ACS Infectious Diseases最新文献

{"title":"BDF6 Deficiency Severely Compromises Intracellular Amastigote Development and Infectivity of Trypanosoma cruzi","authors":"Victoria Boselli Raposo,&nbsp;Virginia Perdomo,&nbsp;Florencia Pacini,&nbsp;Juan A. Cueto,&nbsp;Alejandro Pezza,&nbsp;Patricia S. Romano,&nbsp;Roberto Docampo,&nbsp;Ana Rosa Perez,&nbsp;Esteban Serra","doi":"10.1021/acsinfecdis.6c00417","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00417","url":null,"abstract":"<p><em>Trypanosoma cruzi</em>, the causative agentof Chagasdisease, relies on complex gene regulatory mechanisms to adapt toits diverse host environments. In recent years, it has been establishedthat epigenetics plays an essential role in these mechanisms via theregulation of chromatin structure. Bromodomain-containing factors(BDFs), known for recognizing acetylated lysines on histones, haveemerged as key factors in chromatin remodeling complexes. Among theeight predicted BDFs in <em>T. cruzi</em>, BDF6 is part ofa TINTIN-like complex with MRGx and MRGBP, homologous to componentsof the NuA4/TIP60 chromatin remodeling complex. We generated knockout(KO) parasites for the <em>bdf6</em> gene using CRISPR/Cas9gene editing. BDF6 KO epimastigotes exhibit normal morphology butdecreased size and growth, and the resulting metacyclic trypomastigotesdisplayed drastically reduced infectivity. Strikingly, once insidehost cells, KO parasites differentiated into amastigotes but failedto replicate. This intracellular arrest was reversed by episomal complementationof BDF6. Consistently, KO parasites also exhibited impaired infectivityin mice, a defect that was also rescued in the addback parasite lines.Our findings highlight BDF6 as a critical regulator of intracellularparasite development, operating in stages beyond epimastigotes whereepigenetic plasticity is essential for host adaptation. This strikingstage-specific phenotype of BDF6 KO underscores its functional importanceand highlights the relevance of epigenetic regulators along <em>T. cruzi</em> life cycle.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3430–3444"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149153049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advancing Tuberculosis Antigen Detection by Selecting Sandwich-Compatible Aptamers Against the Mycobacterium tuberculosis ESAT-6/CFP-10 Heterodimer Using Library-Immobilized Strategies","authors":"Shaira Jane Acosta,&nbsp;M. Carmen Bermudo Redondo,&nbsp;Lucie Mužíková Čechová,&nbsp;Marek Ondruš,&nbsp;Vasso Skouridou,&nbsp;Ciara K. O’Sullivan","doi":"10.1021/acsinfecdis.6c00529","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00529","url":null,"abstract":"<p>Tuberculosis (TB) represents the world’s leadinginfectiousdisease killer, surpassing COVID-19. Despite being preventable andcurable, a substantial proportion of cases remain undiagnosed or unreported,sustaining transmission. The detection of antigens such as the <em>Mycobacterium tuberculosis</em> ESAT-6/CFP-10 heterodimer,which are secreted at different stages of infection, is a promisingscreening strategy. Traditional antigen tests relying on antibodiessuffer from significant problems of specificity and sensitivity. Aptamers,selected in an <em>in vitro</em> technique termed SELEX (<strong>S</strong>ystematic <strong>E</strong>volution of <strong>L</strong>igandsby <strong>EX</strong>ponential enrichment), are short, single-strandedoligonucleotides that fold into unique 3D structures, allowing themto bind to specific targets, including proteins, cells, or small molecules,with high affinity and specificity. However, conventional SELEX methodsoften yield suboptimal aptamers partly due to target immobilizationthat can promote nonspecific interactions and distort protein conformation.Here, we employed two library-immobilized strategies to generate aptamersagainst the <em>M. tuberculosis</em> ESAT-6/CFP-10 heterodimer.We first performed capture-SELEX to select aptamers against the unconstrained,conformationally native heterodimer target in solution. These selectedaptamers were then exploited in a second SELEX, where aptamer-targetcomplexes were “baited” by a target-specific antibodyto isolate sandwich-compatible aptamers for downstream assay development.The selected aptamers exhibited high nanomolar affinity regardlessof library and selection strategy employed, with bait-SELEX-derivedcandidates emerging after fewer selection rounds. These aptamers weresuccessfully applied in antibody/aptamer hybrid sandwich assays, supportingtheir potential for TB antigen detection in nonsputum samples.