Journal of microbiological methods最新文献_第9页

Disk-based method for rapid determination of primary antituberculosis drug susceptibility in Mycobacterium tuberculosis 基于圆盘法快速测定结核分枝杆菌原发性抗结核药物敏感性。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-07 DOI: 10.1016/j.mimet.2025.107322

Cemilenur Atas , Kubra Yildirim , Ahmet Yilmaz Coban

Rapid and accurate determination of Mycobacterium tuberculosis susceptibility to primary drugs is critical for the timely initiation of effective therapy and the control of resistant tuberculosis (TB). This study aims to evaluate disk-based method for determining primary anti-TB drug susceptibility of M. tuberculosis isolates. A total of 67 M. tuberculosis isolates were used, of which 5 reference (M. tuberculosis H37Rv, ATCC-35822, ATCC-35838, ATCC-35820, ATCC-35837). Isolates were tested simultaneously with the nitrate reductase assay (NRA) and resazurin tube assay (RTA) versions in the newly developed disk-based method. Drug susceptibility test (DST) results were compared separately with the propotion method and BACTEC MGIT 960. When comparing DST results determined by disk-based NRA with MGIT 960, the agreement was 93.33 % for INH, 91.67 % for RIF, 78.33 % for STR, and 88.33 % for EMB; and when comparing with the proportion method, the agreement was 93.33 % for INH, 96.67 % for RIF, 91.67 % for STR, and 88.33 % for EMB. When comparing DST results determined by disk-based RTA with MGIT 960, the agreement was 95.16 % for INH, 91.94 % for RIF, 75.81 % for STR, and 91.94 % for EMB; and when comparing with the proportion method, the agreement was 95.16 % for INH, 96.77 % for RIF, 87.10 % for STR, and 88.71 % for EMB. The newly developed disk-based drug susceptibility method gives faster results than existing methods since it is applied together with colorimetric tests. It is more advantageous than other methods because it is disk-based in maintaining antibiotic stability. More studies are needed to evaluate its performance.

快速准确地测定结核分枝杆菌对主要药物的敏感性对于及时开始有效治疗和控制耐药结核病至关重要。本研究旨在探讨基于圆盘法测定结核分枝杆菌原抗结核药物敏感性的方法。一共67个 M。选取结核分枝杆菌H37Rv、ATCC-35822、ATCC-35838、ATCC-35820、ATCC-35837 5株作为参考。同时采用硝酸还原酶法（NRA）和瑞祖脲管法（RTA）对分离菌株进行检测。药敏试验（DST）结果分别与propotion法和BACTEC MGIT 960进行比较。将基于磁盘的NRA测定的DST结果与MGIT 960进行比较，INH的一致性为93.33 %，RIF的一致性为91.67 %，STR的一致性为78.33 %，EMB的一致性为88.33 %；与比例法比较，INH符合率为93.33 %，RIF符合率为96.67 %，STR符合率为91.67 %，EMB符合率为88.33 %。将基于磁盘的RTA测定的DST结果与MGIT 960进行比较，INH的一致性为95.16 %，RIF的一致性为91.94 %，STR的一致性为75.81 %，EMB的一致性为91.94 %；与比例法比较，INH符合率为95.16 %，RIF符合率为96.77 %，STR符合率为87.10 %，EMB符合率为88.71 %。新开发的基于磁盘的药物敏感性方法由于与比色试验一起应用，因此比现有方法更快地得出结果。它比其他方法更有利，因为它在维持抗生素稳定性方面是基于磁盘的。需要更多的研究来评估其性能。

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引用次数: 0

Dioscorea tokoro Makino extract suppresses the growth of Saccharomyces cerevisiae INVSc1 by disrupting the endolysosomal pathway 牧野薯蓣提取物通过破坏内溶酶体途径抑制酿酒酵母INVSc1的生长。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-05 DOI: 10.1016/j.mimet.2025.107321

