU/mL(9 pg/μL)。自制 PCR 和 FBC 检测脑膜炎双球菌的 LOD 分别为 4.49 CFU/mL(1.1 pg/μL)和 13 × 103 CFU/mL(30 pg/μL)。我们的结果证实,自制 PCR 和 FBC 检测流感嗜血杆菌的 LOD 分别为 15.1 CFU/mL(30 fg/μL)和 41 × 102 CFU/mL(90 pg/μL)。这两种技术在检测肺炎双球菌、脑膜炎双球菌和流感嗜血杆菌方面都具有适当的灵敏度和特异性。
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引用次数: 0
Flow cytometry: Unravelling the real antimicrobial and antibiofilm efficacy of natural bioactive compounds 流式细胞仪:揭示天然生物活性化合物的真正抗菌和抗生物膜功效
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-15 DOI: 10.1016/j.mimet.2024.106956
Valeria Poscente , Luciana Di Gregorio , Manuela Costanzo , Roberta Bernini , Annamaria Bevivino

Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring system for preventing the spread risk of foodborne disease. The present work used a combined culture-based and FCM approach to assess the in vitro efficacy of essential oils (EOs) from condiment plants commonly used in Mediterranean Europe (i.e., thyme EO, oregano EO, basil EO, and lemon EO) against planktonic and sessile cells of food-pathogenic Listeria monocytogenes 56 LY, and contaminant and alterative species Escherichia coli ATCC 25922 and Pseudomonas fluorescens ATCC 13525. Evaluation of the bacterial response to the increasing concentrations of natural compounds posed FCM as a crucial technique for the quantification of the live/dead, and viable but non-culturable (VBNC) cells when antimicrobial agents exert no real bactericidal action. Furthermore, the FCM results displayed higher numbers of viable bacteria expressed as Active Fluorescent Units (AFUs) with a greater level of repeatability compared with outcomes of the plate-count method. Overall, accurate counting of viable microbial cells is a critically important parameter in food microbiology, and flow cytometry provides an innovative approach with high-throughput potential for applications in the food industry as “flow microbiology”.

流式细胞术(FCM)可提供有关细菌存活率和生理机能的独特信息,从而建立实时预警的抗菌和抗生物膜监测系统,防止食源性疾病的传播风险。本研究采用基于培养和 FCM 的综合方法,评估来自欧洲地中海地区常用调味品植物的精油(即百里香精油、牛至精油、罗勒精油和柠檬精油)对单核细胞增生李斯特菌 56 LY 的浮游和无柄细胞、大肠埃希氏菌 ATCC 25922 和荧光假单胞菌 ATCC 13525 的污染和改变物种的体外药效。在抗菌剂没有真正发挥杀菌作用的情况下,评估细菌对天然化合物浓度增加的反应,使 FCM 成为量化活/死细胞和可存活但不可培养(VBNC)细胞的关键技术。此外,与平板计数法的结果相比,以活性荧光单位(AFUs)表示的 FCM 结果显示了更高的存活细菌数量和更高的重复性。总之,对有活力的微生物细胞进行精确计数是食品微生物学中一个极其重要的参数,而流式细胞仪提供了一种创新方法,具有高通量潜力,可作为 "流式微生物学 "应用于食品工业。
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引用次数: 0
Objectification of evaluation criteria in microscopic agglutination test using deep learning 利用深度学习实现显微凝集试验评价标准的客观化
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-14 DOI: 10.1016/j.mimet.2024.106955
Risa Nakano , Yuji Oyamada , Ryo Ozuru , Michinobu Yoshimura , Kenji Hiromatsu

We aim to objectify the evaluation criteria of agglutination rate estimation in the Microscopic Agglutination Test (MAT). This study proposes a deep learning method that extracts free leptospires from dark-field microscopic images and calculates the agglutination rate. The experiments show the effect of objectification with real pictures.