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3472–3486"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149152985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Targeted Inhibition of Intracellular Coxiella burnetii by Overcoming the Vacuolar Barrier with Autologous Exosomal Doxycycline","authors":"Yuchen Zhang,&nbsp;Xiaoxiao Chen,&nbsp;Jinru Lin,&nbsp;Jun Jiao,&nbsp;Yonghui Yu,&nbsp;Wenbo Wei,&nbsp;Shan Zhang,&nbsp;Yufei Jiang,&nbsp;Yinhui Lin,&nbsp;Ziyuan Zhang,&nbsp;Shaoya Liu,&nbsp;Hui Yue,&nbsp;Xuan OuYang,&nbsp;Yajun Song","doi":"10.1021/acsinfecdis.6c00459","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00459","url":null,"abstract":"<p><em>Coxiella burnetii</em>, the causativeagent of Q fever, is an obligate intracellular bacterium that replicateswithin the <em>Coxiella</em>-containing vacuole (CCV), a lysosome-likecompartment. The acidic microenvironment within the CCV impairs theactivity of weak-base antibiotics such as doxycycline, necessitatingprolonged, high-dose therapy. To overcome this fundamental deliverybarrier, we engineered a biomimetic nanoplatform by encapsulatingdoxycycline (DOX) within exosomes derived from THP-1 cell-derivedmacrophages (tExo). DOX@tExo nanoparticles displayed characteristicexosomal morphology and protein markers and demonstrated favorablebiocompatibility both <em>in vitro</em> and <em>in vivo</em>. Importantly, DOX@tExo were efficiently internalized by macrophages,trafficked specifically to lysosomes, and accumulated within CCVs.This precise targeting resulted in an enhancement of antibacterialpotency, reducing the minimal effective concentration for marked inhibitionof intracellular bacterial growth to 0.015 μg/mL, more thanan order of magnitude lower than that of free DOX. Beyond direct drugdelivery, DOX@tExo treatment concurrently reduced autophagic cargodelivery, as indicated by decreased p62 accumulation, thereby potentiallydisrupting a host process coopted by the pathogen. In a murine modelof pulmonary infection, DOX@tExo treatment significantly reduced bacterialburdens in multiple organs. Collectively, this work establishes ahomologous exosome platform derived from THP-1 cells that combinestargeted antibiotic delivery with host-directed immunomodulation,offering a therapeutic strategy against refractory intracellular bacterialinfections.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3445–3461"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149153004","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AX-35 Targets the Cytochrome bc1 Complex in Mycobacterium abscessus and Reveals a Novel Resistance Mechanism","authors":"Andréanne Lupien,&nbsp;Thomas Dessureault,&nbsp;Caroline Shi-Yan Foo,&nbsp;Maryline Kienle,&nbsp;Anthony Vocat,&nbsp;Andrej Benjak,&nbsp;Iain Roe,&nbsp;Karl-Heinz Altmann,&nbsp;Stewart T. Cole,&nbsp;Marcel A. Behr","doi":"10.1021/acsinfecdis.6c00313","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00313","url":null,"abstract":"<p>In patients with cystic fibrosis,nontuberculous mycobacteriaareopportunistic, intracellular and extracellular pulmonary pathogensthat contribute to deterioration of lung function and, ultimately,morbidity from respiratory failure. Among nontuberculous mycobacterialrespiratory pathogens, members of the <em>Mycobacterium abscessus</em> complex pose additional treatment complicationsas they are notoriously drug-resistant. As part of our routine screeningagainst mycobacterial species, one of the lead antituberculosis compounds,the cytochrome <em>bc</em>₁ inhibitor AX-35, wasfound to be active against isolates from the <em>Mycobacterium abscessus</em> complex <em>in vitro</em> (MIC ≤15 μg/mL) and <em>ex vivo</em> in infected macrophages.Isolation and characterization of AX-35-resistant mutants in <em>M. abscessus</em> ATCC 19977 (MIC AX-35 &gt; 100 μg/mL)revealed the presence of a chimeric gene resulting from the recombinationof 737 bps of <em>MAB_2467</em> into <em>MAB_1966c</em>. In <em>M. abscessus</em>, both genes encodefor the <em>b</em> subunit of the cytochrome <em>bc</em>₁ oxidase, also known as QcrB. Further analysis, comprisinggene inactivation, ATP-depletion assays, AlphaFold3-based structuralmodeling, and molecular docking, suggested that the product of <em>MAB_1966c</em> is the target of AX-35. Interestingly, transcriptomicanalysis of <em>M. abscessus</em> exposed toAX-35 revealed that the compound induced expression of <em>MAB_2467</em> and the alternate terminal oxidase, <em>cytochrome bd</em>. In <em>M. tuberculosis</em>, in the absenceof the cytochrome bd oxidase, QcrB inhibitors, including AX-35, arebactericidal. However, in <em>M. abscessus</em>, AX-35 had bacteriostatic activity. These results suggested that,in the presence of AX-35, <em>MAB_2467</em> may compensatefor the absence of the cytochrome <em>bd</em> oxidase andthat <em>M. abscessus</em> ATCC 19977 genomeencodes two QcrB paralogs.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3362–3372"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149153012","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Artificial Intelligence-Integrated Proteome-Wide Target Prioritization for Drug Discovery against Multidrug-Resistant Neisseria gonorrhoeae","authors":"Ravi Kant,&nbsp;Saurabh Mazumdar,&nbsp;Ananya Mahajan,&nbsp;Myron Christodoulides","doi":"10.1021/acsinfecdis.6c00521","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00521","url":null,"abstract":"<p><em>Neisseria gonorrhoeae</em> causes ∼82.4 millioninfections annually and is rapidly approaching an untreatable status.The pathogen has developed resistance to every antibiotic class introducedsince the sulfonamide era. Ceftriaxone, the last WHO- and CDC-recommendedempirical therapy, is now compromised by high-level resistance inextensively drug-resistant lineages reported across multiple continents.No licensed vaccine exists, and no antibiotic with a novel mechanismhas been introduced for this pathogen in over 40 years. Conventionaldrug discovery is too slow and insufficiently resistance-aware toaddress this escalating threat. In this review, we examine in silicoproteome-wide target fishing as a rapid, systematic approach. Theapproach applies six biologically informed filters to the <em>N. gonorrhoeae</em> core proteome: core-genome definition, host-homologyexclusion, essentiality screening, metabolic chokepoint identification,subcellular localization, and resistance-aware prioritization. Acrossdata sets spanning 12–69 clinical strains, this pipeline reduces2000–12,300 protein-coding sequences to 12–30 high-confidencetargets. An integrated artificial intelligence (AI)/machine learning(ML) scoring layer further ranks candidates using sequence features,AlphaFold2 structures, protein–protein interaction networks,and genotype–phenotype data from ∼20,000 clinical isolates.Five targets showed consistent cross-study prioritization: LpxC, MurA,FabI, DapD, and NGFG_RS03485. Of these, only LpxC has experimentalinhibitor validation against multidrug-resistant gonococci; the remainingcandidates are supported computationally but lack biochemical confirmation.Bridging this validation gap remains the critical bottleneck. However,advances in pan-genome analysis, structural prediction, and generativeAI for compound design provide a more tractable route to novel therapeuticsthan previously available.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3296–3308"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149153038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ag85A-Dependent Surface Lipid Remodeling Impairs Adhesion and Infection of Therapeutic Mycobacteriophage","authors":"Morgane Illouz,&nbsp;Valentin Wasselin,&nbsp;Adrián Pál,&nbsp;Colin Lewis,&nbsp;Maëlle Foubert,&nbsp;Aurore Briand,&nbsp;Silke Malmsheimer,&nbsp;Christian Chalut,&nbsp;Jana Korduláková,&nbsp;Graham Hatfull,&nbsp;Laurent Kremer","doi":"10.1021/acsinfecdis.6c00626","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00626","url":null,"abstract":"<p>Patients infected with mycobacteria,particularly <em>Mycobacterium abscessus</em>, frequently experience antibiotictreatment failure. Phage therapy, used under compassionate care, offersa promising alternative and has already