Sen Takeshita, Hideki Inoue, Yasuhiro Iida

Exploring novel antifungal mechanisms is needed because of the side effects of existing treatments and the increasing prevalence of drug resistance. We recently reported that inhibition of the endocytic pathway via a lysosome inhibitor or Fab1/Vac14 suppression enhances the localization of emerald green fluorescent protein-bound β-1,3-glucanase (BGL2-EmGFP) at the tip in Saccharomyces cerevisiae INVSc1, used as a non-pathogenic model. Overexpression of wild-type BGL2-EmGFP, but not an inactive mutant, completely abolished cell proliferation, suggesting that impairing tip growth through dysregulated glucanase activity represents a novel antifungal mechanism. In this study, using a phenotypic screening strategy combining fluorescein isothiocyanate (FITC)–dextran endocytosis/exocytosis assays with BGL2-EmGFP localization analysis, Dioscorea tokoro Makino extract was found to induce dysregulation of the endocytic pathway and promote BGL2-EmGFP localization at the tip, accompanied by growth inhibition in S. cerevisiae INVSc1. These findings suggest that this phenotypic screening strategy provides a promising approach based on a novel antifungal mechanism involving endocytic dysregulation and enhanced BGL2 localization at the tip.

由于现有治疗方法的副作用和日益普遍的耐药性，探索新的抗真菌机制是必要的。我们最近报道，通过溶酶体抑制剂或Fab1/Vac14抑制内噬途径，可以增强酿酒酵母（Saccharomyces cerevisiae） INVSc1中祖母绿荧光蛋白结合的β-1,3-葡聚糖酶（BGL2-EmGFP）在顶端的定位，作为非致病性模型。过度表达野生型BGL2-EmGFP，而不是失活突变体，完全破坏细胞增殖，表明通过葡聚糖酶活性失调来损害尖端生长是一种新的抗真菌机制。本研究采用异硫氰酸荧光素(FITC)-葡聚糖内吞/胞外作用与BGL2-EmGFP定位相结合的表型筛选策略，发现野山山药提取物可诱导酿酒葡萄球菌（S. cerevisiae） INVSc1的内吞途径失调，促进BGL2-EmGFP在尖端的定位，并伴有生长抑制。这些发现表明，这种表型筛选策略提供了一种基于新型抗真菌机制的有前途的方法，该机制涉及内吞失调和BGL2在尖端的定位增强。

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引用次数: 0

Rapid qualitative identification and application of six common lactic acid bacteria based on surface enhanced Raman spectroscopy 基于表面增强拉曼光谱的六种常见乳酸菌快速定性鉴定及应用。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-03 DOI: 10.1016/j.mimet.2025.107312

Huihui Zhi , Shijie Liu , Yueyu Bai , Miaoyun Li , Dong Liang , Lijun Zhao , Jong-Hoon Lee , Zihou Liu , Qian Ding , Manhong Zhan , Lingxia Sun , Yangyang Ma , Yaodi Zhu

To achieve rapid identification and detection of lactic acid bacteria (LAB) from different genera and species, six types of lactic acid bacteria were selected as the research objects, including Lactobacillus plantarum L1, Lactobacillus gasseri Y2, Lactobacillus sakei C3, Pediococcus pentosaceus W6, Streptococcus lactis S18 and Pediococcus lactis R9. Silver nanoparticle (AgNPs) were prepared as the SERS substrate material. The SERS spectra of the six LAB strains were analyzed, and a classification and identification model was constructed.The results showed that AgNPs, as the SERS substrate material, exhibited good signal enhancement performance and excellent reproducibility (RSD < 6 %), which could clearly distinguish the differences in SERS spectral peaks among the six LAB strains. Through Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA), Various types of lactic acid bacteria can be fully visualized for classification and identification, with a model accuracy rate of 100 %. This study proposes a potential technical solution for quick qualitative identification of lactic acid bacteria.

