Journal of microbiological methods最新文献

{"title":"Selective anti-adhesion and anti-biofilm action of D-phenylalanine chiral nanofilms against Staphylococcus aureus","authors":"Xinglei Guo,&nbsp;Xiao Feng,&nbsp;Jianhao Zhang,&nbsp;Hao Zhang,&nbsp;Wenjing Yan","doi":"10.1016/j.mimet.2025.107353","DOIUrl":"10.1016/j.mimet.2025.107353","url":null,"abstract":"<div><div>Biofilm formed by foodborne pathogens on contact surface are notoriously resistant to conventional disinfection, leading to equipment failure and persistent contamination. Therefore, modulating initial bacterial adhesion offers a promising strategy to prevent biofilm formation. Herein, we developed phenylalanine-functionalized gold nanoparticles (D/L-Phe Au NPs) by conjugating D- or <em>L</em>-phenylalanine onto gold nanoparticle surfaces. Using liquid-liquid interfacial self-assembly, we fabricated chiral nanofilms (D/L-Phe Au NP films). Short-term anti-adhesion experiments showed that D-Phe and L-Phe Au NP films reduced initial adhesion of <em>Staphylococcus aureus</em> (<em>S. aureus</em>) by 68.7 % and 57.5 %, respectively. In long-term antibiofilm assays, dense biofilms developed on polydimethylsiloxane (PDMS) and unmodified Au NP film, whereas D/L-Phe Au NP films demonstrated minimal bacterial adhesion with OD₆₀₀ values consistently below 0.5. Live/dead staining indicated that the average fluorescence intensity of green (red) on D/L-Phe Au NP films was reduced to 4.47 % (7.89 %) and 3.18 % (5.92 %) compared to PDMS, respectively. Furthermore, bacterial metabolic activity decreased significantly, accompanied by structural damage and reduced secretion of extracellular polysaccharides and proteins. These results demonstrate that D/L-Phe Au NP films, particularly the D-enantiomer, are a potent surface treatment strategy to combat biofilm formation in food-processing environments.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107353"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145681923","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}

{"title":"Comparative bioremediation of petroleum-contaminated soil by live bacterial isolates and crude biosurfactant supernatants","authors":"Lina Adnan Abis ,&nbsp;Omar M. Hassan ,&nbsp;Khalil T. Hassan","doi":"10.1016/j.mimet.2025.107345","DOIUrl":"10.1016/j.mimet.2025.107345","url":null,"abstract":"<div><div>Petroleum-contaminated soils are difficult to remediate rapidly because early removal is limited by hydrocarbon bioavailability. We compared live bioaugmentation using three isolates, <em>Pseudomonas aeruginosa</em>, <em>Bacillus subtilis</em>, and <em>Pseudomonas putida,</em> with their crude biosurfactant containing cell-free supernatants (CFS) under harmonized 14-day microcosm conditions. Total petroleum hydrocarbons (TPH) were quantified by GC–FID, and apparent first-order kinetics were estimated. Biosurfactant function was assessed by oil-spreading, emulsification index (E24), drop-collapse, and critical micelle concentration (CMC). Across all strains, CFS matched or outperformed live cells for early TPH reduction: 14-day removal in bio-amended treatments ranged 35–54 % versus 12 % in the abiotic control, with <em>P. aeruginosa</em> CFS giving the highest effect (54 %). The kinetic constant for <em>P. aeruginosa</em> CFS was 0.031 day⁻¹ (half-life 22.7 days), compared with 0.026 day⁻¹ (26.3 days) for <em>P. aeruginosa</em> live cells and 0.009 day⁻¹ (76 days) in the control. Trends in removal and k values aligned with biosurfactant metrics (lower CMC, larger oil-spreading diameters, higher E24), indicating that interfacial mass transfer rather than microbial growth governed early-phase performance. One-way ANOVA with Tukey's HSD confirmed significant differences among treatments (<em>p</em> &lt; 0.05). Operationally, crude CFS are batch-producible, do not introduce live organisms, and can be quality-controlled using simple functional assays. These features, together with the observed early gains in TPH removal and shorter half-lives, support CFS dosing as a practical, lower-risk alternative or complement to live bioaugmentation in rapid cleanup windows. We recommend pilot-scale testing across soil textures and longer horizons to define when in-situ biosurfactant production by live degraders narrows the CFS–live gap. These findings support the potential use of crude biosurfactant supernatants as a practical, low-risk alternative to live bioaugmentation for the remediation of petroleum-contaminated soils.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107345"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145616034","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}