demonstrated clinical efficacy.However, phage-bacteria interactions and their coevolution remaincritical factors that can compromise therapeutic success. Trehalosepolyphleates (TPPs), complex lipids of the outer membrane, play akey role in the adhesion of the therapeutic mycobacteriophage BPsΔ<em>33</em>HTH_HRM10, and loss of TPP synthesis confersbacterial resistance to the phage. Herein, we found that deletionof the mycolyltransferase-encoding gene <em>MSMEG_6398</em>, but not of its homologues <em>MSMEG_6396</em> or <em>MSMEG_6399,</em> all of which encode membersof the Ag85 complex, increased resistance to BPsΔ<em>33</em>HTH_HRM10 on solid and liquid media.Alterations in the mycomembrane, the first barrier encountered byphages, were revealed by lipid profiling, while adhesion assays andflow cytometry indicated a defect at the binding stage. This effectwas related to the accumulation of trehalose dimycolate (TDM) in the <em>MSMEG_6398</em> mutant. The role of TDM insusceptibility/resistance to BPsΔ<em>33</em>HTH_HRM10was then demonstrated with a knockdown mutant of the arabinosyltransferaseAftB involved in a terminal step of cell wall arabinan biosynthesisassociated with TDM accumulation and further validated using ethambutol,a drug that induces TDM accumulation. Collectively, these findingssuggest that TPPs may be masked by excess trehalose-bound lipids,reducing phage adsorption and preventing subsequent phage infection.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3530–3542"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149152977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biopolymer-Antigen Conjugate Vaccines for Prevention of Tularemia","authors":"Gayathri Sam,&nbsp;Charles T. Spencer,&nbsp;Bernd H. A. Rehm","doi":"10.1021/acsinfecdis.6c00694","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00694","url":null,"abstract":"<p><em>Francisella tularensis</em>, the causativeagent of tularemia, is a facultative intracellular pathogen categorizedas a tier I bioterrorism agent. The current live vaccine strain (LVS)vaccine poses safety and virulence concerns, advocating the need fora safer alternative. In this study, we demonstrated a combinationof antigens FopA, Tul4, SucB, and DnaK conjugated to biopolymer particles(BPs) as a multivalent subunit vaccine that not only induces LVS andSchuS4-specific antibody titers but also confers protective immunitywith differential cytokine profiles upon LVS challenge. This multivalentvaccine conferred 80% survival in mice, with antibodies of opsonizingability and rapid recovery from weight loss. The production of thesebiopolymer conjugate vaccines was achieved in one step inside engineered <em>Escherichia coli</em> and is not only cost-effective butalso leads to thermostable formulations, eliminating the need forcold-chain transport and storage. <em>E. coli</em> fermentation-based production avoids the need for high-containmentfacilities. Collectively, the FopA-BP-Tul4, SucB-BP, and Dnak-BP combinedformulations hold the promise for the development of a robust subunitvaccine for the prevention of tularemia.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3543–3558"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149153153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Performance of a Group II Intron Reverse Transcriptase from Anoxybacillus flavithermus in Duplex RT-LAMP for SARS-CoV-2 Detection","authors":"Igor P. Oscorbin,&nbsp;Elizaveta D. Baranova,&nbsp;Maxim L. Filipenko","doi":"10.1021/acsinfecdis.6c00293","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00293","url":null,"abstract":"<p>Reverse transcriptases from bacterial group II introns(GII RTs)demonstrate superior thermostability and processivity compared withretroviral counterparts. While GII RTs have proven advantageous forRNA-seq, their performance in other applications, such as reversetranscription loop-mediated isothermal amplification (RT-LAMP), remainspoorly studied. RT-LAMP is a widely used method suitable for point-of-carediagnostics of RNA viruses and other pathogens. In this study, wecompared the performance in RT-LAMP of GII RT from the thermophilicbacterium <em>Anoxybacillus flavithermus</em> (Afl RT) with that of the retroviral RNase H-deficient reverse