为实现对不同属种乳酸菌的快速鉴定和检测，选取植物乳杆菌L1、产气乳杆菌Y2、酒井乳杆菌C3、戊糖Pediococcus ptosaceus W6、乳酸链球菌S18和乳酸Pediococcus lactis R9 6种乳酸菌作为研究对象。制备了纳米银作为SERS衬底材料。对6株LAB菌株的SERS光谱进行分析，并建立分类鉴定模型。结果表明，AgNPs作为SERS衬底材料，具有良好的信号增强性能和良好的重现性（RSD）

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引用次数: 0

Suppression/competition PCR: A novel method to minimize unwanted amplicons in metabarcoding, with applications to parasite detection in fecal samples 抑制/竞争PCR：一种在元条形码中减少不需要扩增子的新方法，应用于粪便样本中的寄生虫检测。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-03 DOI: 10.1016/j.mimet.2025.107320

Justin Mark Carpani , John R. Barta , Rebecca A. Guy

Metabarcoding is widely used for detecting microorganisms in fecal samples, but its effectiveness is often limited by the co-amplification of abundant non-target DNA. In this study, a novel metabarcoding assay was developed to amplify a near-complete 18S rRNA gene fragment suitable for long-read nanopore sequencing, enhancing taxonomic resolution. The primers were optimized to maximize detection of parasitic taxa while minimizing off-target amplification of bacterial and archaeal sequences, thereby improving assay specificity. In this study, the 18S metabarcoding assay worked well on clinical fecal samples containing clinically relevant levels of parasites. However, analysis of ungulate fecal samples revealed that fungal and plant sequences vastly outnumbered other eukaryotic taxa in many samples, obscuring the detection of low-abundance protozoan and helminth parasites. To address this, Suppression/Competition PCR was developed, a novel method that selectively reduces amplification of unwanted DNA. This approach reduced fungal and plant reads by over 99 %, enabling sequences from other taxa to comprise an average of over 98 % of total reads as opposed to an initial 36 %. Utilizing this newly-developed metabarcoding assay in either the standard or Suppression/Competition configuration on fecal DNA extracts from a range of host species, parasites of interest such as Cryptosporidium sp., Cyclospora cayetanensis, Blastocystis sp., Entamoeba sp., Eimeria sp., Ancylostoma sp., and Toxocara sp. were detected, demonstrating its broad applicability.

元条形码被广泛用于检测粪便样品中的微生物，但其有效性往往受到大量非靶DNA共扩增的限制。在这项研究中，开发了一种新的元条形码分析方法来扩增一个接近完整的18S rRNA基因片段，适用于长读纳米孔测序，提高了分类分辨率。优化引物以最大限度地检测寄生类群，同时最大限度地减少细菌和古细菌序列的脱靶扩增，从而提高检测特异性。在本研究中，18S元条形码分析在含有临床相关水平寄生虫的临床粪便样本中效果良好。然而，对有蹄类粪便样本的分析显示，真菌和植物序列在许多样本中远远超过其他真核生物分类群，模糊了低丰度的原生动物和蠕虫寄生虫的检测。为了解决这个问题，抑制/竞争PCR被开发出来，这是一种选择性地减少不需要的DNA扩增的新方法。这种方法将真菌和植物的reads减少了99% %以上，使来自其他分类群的序列平均占总reads的98% %以上，而不是最初的36% %。利用这种新开发的元条形码分析方法，在标准或抑制/竞争配置下，对来自一系列宿主物种的粪便DNA提取物进行了检测，检测到感兴趣的寄生虫，如隐孢子虫、卡耶坦环孢子虫、囊虫、内阿米巴、艾美球虫、钩虫和弓形虫，证明了其广泛的适用性。

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引用次数: 0

A rapid and visual detection for canine Adenovirus-2 using CRISPR-Cas13a-based SHERLOCK technology 基于crispr - cas13的SHERLOCK技术对犬腺病毒2的快速和视觉检测

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-01 DOI: 10.1016/j.mimet.2025.107314

Boyu Liu , Yifan Li , Zaixing Yang , Jingqi Wu , Yuxuan Jiang , Lili Zhao , Junwei Ge