{"title":"Unmasking metabolic endotoxemia: Proteinase K–enhanced detection of free and protein-associated endotoxin in human serum","authors":"Alejandra Vargas-Caraveo ,&nbsp;Keren E. Tovar-Presas ,&nbsp;Dalia S. Flores-Molina ,&nbsp;Diego J. Hernandez-Castro ,&nbsp;Katya A. Carrasco-Urrutia ,&nbsp;Ana L. Arellano-Ortiz","doi":"10.1016/j.mimet.2025.107329","DOIUrl":"10.1016/j.mimet.2025.107329","url":null,"abstract":"<div><div>Metabolic endotoxemia is a low-grade endotoxin exposure that leaks from the gut into the bloodstream, contributing to low-grade chronic inflammation. The most used method to quantify endotoxins is the Limulus Amebocyte Lysate (LAL) assay; however, its accuracy is limited due to the strong affinity of endotoxins for plasma proteins like albumin and lipoproteins, which can mask them and interfere with detection by factor C.</div><div>The aim of this study was to evaluate the use of proteinase K pretreatment to unmask protein-bound endotoxins in human serum, allowing quantification of total, free, and bound fractions using the LAL assay. Participants were classified as normal-weight or obese based on anthropometric and biochemical criteria. Blood samples were obtained under fasting conditions and 180 min after a balanced meal. Each sample was split into two aliquots: one untreated and the other digested with proteinase K. Protein degradation was confirmed by SDS-PAGE, and a formula was applied to estimate the percentage of protein-bound endotoxin.</div><div>Results showed an approximately fivefold increase in detectable endotoxin levels after proteolysis under fasting conditions in both groups, and a smaller, though significant, postprandial increase. Obese participants showed a lower postprandial percentage of protein-bound endotoxin than normal-weight individuals, despite similar endotoxin after proteolysis levels in both groups.</div><div>These findings highlight the need for proteolysis in accurately measuring endotoxin concentrations. Furthermore, the proportion of protein-bound endotoxin may serve as a marker of physiological detoxification capacity. The study suggests that inflammation risk is more closely tied to endotoxin bioavailability than total circulating levels.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107329"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145522780","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}

{"title":"Utility of a commercially available transwell system for assessment of MSC-mediated mitigation of biofilms","authors":"Sarah M. Khatibzadeh ,&nbsp;Linda A. Dahlgren ,&nbsp;Clayton C. Caswell ,&nbsp;William A. Ducker ,&nbsp;Stephen R. Werre ,&nbsp;Sophie H. Bogers","doi":"10.1016/j.mimet.2025.107325","DOIUrl":"10.1016/j.mimet.2025.107325","url":null,"abstract":"<div><div>Mesenchymal stromal cells (MSC) reduce and prevent biofilm infections in a paracrine fashion by secretion of antimicrobial proteins. However, studies to-date have used conditioned cell culture medium alone rather than allowing real-time MSC-biofilm interaction. A co-culture method to evaluate MSC efficacy in reducing established biofilms is needed to facilitate MSC-bacterial interactions while enabling biofilm separation for imaging and quantification after co-culture. We describe the development of a novel in vitro method to co-culture established <em>S. aureus</em> and <em>E. coli</em> biofilms with equine bone marrow-derived MSC ± amikacin sulfate using a commercially available transwell plate system. The system was used to co-culture MSC from 6 horses with mature biofilms and perform analysis of biomass by crystal violet assay, bioburden by colony forming unit analysis and biofilm size by quantitative photographic analysis using Image J. Our method allowed downstream quantification of biofilm biomass, live bacterial counts, and photographic analysis of biofilm size and may be used for high-throughput evaluation of MSC as a treatment for biofilm infections.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107325"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145523288","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}