我们旨在为显微镜凝集试验(MAT)中凝集率估计的评价标准制定客观标准。本研究提出了一种深度学习方法,可从暗视野显微图像中提取游离钩端螺旋体并计算凝集率。实验显示了真实图片的物化效果。
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引用次数: 0
Meta-analysis of wastewater microbiome for antibiotic resistance profiling 用于抗生素耐药性分析的废水微生物组元分析。
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-14 DOI: 10.1016/j.mimet.2024.106953
Sakina Bombaywala , Abhay Bajaj , Nishant A. Dafale

The microbial composition and stress molecules are main drivers influencing the development and spread of antibiotic resistance bacteria (ARBs) and genes (ARGs) in the environment. A reliable and rapid method for identifying associations between microbiome composition and resistome remains challenging. In the present study, secondary metagenome data of sewage and hospital wastewaters were assessed for differential taxonomic and ARG profiling. Subsequently, Random Forest (RF)-based ML models were used to predict ARG profiles based on taxonomic composition and model validation on hospital wastewaters. Total ARG abundance was significantly higher in hospital wastewaters (15 ppm) than sewage (5 ppm), while the resistance towards methicillin, carbapenem, and fluoroquinolone were predominant. Although, Pseudomonas constituted major fraction, Streptomyces, Enterobacter, and Klebsiella were characteristic of hospital wastewaters. Prediction modeling showed that the relative abundance of pathogenic genera Escherichia, Vibrio, and Pseudomonas contributed most towards variations in total ARG count. Moreover, the model was able to identify host-specific patterns for contributing taxa and related ARGs with >90% accuracy in predicting the ARG subtype abundance. More than >80% accuracy was obtained for hospital wastewaters, demonstrating that the model can be validly extrapolated to different types of wastewater systems. Findings from the study showed that the ML approach could identify ARG profile based on bacterial composition including 16S rDNA amplicon data, and can serve as a viable alternative to metagenomic binning for identification of potential hosts of ARGs. Overall, this study demonstrates the promising application of ML techniques for predicting the spread of ARGs and provides guidance for early warning of ARBs emergence.

微生物组成和压力分子是影响环境中抗生素耐药细菌(ARBs)和基因(ARGs)发展和传播的主要驱动因素。用一种可靠而快速的方法来确定微生物组组成与耐药性组之间的关联仍然具有挑战性。在本研究中,对污水和医院废水的二次元基因组数据进行了差异分类和 ARG 分析评估。随后,使用基于随机森林(RF)的 ML 模型来预测基于分类组成的 ARG 图谱,并对医院废水进行模型验证。医院废水中 ARG 的总丰度(15 ppm)明显高于污水(5 ppm),而对甲氧西林、碳青霉烯类和氟喹诺酮类药物的耐药性则占主导地位。虽然假单胞菌占主要部分,但链霉菌、肠杆菌和克雷伯氏菌是医院废水的特征。预测模型显示,埃希氏菌、弧菌和假单胞菌等致病菌属的相对丰度对 ARG 总计数的变化影响最大。此外,该模型还能识别宿主特有的致病类群和相关 ARG 的模式,预测 ARG 亚型丰度的准确率大于 90%。对医院废水的预测准确率超过了 80%,这表明该模型可以有效地推广到不同类型的废水系统中。研究结果表明,ML 方法可以根据细菌组成(包括 16S rDNA 扩增子数据)识别 ARG 剖面,可以作为元基因组分选的一种可行替代方法,用于识别 ARGs 的潜在宿主。总之,本研究证明了应用 ML 技术预测 ARGs 传播的前景,并为 ARBs 出现的早期预警提供了指导。
{"title":"Meta-analysis of wastewater microbiome for antibiotic resistance profiling","authors":"Sakina Bombaywala ,&nbsp;Abhay Bajaj ,&nbsp;Nishant A. Dafale","doi":"10.1016/j.mimet.2024.106953","DOIUrl":"10.1016/j.mimet.2024.106953","url":null,"abstract":"<div><p>The microbial composition and stress molecules are main drivers influencing the development and spread of antibiotic resistance bacteria (ARBs) and genes (ARGs) in the environment. A reliable and rapid method for identifying associations between microbiome composition and resistome remains challenging. In the present study, secondary metagenome data of sewage and hospital wastewaters were assessed for differential taxonomic and ARG profiling. Subsequently, Random Forest (RF)-based ML models were used to predict ARG profiles based on taxonomic composition and model validation on hospital wastewaters. Total ARG abundance was significantly higher in hospital wastewaters (15 ppm) than sewage (5 ppm), while the resistance towards methicillin, carbapenem, and fluoroquinolone were predominant. Although, <em>Pseudomonas</em> constituted major fraction, <em>Streptomyces, Enterobacter</em>, and <em>Klebsiella</em> were characteristic of hospital wastewaters. Prediction modeling showed that the relative abundance of pathogenic genera <em>Escherichia, Vibrio,</em> and <em>Pseudomonas</em> contributed most towards variations in total ARG count. Moreover, the model was able to identify host-specific patterns for contributing taxa and related ARGs with &gt;90% accuracy in predicting the ARG subtype abundance. More than &gt;80% accuracy was obtained for hospital wastewaters, demonstrating that the model can be validly extrapolated to different types of wastewater systems. Findings from the study showed that the ML approach could identify ARG profile based on bacterial composition including 16S rDNA amplicon data, and can serve as a viable alternative to metagenomic binning for identification of potential hosts of ARGs. Overall, this study demonstrates the promising application of ML techniques for predicting the spread of ARGs and provides guidance for early warning of ARBs emergence.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"223 ","pages":"Article 106953"},"PeriodicalIF":2.2,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957586","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Novel cereal bran based low-cost liquid medium for enhanced growth, multifunctional traits and shelf life of consortium biofertilizer containing Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp. 基于谷物麸皮的新型低成本液体培养基,用于提高含氮球菌、枯草芽孢杆菌和假单胞菌的联合体生物肥料的生长、多功能性状和保质期。
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-11 DOI: 10.1016/j.mimet.2024.106952
Akashdeep , Suman Kumari , Neeraj Rani

