Search Results (71)

Bloodstream infections (BSI) carry high mortality, but traditional blood culture is too slow for urgent clinical needs. This study aims to develop a rapid point-of-care testing assay based on one-tube multiplex nested polymerase chain reaction (PCR) (OM-NPCR-POCT) for early diagnosis of three pathogens in bloodstream infection patients: Escherichia coli (ECO), Klebsiella pneumoniae (KPN), and Streptococcus pneumoniae (SPN). The analytical sensitivity of the one-tube multiplex nested PCR (OM-NPCR) was evaluated using recombinant bacterial plasmids. The analytical sensitivity of the OM-NPCR-POCT assay was assessed using simulated samples. The clinical performance was evaluated in 90 clinical blood samples, with results compared to conventional quantitative PCR (qPCR). Finally, the agreement between the two detection methods was assessed via Kappa analysis. The limits of detection (LODs), calculated from serial dilution experiments, were 4, 2, and 1 copies/μL for plasmids ECO, KPN, and SPN, respectively. The OM-NPCR-POCT assay achieved LODs of 20, 10, and 10 CFU/mL for ECO, KPN, and SPN in simulated samples, with a total testing time of approximately 2 h. The clinical evaluation of OM-NPCR-POCT demonstrates consistency with conventional qPCR while exhibiting higher sensitivity. This method has potential as a rapid diagnostic tool for early bloodstream infection detection. Full article

Background: Rapid multiplex PCR assays promise faster and broader detection of uropathogens and resistance markers than conventional quantitative urine culture and susceptibility testing (C&S), but trial evidence linking PCR-guided management to patient-centered outcomes and the mechanisms of any benefit is limited. We performed an ad hoc analysis of the randomized, multicenter NCT06996301 trial to evaluate whether PCR-guided diagnostic management improves clinical symptom resolution in complicated urinary tract infection (cUTI) and to quantify mediation by time-to-antibiotic start and antibiotic appropriateness. Methods: Paired PCR and C&S were collected for all participants; treating investigators received and acted on randomized results from one diagnostic modality and remained blinded to the comparator. The modified intention-to-treat (Mod-ITT) cohort at end-of-study (EOS) included 362 participants (PCR n = 193; C&S n = 169). The primary outcome was complete clinical cure at EOS (absence of all baseline symptoms). Secondary outcomes included partial cure (≥50% symptom reduction) and per-symptom changes. We used mixed-effects logistic regression (site random intercept) to estimate associations, and causal mediation analysis with nonparametric bootstrap (B = 2000) to decompose PCR’s total effect into indirect effects via time-to-antibiotic (log-transformed) and antibiotic appropriateness (binary, adjudicated at EOS) for complete clinical cure and partial cure. Results: Median time-to-first antibiotic was substantially shorter in the PCR arm (20 h; IQR 12–36) than in the C&S arm (52 h; IQR 30–66; p < 0.001). Antibiotic appropriateness was higher after PCR-guided care (161/193; 83.4%) versus C&S (105/169; 62.1%; p < 0.001). Complete clinical cure occurred in 143/193 (74.1%) PCR versus 106/169 (62.7%) C&S (p = 0.020); partial cure in 161/193 (83.4%) versus 121/169 (71.6%; p = 0.014). In a total-effect mixed model (no mediators), PCR assignment was associated with higher odds of cure (adjusted OR 1.95; 95% CI 1.12–3.39; p = 0.018). In the mechanistic model including mediators, antibiotic appropriateness (OR 2.48; 95% CI 1.45–4.24; p = 0.001), and time-to-antibiotic (per 1 h, OR 0.95; 95% CI 0.926–0.975; p < 0.001) were independently predictive, while the direct arm effect was attenuated (OR 1.10; 95% CI 0.33–3.71). Mediation analysis estimated a statistically significant combined indirect effect (ACME) of 0.0648 (95% CI 0.0343–0.0977), ADE 0.0207 (95% CI −0.0282–0.0784), total effect 0.0796 (95% CI 0.0419–0.1225), and proportion mediated ≈ 74%. Both time-to-antibiotic and appropriateness contributed, with ACME_time ≈ 0.046 and ACME_appropriateness ≈ 0.019. Exploratory analysis using partial cure as the outcome confirmed the robustness and internal validity of the complete-cure findings. Conclusions: In this ad hoc analysis of a randomized trial, PCR-guided management of cUTI improved patient-centered symptom outcomes compared with culture-guided care. Most of the benefit was mediated through faster initiation of antibiotics and, to a lesser extent, increased probability of an appropriate initial antibiotic. These results support stewardship-integrated, rapid molecular diagnostics (used alongside culture) to shorten time-to-effective therapy and improve clinical outcomes in cUTI. Full article