{"title":"Production, optimization and characterization of cellulase produced from Aspergillus niger in submerged fermentation through response surface methodology","authors":"Kayynat Shoukat ,&nbsp;Irfan Ahmad ,&nbsp;Hafiz Abdullah Shakir ,&nbsp;Muhammad Khan ,&nbsp;Marcelo Franco ,&nbsp;Muhammad Irfan","doi":"10.1016/j.mimet.2025.107357","DOIUrl":"10.1016/j.mimet.2025.107357","url":null,"abstract":"<div><div>Cellulase is considered as the most significant enzymes, widely studied owing to its extensive industrial applications and the increasing worldwide research focus on its production. In this research, mango peels were used as a source of carbon to produce cellulase by <em>Aspergillus niger</em>. The enzyme was produced, characterized under different parameters to optimize its activity, and its application potential was also evaluated. Initially, OFAT (one-factor-at-a-time) optimization maximized cellulase yield. The peak activity was noted after a 72-h incubation, when both the substrate concentration and the inoculum were set to 3.5 %, a 3.8-fold increase as compared to the unoptimized conditions (0.5 % substrate and 24 h). Different types of nutritional components, were screened through Plackett-Burman Design (PBD) and selected parameters were optimized by Central Composite Design (CCD) of Response Surface Methodology (RSM). Among these nutritional components, the combination that yielded the highest enzyme activity included 0.1 % yeast extract, 0.5 % MgSO₄, 0.275 % KH₂PO₄, and 0.5 % (NH₄)₂SO₄. The CMCase (carboxymethyl cellulase) activity increased 1.18-fold (17.9 %), FPase (filter paper cellulase) activity showed a more substantial improvement, corresponding to a 1.87-fold (86.8 %) enhancement. Characterization of enzyme showed that maximum activity and stability of CMCase was at temperature 60 °C and 50 °C for FPase and pH 5. Enzyme showed peak activity in the presence of NH₄Cl, KCl, FeSO₄, and CuSO₄. Solvents like SDS, Tween-80, butanol, n-hexane, ethyl alcohol, and isopropanol enhanced the stability of enzyme. Using Arrhenius plot, the values of activation energy (Ea) were calculated as −26.48 kJ/mol and − 20.54 kJ/mol for CMCase and FPase respectively. Enthalpy (∆H) and entropy (∆S) of the system were also calculated and evaluation revealed that the enthalpy for CMCase and FPase were 23.5 kJ/mol and 17.4 kJ/mol whereas the values of entropy were − 19.37 kJ/mol and − 16.3 kJ/mol respectively. Using the Lineweaver-Burk plot the values of K_m and V_max were estimated as K_m = 7.45 mM and V_max = 81.7 U/ml/min for CMCase, and K_m = 1.168 mM and V_max = 158.07 U/ml/min for FPase. Additionally, this study also indicated that <em>Aspergillus niger</em> could be useful for saccharification processes. Maximum level (11 %) of saccharification was observed at duration of 28 h.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107357"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145701310","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}

{"title":"Colorimetric methods for detecting antimicrobial resistant mycobacteria: An overview","authors":"Mariana Quaresma de Souza ,&nbsp;Ana Júlia Reis ,&nbsp;Andrea von Groll ,&nbsp;Pedro Eduardo Almeida da Silva ,&nbsp;Ivy Bastos Ramis","doi":"10.1016/j.mimet.2025.107341","DOIUrl":"10.1016/j.mimet.2025.107341","url":null,"abstract":"<div><div>Phenotypic culture-based methods continue to be recommended for identification of the susceptibility profile to antimicrobials in mycobacterial isolates. For this, in-house colorimetric methods have been developed for detection of <em>Mycobacterium tuberculosis</em> and nontuberculous mycobacteria resistant strains, associated with an easier interpretation of results. Most existing colorimetric methods are based on the use of redox colorimetric indicators, such as resazurin and tetrazolium salts. Other tests require revealing solutions to generate color, while there are also tests that target the loss of color as a way of indicating bacterial growth. This review aimed to elucidate the available colorimetric methods to detect resistant mycobacterial strains, including their evaluation and application. Despite varying in terms of the format used, the methods presented in this review are relatively simple to perform and cheaper than molecular and semi-automated phenotypic methods, and can be implemented in different scenarios, including in limited resource settings.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107341"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145616032","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}