The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of bran-based liquid media formulations were evaluated for the growth of consortium biofertilizer cultures (Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.). Among the bran-based formulations, wheat bran-based formulation WB5, exhibited the highest viable cell of 10.68 ± 0.09 Log10 CFU/ml and 12.63 ± 0.04 Log10 CFU/ml for Azotobacter chroococcum and Bacillus subtilis whereas for Pseudomonas sp., rice bran based bioformulation RB5 recorded maximum viability (12.71 ± 0.05 Log10 CFU/ml) after 72 h of incubation. RB51 and WB52liquid formulations were further optimized for enhanced shelf life using 5, 10 and 15 mM of trehalose, 0.05 and 0.1% carboxymethyl cellulose, and 0.5 and 1.0% glycerol. Following the peak growth at 72 h of incubation, a gradual decrease in the viable population of consortium biofertilizer cultures was observed in all the liquid formulations. The WB5 and RB5 formulations with 15 mM trehalose and 0.1% CMC, not only recorded significantly highest cell count of consortium biofertilizer cultures, but also maximally supported multi-functional traits i.e., phosphate and zinc solubilization, ammonia and IAA production up to 150 days. Further evaluation of seedling emergence and growth of wheat (PBW 826) under axenic conditions recorded WB5 amended with 15 mM trehalose-based consortium bioformulation to exhibit maximum emergence and growth of wheat seedlings. This low-cost liquid formulation can be used for large-scale biofertilizer production as a cost-effective liquid biofertilizer production technology.

本研究旨在利用谷物(大米和小麦)麸皮来开发低成本的液体复合生物配方。对不同浓度的麦麸基液体培养基配方进行了评估,以促进联合体生物肥料培养物(绿球菌、枯草芽孢杆菌和假单胞菌)的生长。在以麦麸为基础的制剂中,以麦麸为基础的制剂 WB5 在培养 72 小时后对根瘤酵母菌和枯草芽孢杆菌表现出最高的存活细胞数(10.68 ± 0.09 Log10 CFU/ml)和(12.63 ± 0.04 Log10 CFU/ml),而对假单胞菌来说,以米糠为基础的生物制剂 RB5 则表现出最高的存活细胞数(12.71 ± 0.05 Log10 CFU/ml)。对 RB51 和 WB52 液体配方进行了进一步优化,使用 5、10 和 15 毫摩尔的三卤糖,0.05 和 0.1%的羧甲基纤维素,以及 0.5 和 1.0%的甘油,以提高保质期。在培养 72 小时达到生长高峰后,在所有液体配方中都观察到生物肥料联合体培养物的存活数量逐渐减少。含有 15 毫摩尔三卤糖和 0.1% CMC 的 WB5 和 RB5 配方不仅能显著提高生物肥料联合体培养物的细胞数,而且还能最大限度地支持多功能特性,即磷酸盐和锌的溶解、氨和 IAA 的产生,直至 150 天。在轴生条件下对小麦(PBW 826)的出苗和生长进行的进一步评估表明,添加了 15 mM 曲哈糖的 WB5 生物复合制剂能最大程度地促进小麦出苗和生长。这种低成本液体配方可用于大规模生物肥料生产,是一种经济高效的液体生物肥料生产技术。
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引用次数: 0
Novel method for screening probiotic candidates tolerant to human gastrointestinal stress 筛选耐受人类胃肠道压力的候选益生菌的新方法。
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-08 DOI: 10.1016/j.mimet.2024.106945
Takuma Kozawa , Hideki Aoyagi