Canine adenovirus type 2 (CAdV-2) is an important pathogen causing infectious tracheobronchitis (ITB) and viral enteritis in puppies, often exacerbating clinical symptoms through co-infection with other viruses. However, existing diagnostic methods for CAdV-2 exhibit notable limitations. Specifically, they are time-consuming, require additional nucleic acid purification steps, depend on expensive detection equipment, and necessitate operation by professional personnel. Collectively, these limitations prevent the achievement of rapid and accurate CAdV-2 detection in resource-limited settings. In this study, we established a novel CAdV-2 detection method by integrating CRISPR/Cas13a collateral cleavage activity with HUDSON rapid nucleic acid extraction, recombinase-aided amplification (RAA), and a lateral flow strip. This isothermal assay allows for visual, naked-eye result interpretation and achieves a sensitivity of 10² copies/μL as read by lateral flow strips (corresponding to approximately 750 copies per reaction). It showed excellent specificity with no cross-reactivity observed against five other major canine viruses. When tested on 20 clinical samples, the assay demonstrated a 95 % concordance rate with the conventional simplex PCR results. The entire detection process is simple to perform, requires only basic equipment, and delivers results within 90 min. The developed CRISPR/Cas13a-based detection method exhibits significant application potential for CAdV-2 detection. This study develops a CRISPR/Cas13a-based point-of-care diagnostic tool for CAdV-2, delivering rapid, sensitive, and visual detection that significantly facilitates field-based pathogen surveillance and control efforts, while advancing the application of CRISPR diagnostics in veterinary infectious diseases.

犬腺病毒2型（Canine adenovirus 2, cadv2）是引起幼犬感染性气管支气管炎（ITB）和病毒性肠炎的重要病原体，常与其他病毒共感染而加重临床症状。然而，现有的cadv2诊断方法存在明显的局限性。具体来说，它们耗时，需要额外的核酸纯化步骤，依赖于昂贵的检测设备，并且需要专业人员操作。总的来说，这些限制阻碍了在资源有限的情况下实现快速准确的cadv2检测。在本研究中，我们将CRISPR/Cas13a侧链裂解活性与HUDSON快速核酸提取、重组酶辅助扩增（recombinase-aided amplification， RAA）和侧流条带结合，建立了一种新的cadv2检测方法。该等温分析允许视觉，裸眼结果解释，并达到102拷贝/μL的灵敏度，通过横向流动条读取（相当于每次反应约750拷贝）。对其他5种主要犬病毒均无交叉反应，具有良好的特异性。当对20个临床样本进行测试时，该分析显示与传统单一PCR结果的一致性率为95% %。整个检测过程操作简单，只需要基本的设备，并在90 min内提供结果。所开发的基于CRISPR/ cas13的检测方法在cadv2检测中具有重要的应用潜力。本研究开发了一种基于CRISPR/ cas13的cadv2即时诊断工具，提供快速、敏感和视觉检测，极大地促进了现场病原体监测和控制工作，同时推进了CRISPR诊断在兽医传染病中的应用。

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引用次数: 0

Comparison of three commercial DNA extraction kits and assemblers for AMR determinant detection in Pseudomonas aeruginosa and Enterobacter cloacae using long-read sequencing 铜绿假单胞菌和阴沟肠杆菌AMR决定因子长读序列检测的三种商用DNA提取试剂盒和组装器的比较

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-10-31 DOI: 10.1016/j.mimet.2025.107317

Anurag Kumar Bari , Basil Britto Xavier , Tim Severs , Bhanu Sinha , John W.A. Rossen , on behalf of the DRAIGON Consortium

Background

Pseudomonas aeruginosa and Enterobacter cloacae, both members of the ESKAPE group, are multidrug-resistant pathogens that pose significant challenges in clinical care. The high GC-content (∼67 %) of the P. aeruginosa genome complicates DNA extraction and long-read sequencing, with downstream effects on genome assembly and analyses. We compared three commercial extraction kits for isolating high-quality DNA suitable for Oxford Nanopore sequencing from clinical isolates of P. aeruginosa and E. cloacae.