{"title":"Rapid microbiological method for analysis of clindamycin-based product potency and its equivalence to UV and HPLC methods","authors":"Isadora Alves Lustosa,&nbsp;Ana Carolina Kogawa","doi":"10.1016/j.mimet.2025.107338","DOIUrl":"10.1016/j.mimet.2025.107338","url":null,"abstract":"<div><div>Clindamycin is an antimicrobial sold in capsules, injectable solution, or gel for topical use. Despite its wide use, current official compendia and literature lack microbiological methods to assess its potency, relying mainly on physicochemical assays such as high-performance liquid chromatography (HPLC). These techniques, however, do not evaluate the biological activity of the drug. In this context, the present study proposes, for the first time, aimed to develop and validate a rapid microbiological method to determine the potency of clindamycin in capsule form and to compare its results with those obtained from UV and HPLC methods. The method employed <em>Escherichia coli</em> ATCC 8739, BHI broth as the growth medium, and a hydroalcoholic diluent (ethanol: water, 1:1 <em>v</em>/v) for solution preparation. Concentrations tested were 8, 16, and 32 μg mL⁻¹, with incubation at 35 ± 2 °C for 4 h under agitation (70 rpm), followed by absorbance measurement at 530 nm. The method exhibited linearity (<em>r</em> &gt; 0.99), selectivity, precision (RSD 3.16 %, 3.57 %, 3.14 %), accuracy (average recovery of 99.47 %), and robustness under most tested conditions, as percentage of inoculum, brand of BHI broth, and volume of BHI broth used in the tube. Statistical comparison confirmed the equivalence of the proposed method with UV and HPLC (F_cal 0.68 &lt; F_tab 5.14). This validated microbiological method offers a fast, reliable, and biologically relevant alternative for routine potency determination of clindamycin capsules, addressing a major analytical gap in the quality control of antimicrobial products.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107338"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145587948","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}

{"title":"Different media affect the growth and antimicrobial minimum inhibitory concentrations of Mycoplasma gallisepticum and Mycoplasma synoviae","authors":"Hossein Taiyari ,&nbsp;Jalila Abu ,&nbsp;Zunita Zakaria ,&nbsp;Sharina Omar ,&nbsp;Chan Zi Wen ,&nbsp;Nik Mohd Faiz","doi":"10.1016/j.mimet.2025.107350","DOIUrl":"10.1016/j.mimet.2025.107350","url":null,"abstract":"<div><div>The main pathogenic <em>Mycoplasma</em> spp. infecting chickens are <em>Mycoplasma gallisepticum</em> (MG) and <em>Mycoplasma synoviae</em> (MS). The MG and MS play an etiological role in chronic respiratory disease. One of the methods to control avian mycoplasmosis is antibiotic therapy. The microdilution minimum inhibitory concentration (MIC) assay is one of the most commonly used tests to assess the antibiotic susceptibility profile of isolates. The effect of type of medium used in the MIC assay of MG and MS has not been evaluated. Therefore, the aim of this study is to determine the impact of two commonly used avian mycoplasmas growth media, Frey medium and Pleuropneumonia-like Organisms (PPLO) medium, on growth rate and the MIC values of MG and MS. The comparison of growth rates and MIC values between two media was conducted in three replicates, and the mean values were used for any further analysis. First, the growth rates of MG and MS were compared in both Frey and PPLO media. Then, antimicrobial MIC values were determined using the microbroth dilution method. The results show that MG and MS isolates had faster growth rates in Frey compared to PPLO (<em>p</em> ≤ 0.05). The antimicrobial MIC values of MG and MS isolates were different in Frey and PPLO media. This study indicates the significant effect of the type of medium used in the determination of antimicrobial MIC values of MG and MS, and can be used as a fundamental source for preparation of a more comprehensive MG and MS MIC protocol.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107350"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145648899","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}