Tolerance to human gastrointestinal stressors is crucial for probiotics to exhibit their health benefits; however, there is no standardised method for screening their stress tolerance. In this study, we proposed a novel method for screening probiotic candidates tolerant to human gastrointestinal stress—gastrointestinal tolerance assay and culture (GTA-C) method—using black polyethylene terephthalate (PET) non-woven fabric as a scaffold to modify the specialized cellulose film (SCF) method. The modified SCF method showed excellent pH-based diffusion of medium components, had minimal effect on the growth of Escherichia coli K12, and improved the visibility of the colonies. Analysis of kimchi samples cultured using the SCF and modified SCF methods revealed that the modified method diversified the cultured bacteria. GTA in a simulated human fasting state using the modified SCF method showed that acid stress significantly affected the growth of four bacteria used as probiotics and that tolerance to acid stress may be species-dependent. Screening of probiotics in kimchi samples resulted in the identification of lactic acid bacteria tolerant to human gastrointestinal stress during fasting. Our results indicate that the modified SCF method (GTA-C method) is useful for screening probiotics resistant to the gastrointestinal environment during fasting.

对人类胃肠道应激源的耐受性是益生菌发挥其健康益处的关键;然而,目前还没有筛选其应激耐受性的标准化方法。在这项研究中,我们提出了一种筛选耐受人类胃肠道应激的候选益生菌的新方法--胃肠道耐受性检测和培养(GTA-C)法--使用黑色聚对苯二甲酸乙二醇酯(PET)无纺布作为支架,对专用纤维素膜(SCF)法进行了改进。改良后的 SCF 法显示出培养基成分在 pH 值基础上的良好扩散性,对大肠杆菌的生长影响极小,并提高了菌落的可见度。对使用 SCF 法和改良 SCF 法培养的泡菜样品进行的分析表明,改良 SCF 法使培养的细菌更加多样化。使用改良 SCF 方法在模拟人体空腹状态下进行的 GTA 显示,酸应激显著影响了用作益生菌的四种细菌的生长,而且对酸应激的耐受性可能与物种有关。通过对泡菜样品中的益生菌进行筛选,发现了对禁食期间人类胃肠道压力具有耐受性的乳酸菌。我们的结果表明,改良 SCF 法(GTA-C 法)可用于筛选对禁食期间胃肠道环境有耐受性的益生菌。
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引用次数: 0
Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato 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。
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-05 DOI: 10.1016/j.mimet.2024.106941
Sébastien Masséglia , Magalie René-Martellet , Maxime Rates , Cecilia Hizo-Teufel , Volker Fingerle , Gabriele Margos , Xavier Bailly

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.

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。
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引用次数: 0
Serum exosome tRFs as a promising biomarker for active tuberculosis and latent tuberculosis infection 血清外泌体 tRFs 作为活动性肺结核和潜伏性肺结核感染的有望生物标记物。
IF 2.2 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-04 DOI: 10.1016/j.mimet.2024.106944
Xiangyu Xi , Binghua Wang , Ruimei Zhang , Chunhua Ling

Objective

To analyse the expression profiles of serum exosome tRFs/tiRNAs and to explore their diagnostic value in tuberculosis (TB) activity.

Methods

The serum exosome tRF/tiRNA profile was analysed using high-throughput sequencing technology in 5 active tuberculosis (ATB) patients, 5 latent tuberculosis infection (LTBI) patients and 5 healthy controls (HCs). Then, serum exosome tRFs/tiRNAs were validated by quantitative real-time polymerase chain reaction (qRT–PCR), and their diagnostic value was evaluated by receiver operating characteristic curve (ROC) and area under the curve (AUC). Finally, bioinformatics analysis was performed to explore and identify the potential biological pathways induced by tRFs/tiRNAs.

Results

The sequencing results revealed that serum exosome tRF/tiRNA expression profiles were different among ATB patients, LTBI patients and HCs. Three tRFs (tRF-56:75-Trp-CCA-4, tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2) were selected for qRT–PCR validation. The results demonstrated that the expression level of tRF-1-22-chrM.Ser-GCT was upregulated in ATB patients, while tRF-56-75-Trp-CCA-4 was downregulated, which was consistent with the sequencing data. The AUCs of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM. Ser-GCT were 0.824 and 1.000, respectively, which have significant values in the diagnosis of ATB patients. Moreover, the expression levels of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2 in ATB patients and LTBI were different, which indicated that these three tRFs could effectively distinguish ATB patients and LTBI patients.