Methods

Genomic DNA was extracted from P. aeruginosa (n = 63; 3 × 21) and E. cloacae (n = 96; 3 × 32) clinical isolates using the MagAttract HMW DNA Kit (Qiagen), the DNeasy UltraClean Microbial Kit (Qiagen), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (ThermoFisher). DNA quantity/quality was assessed by spectrophotometry (NanoDrop, Thermo Fisher Scientific), fluorometry (Qubit, ThermoFisher), and capillary electrophoresis (TapeStation 2200, Agilent). Libraries were prepared and sequenced using Oxford Nanopore Technologies platforms. Assemblies were generated with Unicycler (v.0.5.1) and Flye (v.2.9.6); quality was assessed with QUAST (v.5.3). Genome completeness was evaluated by CheckM (v.1.1.6). Antimicrobial resistance determinants were identified with AMRFinderPlus (v.4.1.19).

Results

DNeasy yielded up to 4.7× higher DNA and ∼ 50 % higher sequencing output than MagAttract, while MagAttract produced higher DNA integrity and more contiguous assemblies. The choice of assembly had a greater impact on the detection of AMR determinants than the extraction method alone. Across workflows, Flye outperformed Unicycler, increasing detection by 2–14 percentage points. The best-performing combination (DNeasy + Flye) achieved 95.2 % AMR determinants, compared to 67.8 % for MagMAX + Unicycler, with the most difference (37.5 %) in efflux pump genes.

Conclusions

This systematic comparison highlights trade-offs between DNA yield, integrity, and downstream assembly performance, demonstrating that assembler choice critically impacts the detection of AMR determinants. These findings provide practical guidance for optimizing long-read-based sequencing workflows to support AMR surveillance and genomic epidemiology.

背景：铜绿假单胞菌和阴沟肠杆菌都是ESKAPE组的成员，是多重耐药病原体，对临床护理构成重大挑战。P. aeruginosa基因组的高gc含量（~67 %）使DNA提取和长读测序复杂化，并对基因组组装和分析产生下游影响。我们比较了从铜绿假单胞菌和阴沟肠杆菌临床分离株中分离高质量DNA的三种商业提取试剂盒。方法：采用MagAttract HMW DNA试剂盒（Qiagen）、DNeasy UltraClean微生物试剂盒（Qiagen）和MagMAX™微生物组超核酸分离试剂盒（ThermoFisher）对P. aeruginosa （n = 63;3 × 21）和E. cloacae （n = 96;3 × 32）临床分离株进行基因组DNA提取。采用分光光度法（NanoDrop, ThermoFisher Scientific）、荧光法（Qubit, ThermoFisher）和毛细管电泳法（TapeStation 2200, Agilent）评估DNA的数量/质量。利用Oxford Nanopore Technologies平台制备文库并进行测序。程序集是用Unicycler （v.0.5.1）和fly （v.2.9.6）生成的；用QUAST （v.5.3）评价质量。使用CheckM （v.1.1.6）评估基因组完整性。使用AMRFinderPlus （v.4.1.19）鉴定抗微生物药物耐药性决定因素。结果：与magattraction相比，DNeasy的DNA产量高出4.7倍，测序产量提高 ~ 50 %，而magattraction的DNA完整性更高，序列更连续。组装体的选择比单独的提取方法对AMR决定因素的检测有更大的影响。在整个工作流程中，fly的表现优于Unicycler，将检测率提高了2-14个百分点。表现最好的组合（DNeasy + Flye）获得了95.2 %的AMR决定因子，而MagMAX + Unicycler获得了67.8% %的AMR决定因子，其中外排泵基因差异最大（37.5% %）。结论：这一系统比较突出了DNA产率、完整性和下游组装性能之间的权衡，表明组装者的选择对AMR决定因素的检测有重要影响。这些发现为优化基于长读的测序工作流程以支持AMR监测和基因组流行病学提供了实用指导。

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引用次数: 0

A rapid and efficient method for visualisation of microbial biofilms on natural, and industrially- and medically-relevant surfaces, using field emission- scanning electron microscopy 使用场发射扫描电子显微镜，快速有效地可视化天然、工业和医学相关表面上的微生物生物膜。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-10-30 DOI: 10.1016/j.mimet.2025.107315