{"title":"Development of a rapid molecular assay for specific identification of Mycobacterium avium and Mycobacterium intracellulare","authors":"Takafumi Yoshikane ,&nbsp;Masashi Michibuchi ,&nbsp;Chihiro Ogawa ,&nbsp;Takahiro Azuma ,&nbsp;Tomomi Yamazaki ,&nbsp;Shinji Hatakeyama ,&nbsp;Masaki Takanashi ,&nbsp;Kinuyo Chikamatsu ,&nbsp;Akio Aono ,&nbsp;Akiko Takaki ,&nbsp;Satoshi Mitarai ,&nbsp;Hiromichi Suzuki","doi":"10.1016/j.mimet.2025.107359","DOIUrl":"10.1016/j.mimet.2025.107359","url":null,"abstract":"<div><div>In genetic testing of non-tuberculous mycobacteria, false positives due to cross-reactions and differences in detection performance among strains are problematic. A rapid genetic testing method with high specificity to distinguish only <em>Mycobacterium avium</em> and <em>Mycobacterium intracellulare</em> and high detection performance against genetic diversity among strains is desirable. We identified completely novel genes (MAA44156_02837 for <em>M. avium</em> and KN251_15665 for <em>M. intracellulare</em>) showing strong identity with <em>M. avium</em> and <em>M. intracellulare</em> respectively. We then developed a new <em>M. avium</em>/<em>M. intracellulare</em> simultaneous detection assay targeting these genes and evaluated their specificity and sensitivity. To evaluate cross-reactivity, the newly developed assay correctly identified all <em>Mycobacterium</em> spp. type strains, except for one examination of a duplicated evaluation with a <em>Mycobacterium kyorinense</em> type strain as a false positive at McFarland 1.0. All <em>M. avium</em>-positive and <em>M. intracellulare-</em>positive clinical isolates used in this study were accurately detected using the newly developed assay. The limits of detection for the newly developed assay were estimated to be 100 CFU/mL for <em>M. avium</em> and 30 CFU/mL for <em>M. intracellulare</em>. In addition, the detection performance was maintained even in samples containing both <em>M. avium</em> and <em>M. intracellulare</em>. The <em>M. avium</em>/<em>M. intracellulare</em> simultaneous detection assay developed in this study demonstrated high specificity, detection performance, and sensitivity for detecting <em>M. avium</em> and <em>M. intracellulare</em>.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107359"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145743148","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}

{"title":"Construction and application of MS2 bacteriophage virus-like particles for SARS-CoV-2 detection using a single-plasmid system","authors":"Mengjie Liang ,&nbsp;Yongxin Li ,&nbsp;Jingyuan Yang ,&nbsp;Chunyan Liu ,&nbsp;Haojie Lin ,&nbsp;Zhaohui Deng ,&nbsp;Xin Zhang","doi":"10.1016/j.mimet.2025.107344","DOIUrl":"10.1016/j.mimet.2025.107344","url":null,"abstract":"<div><div>Research on the compatibility of MS2 bacteriophage virus-like particles (VLPs), which are used for the detection of a range of RNA viruses, with commercially available detection kits is limited. Here, we aimed to construct a positive control for RNA virus nucleic acid detection using MS2 bacteriophage VLPs and evaluate its compatibility with commercial nucleic acid detection kits to support the standardization of molecular diagnostic quality control systems. We generated recombinant plasmids expressing MS2 bacteriophage maturation enzyme, capsid proteins, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes (<em>ORF1ab</em>, <em>N</em>, and <em>E</em>) using a single-plasmid dual-expression system (pACYCDuet-1). The plasmids were expressed in <em>Escherichia coli</em> BL21, and then MS2 VLPs were purified. The VLPs were characterized, and their performance as a positive control was validated for homogeneity and stability; their detection compatibility with five commercial SARS-CoV-2 nucleic acid detection kits was also examined. We successfully generated MS2 VLPs carrying SARS-CoV-2 target genes, with a uniform particle size of 23–28 nm and target gene copy number of 1.99 × 10¹⁰ copies/μL. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis confirmed the expression of MS2 proteins, whereas functional validation revealed excellent nuclease resistance, batch–batch homogeneity, and stability at 4 °C for ≥20 days. Importantly, the positive control exhibited consistent detection performance across all five commercial kits, confirming its practicality for routine laboratory workflows. The established positive control is readily applicable for standardizing quality control in molecular diagnostics.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107344"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145616467","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}