Conclusion

Our findings indicate that serum exosome tRFs can be used as potential markers for the diagnosis of ATB and LTBI.

目的分析血清外泌体tRFs/tiRNA的表达谱,并探讨其在结核病(TB)活动中的诊断价值:采用高通量测序技术分析了5例活动性肺结核(ATB)患者、5例潜伏肺结核感染(LTBI)患者和5例健康对照(HCs)的血清外泌体tRF/tiRNA表达谱。然后,通过实时定量聚合酶链式反应(qRT-PCR)对血清外泌体 tRFs/tiRNAs 进行了验证,并通过接收者操作特征曲线(ROC)和曲线下面积(AUC)对其诊断价值进行了评估。最后,进行了生物信息学分析,以探索和确定tRFs/tiRNAs诱导的潜在生物通路:结果:测序结果显示,ATB 患者、LTBI 患者和 HCs 的血清外泌体 tRF/tiRNA 表达谱不同。结果:测序结果显示,ATB 患者、LTBI 患者和 HCs 的血清外泌体 tRF/tiRNA 表达谱不同。结果显示,tRF-1-22-chrM.Ser-GCT在ATB患者中表达水平上调,而tRF-56-75-Trp-CCA-4表达水平下调,这与测序数据一致。tRF-56:75-Trp-CCA-4和tRF-1:22-chrM.Ser-GCT的AUC为0.824。Ser-GCT的AUC分别为0.824和1.000,在ATB患者的诊断中具有重要价值。此外,tRF-56:75-Trp-CCA-4和tRF-1:22-chrM.Ser-GCT以及tRF-56:76-Val-TAC-1-M2在ATB患者和LTBI患者中的表达水平不同,这表明这三种tRFs能有效区分ATB患者和LTBI患者:我们的研究结果表明,血清外泌体 tRFs 可作为诊断 ATB 和 LTBI 的潜在标记物。
{"title":"Serum exosome tRFs as a promising biomarker for active tuberculosis and latent tuberculosis infection","authors":"Xiangyu Xi ,&nbsp;Binghua Wang ,&nbsp;Ruimei Zhang ,&nbsp;Chunhua Ling","doi":"10.1016/j.mimet.2024.106944","DOIUrl":"10.1016/j.mimet.2024.106944","url":null,"abstract":"<div><h3>Objective</h3><p>To analyse the expression profiles of serum exosome tRFs/tiRNAs and to explore their diagnostic value in tuberculosis (TB) activity.</p></div><div><h3>Methods</h3><p>The serum exosome tRF/tiRNA profile was analysed using high-throughput sequencing technology in 5 active tuberculosis (ATB) patients, 5 latent tuberculosis infection (LTBI) patients and 5 healthy controls (HCs). Then, serum exosome tRFs/tiRNAs were validated by quantitative real-time polymerase chain reaction (qRT–PCR), and their diagnostic value was evaluated by receiver operating characteristic curve (ROC) and area under the curve (AUC). Finally, bioinformatics analysis was performed to explore and identify the potential biological pathways induced by tRFs/tiRNAs.</p></div><div><h3>Results</h3><p>The sequencing results revealed that serum exosome tRF/tiRNA expression profiles were different among ATB patients, LTBI patients and HCs. Three tRFs (tRF-56:75-Trp-CCA-4, tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2) were selected for qRT–PCR validation. The results demonstrated that the expression level of tRF-1-22-chrM.Ser-GCT was upregulated in ATB patients, while tRF-56-75-Trp-CCA-4 was downregulated, which was consistent with the sequencing data. The AUCs of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM. Ser-GCT were 0.824 and 1.000, respectively, which have significant values in the diagnosis of ATB patients. Moreover, the expression levels of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2 in ATB patients and LTBI were different, which indicated that these three tRFs could effectively distinguish ATB patients and LTBI patients.</p></div><div><h3>Conclusion</h3><p>Our findings indicate that serum exosome tRFs can be used as potential markers for the diagnosis of ATB and LTBI.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106944"},"PeriodicalIF":2.2,"publicationDate":"2024-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140858516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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Journal of microbiological methods
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