Abhinaba Chakraborty, Bomba Dam

A rapid and efficient sample preparation method for visualising surface-associated microbial biofilms using Field Emission-Scanning Electron Microscopy (FE-SEM) was developed by optimising the fixative concentration and dehydration process. First, different concentrations of the fixative, glutaraldehyde (5–50 %), were tested on Escherichia coli biofilms formed on smooth glass surface for a fixation period of 30 min, followed by a 10-min dehydration each in increasing grades (10–90 %, with 10 % increment) of alcohol. The highest (50 %) glutaraldehyde concentration resulted in the sharpest biofilm micrographs. Further, the incubation period in each alcohol grade was reduced to 2 min to lower the sample preparation time. Improved imaging using the developed protocol was quantified using a newly developed metric, ‘Cellular Integrity Index’ (CII), which evaluates the morphological integrity of biofilm-cells. The protocol preserved the cellular integrity of individual biofilm-associated cells of Aeromonas hydrophila, A. salmonicida, Pseudomonas fluorescens, and Bacillus mycoides. The optimised method generated distinctive high-resolution micrographs of E. coli biofilms pre-formed on different medically-, industrially-, and environmentally-relevant surfaces, such as polypropylene plastic, catheter, and paper, all with high CII values (95–97 %) with least deformation. Further, the method was used for visualising naturally-formed biofilms on poultry ceca, plant roots, and rock surfaces with impeccable clarity, even effectively resolving different microorganisms, like fungus, algae and bacteria Thus, the developed method will be a valuable asset for any research dealing with the visualisation of naturally-formed or laboratory-developed biofilms on any sort-of surface, be it of a single individual or mixed species, thus enriching environmental, industrial, and medical research.

通过优化固定液浓度和脱水工艺，建立了一种利用场发射扫描电镜（FE-SEM）快速、高效地观察表面相关微生物生物膜的样品制备方法。首先，在光滑玻璃表面形成的大肠杆菌生物膜上测试不同浓度的固定剂戊二醛（5-50 %），固定时间为30 min，然后以酒精的增加等级（10-90 %，增加10 %）脱水10 min。戊二醛浓度最高（50 %）时，生物膜显微图像最清晰。此外，每个酒精等级的孵育时间减少到2 min，以缩短样品制备时间。使用新开发的指标“细胞完整性指数”（CII）对使用开发的方案改进的成像进行量化，该指标评估生物膜细胞的形态完整性。该方案保留了嗜水气单胞菌、沙门氏菌、荧光假单胞菌和真菌芽孢杆菌个体生物膜相关细胞的细胞完整性。优化后的方法生成了在不同的医学、工业和环境相关表面（如聚丙烯塑料、导管和纸）上预先形成的大肠杆菌生物膜的独特的高分辨率显微照片，这些生物膜都具有高CII值（95-97 %）和最小变形。此外，该方法还被用于观察家禽盲肠、植物根和岩石表面上自然形成的生物膜，清晰度无可置疑，甚至可以有效地分辨不同的微生物，如真菌、藻类和细菌。因此，该方法对于任何研究自然形成或实验室开发的生物膜的可视化，无论是单个物种还是混合物种，都将是一项宝贵的资产，从而丰富了环境、工业、还有医学研究。

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引用次数: 0

A workflow for selective isolation of bacterial RNA from Streptococcus agalactiae during in vivo infection 无乳链球菌体内感染过程中选择性分离细菌RNA的工作流程。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-10-30 DOI: 10.1016/j.mimet.2025.107318

Agata Famà , Germana Lentini , Alessia Berbiglia , Riccardo Galasso , Giuseppe Valerio De Gaetano , Francesco Coppolino , Concetta Beninati