transcriptaseM-MuLV. A duplex RT-LAMP targeting SARS-CoV-2 and MS2 phage genomicRNA (internal control) served as a model. Preheating at 95 °Cand 1.5 M betaine increased the efficiency of RT-LAMP with both reversetranscriptases. Under these conditions, the optimal temperature rangeand time of reverse transcription were similar for both enzymes, 55–60°C and 15 min, respectively. However, high concentrations ofAfl RT (&gt;50 U/reaction) inhibited RT-LAMP. Notably, RT-LAMP withAflRT demonstrated higher sensitivity for detecting both in vitro-transcribedRNA fragments and SARS-CoV-2–positive samples. In validationagainst RT-qPCR using nasal swabs (200 SARS-CoV-2-positive and 200negative samples) from patients with respiratory symptoms, the observedsensitivity of RT-LAMP with Afl RT was 100% (200/200; 95% CI, 98.17–100%),whereas with M-MuLV RT, sensitivity was 97.0% (194/200; 95% CI, 93.58–98.89%).Specificity was 100% for both RTs (200/200; 95% CI, 98.17–100%).These observations demonstrate that Afl RT is suitable for RT-LAMPand may increase the sensitivity of LAMP-based diagnostics.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3351–3361"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149153178","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Honoring Prof. Gerard (Gerry) D. Wright, a Fearless Pioneer in Natural Product Antimicrobial Discovery and the Antibiotic Resistome","authors":"Georgina Cox,&nbsp;Eric Brown,&nbsp;Jayanta Haldar","doi":"10.1021/acsinfecdis.6c00889","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00889","url":null,"abstract":"","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3276–3279"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149152871","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RPA-CRISPR/Cas12a Assay for ALV-J with Broad-Spectrum Detection and Reduced Residual Amplicon Load","authors":"Ruiying Han,&nbsp;Ning Xiao,&nbsp;Zhirui Wu,&nbsp;Jiahua Wang,&nbsp;Xiaoli Wang,&nbsp;Xiaochuan Tang","doi":"10.1021/acsinfecdis.6c00419","DOIUrl":"https://doi.org/10.1021/acsinfecdis.6c00419","url":null,"abstract":"<p>Avian leukosis virus subgroup J (ALV-J)is a retrovirusthat posesa serious threat to the poultry industry. It is prone to genetic variation,leading to the emergence of multiple epidemic strains, which createssubstantial challenges for clinical detection. Although the recombinasepolymerase amplification (RPA)-CRISPR/Cas12a rapid detection assaydemonstrates high sensitivity and specificity, it lacks broad applicabilityacross multiple viral strains and is susceptible to amplicon contamination.In this study, a mixed crRNA-based CRISPR/Cas12a detection systemwas established. Sequence analysis predicts its potential to covermost currently circulating ALV-J variant strains. By optimizing theamount of RPA mix and the buffer conditions, a one-tube RPA-CRISPR/Cas12aassay was developed, with the lowest tested concentration giving 9/9positive reactions determined to be 1 × 10⁰ copies/μL(equivalent to 15 copies/reaction in a 15 μL reaction). To simplifythe sample-handling procedure and reduce detection time, a one-potRPA-CRISPR/Cas12a assay was established by adding heparin sodium,achieving the lowest consistently positive concentration of 1 ×10² copies/μL (1500 copies/reaction). Furthermore,for lateral flow assay (LFA) that requires exposure to the environment,the concentration of amplicons was reduced by restricting RPA amplificationand employing an enhanced Cas12a protein while maintaining a lowesttested concentration of 1 × 10¹ copies/μL (150copies/reaction). Specificity evaluation revealed no cross-reactivitywith nontarget avian viruses, yielding a positive signal only forALV-J. When applied to clinical samples, the one-tube RPA-CRISPR/Cas12aassay exhibited complete agreement with RT-qPCR in terms of positivedetection rates. The multiple CRISPR/Cas12a-RPA assays developed inthis study feature broad strain coverage in principle, high accuracy,and low contamination risk, providing practical tools for rapid ALV-Jdetection across diverse scenarios.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":"12 10","pages":"3408–3418"},"PeriodicalIF":4.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149153063","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}