Studying bacterial gene expression directly in infected tissues is crucial for understanding host–pathogen interactions, however recovery of intact bacterial RNA in vivo remains technically challenging. Contamination with host RNA and the instability of bacterial transcripts often limit downstream applications. Here, we developed an optimized protocol for the selective isolation of Streptococcus agalactiae RNA from infected mouse peritoneal lavage fluids. The method combines differential centrifugation, selective osmotic lysis of eukaryotic cells, and mechanical disruption of bacteria using a high-frequency bead-beating system with 106 μm glass beads. This workflow maximized RNA yield and purity, while minimizing host RNA contamination. Optimization experiments established bead size, homogenization time, and bacterial input as key parameters, enabling the recovery of detectable bacterial RNA from as few as 4 × 10⁵ CFU/mL. In vivo, RNA preparations displayed acceptable purity (A260/280 > 1.8), with moderate integrity (RINe 5.5), yet were suitable for downstream applications. As a proof of concept, quantitative RT-PCR revealed an 11-fold upregulation of the adhesin gene pbsP in vivo compared to in vitro conditions. This protocol provides a reproducible approach for obtaining bacterial RNA from host tissues, facilitating targeted gene expression studies during infection. Although not yet optimal for full transcriptomic profiling, the method provides a practical tool for investigating virulence factor expression in vivo and can be adapted to other bacterial pathogens.

直接研究感染组织中的细菌基因表达对于理解宿主-病原体相互作用至关重要，然而在体内恢复完整的细菌RNA在技术上仍然具有挑战性。宿主RNA的污染和细菌转录物的不稳定性常常限制下游应用。在这里，我们开发了一种优化的方案，用于从感染小鼠腹膜灌洗液中选择性分离无乳链球菌RNA。该方法结合了差速离心，真核细胞的选择性渗透裂解，以及使用106 μm玻璃微珠的高频珠子加热系统对细菌进行机械破坏。该工作流程最大限度地提高了RNA产量和纯度，同时最大限度地减少了宿主RNA污染。优化实验以颗粒大小、均质时间和细菌输入为关键参数，使可检测的细菌RNA回收率低至4 × 105 CFU/mL。在体内，RNA制剂显示出可接受的纯度（A260/280 > 1.8），具有中等的完整性（RINe 5.5），但适合下游应用。作为概念的证明，定量RT-PCR显示，与体外条件相比，粘附素基因pbsP在体内上调了11倍。该方案为从宿主组织中获得细菌RNA提供了一种可重复的方法，促进了感染期间靶向基因表达的研究。虽然该方法还不是最理想的全转录组分析方法，但它为研究体内毒力因子表达提供了一个实用的工具，并且可以适用于其他细菌病原体。

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引用次数: 0

Comparison of diagnostic efficacy of three different oral samples in pulmonary tuberculosis using cepheid gene Xpert® MTB/RIF ultra 造父变星基因Xpert®MTB/RIF ultra对三种不同口腔样本肺结核诊断效果的比较

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-10-30 DOI: 10.1016/j.mimet.2025.107310

Yan Liu, Qianfang Hu, Shuliang Guo

Context

The etiological confirmation of pulmonary tuberculosis typically relies on sputum or bronchoalveolar lavage fluid (BALF). Oral sampling offers a non-invasive and non-sputum alternative, but evidence varies widely due to inconsistent methods. This study employed the Cepheid Gene Xpert® MTB/RIF Ultra (Xpert-Ultra) to compare the diagnostic performance of tongue swabs, pharyngeal swabs, and posterior oropharyngeal saliva (POS) collected from the same patient to identify the optimal option.

Methods

The diagnosis was based on a composite microbiological reference standard. Tongue swabs, pharyngeal swabs, and POS were sequentially collected from each participant. Sputum and BALF underwent acid-fast bacilli smear microscopy, Cepheid Gene Xpert® MTB/RIF (Xpert), and culture, while oral samples were tested with Xpert-Ultra.

Results

All three oral sample types demonstrated 100 % specificity. Compared to tongue swabs and pharyngeal swabs, POS showed the highest sensitivity, both overall and in subgroup analyses, along with the highest bacterial load and the lowest cycle threshold values.

Conclusion

Compared with tongue swabs and pharyngeal swabs, POS exhibits the best diagnostic efficacy, and is more suitable for no-sputum or paucibacillary tuberculosis patients, and shows potential as an alternative to BALF.

背景：肺结核的病原学证实通常依赖于痰液或支气管肺泡灌洗液（BALF）。口腔取样提供了一种非侵入性和非痰液的替代方法，但由于方法不一致，证据差异很大。本研究采用造父变星基因Xpert®MTB/RIF Ultra （Xpert-Ultra）比较从同一患者收集的舌拭子、咽拭子和后口咽唾液（POS）的诊断性能，以确定最佳选择。方法：采用复合微生物参考标准进行诊断。按顺序收集每位参与者的舌拭子、咽拭子和POS。对痰液和BALF进行抗酸杆菌涂片镜检、造父变星基因Xpert®MTB/RIF （Xpert）和培养，同时对口腔样本进行Xpert- ultra检测。结果：所有三种口腔样本类型均具有100% %的特异性。与舌拭子和咽拭子相比，在总体和亚组分析中，POS显示出最高的敏感性，以及最高的细菌负荷和最低的周期阈值。结论：与舌拭子和咽拭子相比，POS的诊断效果最好，更适用于无痰或少菌性肺结核患者，具有替代BALF的潜力。

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引用次数: 0

Development of a bi-epitope peptide as fail-safe internal control for a modified proximity extension assay and implementation in the detection of procalcitonin in human plasma 开发一种双表位肽作为故障安全的内部控制，用于改进的接近扩展试验和实施检测人血浆中的降钙素原。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-10-30 DOI: 10.1016/j.mimet.2025.107319

Bedin Frederic , Benoit Vincent , Rubens Agnes , Lacoux Xavier , Foucault Frederic , Imbaud Pierre

Proximity Extension Assay (PEA) is a simple, minimally invasive assay developed for proteomic applications and biomarker detection in biological samples. The method is the combination of a homogeneous-phase immunoassay with quantitative PCR-based detection. With the aim of providing a fail-safe check for the successive steps of a new PEA-derived protocol, from sample addition to quantitative PCR, an internal control (IC) was designed. The IC consisted of a synthetic peptide made of the concatenation of three Amino Dioxa-octanoic Acid spacers inserted between two epitope-tags. The efficiency of the peptide design was checked in plasma spiked with the IC. It has been shown that the IC can be co-detected with at least one biomarker of diagnostic interest in human plasma specimens (here procalcitonin or PCT), without PCR cross-interference, and with no evidence of major interference with plasma proteins. The dynamic range of the IC peptide detection encompasses 3 orders of magnitude in concentration. At a given concentration, when tested in parallel with PCT on several human samples, Ct values for the IC remained stable while the Ct values for PCT decreased with increasing PCT concentrations. The work shown here represents a preliminary study aimed at establishing proof-of-concept for the use of a peptide containing two epitope tags as a suitable internal control in proximity assays.

PEA是一种简单、微创的检测方法，用于蛋白质组学应用和生物样品中的生物标志物检测。该方法结合了均相免疫分析和基于定量pcr的检测。为了为新的pea衍生方案的连续步骤提供故障安全检查，从样品添加到定量PCR，设计了内部控制（IC）。该IC由插入两个表位标签之间的三个氨基二氧辛酸间隔物连接而成的合成肽组成。在加入IC的血浆中检查了肽设计的效率。研究表明，IC可以与人类血浆标本中至少一种有诊断意义的生物标志物（降钙素原或PCT）共同检测，没有PCR交叉干扰，也没有证据表明对血浆蛋白有主要干扰。IC肽检测的动态范围包括3个数量级的浓度。在给定浓度下，当与PCT同时对几个人体样本进行测试时，IC的Ct值保持稳定，而PCT的Ct值随着PCT浓度的增加而下降。这里所展示的工作代表了一项初步研究，旨在建立概念验证，证明使用含有两个表位标签的肽在接近分析中作为合适的内部控制。

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引用次数: 0