Background/Objectives: The limited specificity of prostate-specific antigen (PSA) drives unnecessary biopsies in prostate cancer (PCa). Urinary extracellular vesicles (uEVs) provide a non-invasive reservoir of tumor-derived nucleic acids and proteins. Aptamers selected by SELEX enable highly specific capture, and artificial intelligence (AI) can accelerate their optimization. This systematic review evaluated AI-assisted SELEX for urine-derived and exosome-enriched aptamer panels in PCa detection. Methods: Systematic searches of PubMed, Scopus, and Web of Science (1 January 2010–24 August 2025; no language restrictions) followed PRISMA 2020 and PRISMA-S. The protocol is registered on OSF (osf.io/b2y7u). After deduplication, 1348 records were screened; 129 studies met the eligibility criteria, including 34 (26.4%) integrating AI within SELEX or downstream refinement. Inclusion required at least one quantitative metric (dissociation constant K_d, SELEX cycles, limit of detection [LoD], sensitivity, specificity, or AUC). Risk of bias was appraised with QUADAS-2 (diagnostic accuracy studies) and PROBAST (prediction/machine learning models). Results: AI-assisted SELEX workflows reduced laboratory enrichment cycles from conventional 12–15 to 5–7 (≈40–55% relative reduction) and reported K_d values spanning low picomolar to upper nanomolar ranges; heterogeneity and inconsistent comparators precluded pooled estimates. Multiplex urinary panels (e.g., PCA3, TMPRSS2:ERG, miR-21, miR-375, EN2) yielded single-study AUCs between 0.70 and 0.92 with sensitivities up to 95% and specificities up to 88%; incomplete 2 × 2 contingency reporting prevented bivariate meta-analysis. LoD reporting was sparse and non-standardized despite several ultralow claims (attomolar to low femtomolar) on nanomaterial-enhanced platforms. Pre-analytical variability and absent threshold prespecification contributed to high or unclear risk (QUADAS-2). PROBAST frequently indicated high risk in participants and analysis domains. Across the included studies, lower K_d and reduced LoD improved analytical detectability; however, clinical specificity and AUC were predominantly shaped by pre-analytical control (matrix; post-DRE vs. spontaneous urine) and prespecified thresholds, so engineering gains did not consistently translate into higher diagnostic accuracy. Conclusions: AI-assisted SELEX is a promising strategy for accelerating high-affinity aptamer discovery and assembling multiplex urinary panels for PCa, but current evidence is early phase, heterogeneous, and largely single-center. Priorities include standardized uEV processing, complete 2 × 2 diagnostic reporting, multicenter external validation, calibration and decision impact analyses, and harmonized LoD and K_d reporting frameworks. Full article

Background: Rapid molecular detection of antimicrobial resistance (AMR) can shorten time to effective therapy in complicated urinary tract infections (cUTI), but the ability of gene presence and quantitative PCR signal (Ct, and ΔCt = Ct_marker − IC_Ct) to predict phenotypic non-susceptibility and clinical outcomes requires rigorous evaluation. We analyzed marker-level concordance, Ct→MIC relationships, and the clinical impact pathway in the randomized NCT06996301 trial. Methods: Marker–phenotype concordance metrics (sensitivity, specificity, PPV, NPV, LR+, LR−, κ) were computed for selected marker × species strata with stable sample sizes. Mixed-effects models (log₂[MIC] ~ ΔCt_marker + IC_Ct + collection_method + prior_abx + (1|site)) assessed quantitative Ct→MIC associations. ROC analyses evaluated ΔCt discrimination of phenotypic non-susceptibility. A pre-specified sensitivity analysis included smaller strata (n ≤ 20) with bootstrap 95% confidence intervals for ΔCt slopes and AUCs. Clinical analyses compared PCR-guided (n = 193) versus culture-guided (n = 169) arms for time-to-antibiotic and treatment success using adjusted logistic regression and causal mediation (time-to-antibiotic as mediator; bootstrap inference). Results: High genotype–phenotype concordance was observed for canonical markers (e.g., bla_CTX-M in E. coli: sensitivity 0.94 [95% CI 0.88–0.97], specificity 0.995 [95% CI 0.990–0.998], κ ≈ 0.93). Mixed models showed modest but significant Ct→MIC associations for select markers (e.g., bla_CTX-M in E. coli: ΔCt slope −0.15 [95% CI −0.27 to −0.02], p = 0.015). The sensitivity analysis (n ≤ 20 strata) confirmed consistent negative directions, with robust bootstrap CIs excluding zero for qnrS (E. coli), tetM (E. coli), blaNDM (Klebsiella), and qnrS (Proteus). ROC AUCs for ΔCt prediction of non-susceptibility ranged from 0.62 to 0.81 (95% CIs ≈ 0.47–0.97). Clinically, PCR guidance shortened median time to antibiotic initiation (20 h vs. 52 h) and increased treatment success (88.1% vs. 78.1%; adjusted OR 1.95 [95% CI 1.12–3.40], p = 0.018). Mediation analysis estimated that 63% (ACME 0.112 [95% CI 0.045–0.178], p = 0.002) of the treatment success benefit was mediated through earlier antibiotic initiation. Conclusions: Binary detection of high-impact AMR genes by multiplex PCR reliably predicts phenotypic non-susceptibility and accelerates effective therapy when integrated with stewardship workflows. Quantitative PCR (ΔCt) provides modest but reproducible information about MIC magnitude and may flag heteroresistant subpopulations. A pragmatic implementation model combining rapid PCR with conventional culture is recommended to optimize clinical benefit while retaining isolate recovery for definitive AST. Full article

Diamorphine (DIM, heroin) is a semi-synthetic opioid that undergoes rapid conversion to 6-monoacetylmorphine and morphine, producing short-lived biomarkers that are difficult to capture during the process. This review critically explores the evolution of analytical techniques for quantitative DIM analysis in biological matrices from 1980 to 2025. It synthesizes findings across blood, plasma, urine, hair, sweat, and postmortem samples, emphasizing matrix-specific challenges and forensic applicability. Unlike previous opioid reviews that primarily focused on metabolites, this work highlights analytical methods capable of successfully detecting diamorphine itself alongside its key metabolites. This review examines 32 studies spanning three decades and compares three core analytical methods: gas chromatography–mass spectrometry (GC–MS), high-performance liquid chromatography (HPLC) with optical detection and liquid chromatography–mass spectrometry (LC–MS). Key performance metrics include sensitivity, sample preparation workflow, hydrolysis control, metabolite coverage, matrix compatibility, automation potential and throughput. GC–MS remains the workhorse for hair and sweat ultra-trace screening after derivatization. HPLC with UV, fluorescence or diode-array detection enables robust quantification of morphine and its glucuronides in pharmacokinetic and clinical settings. LC–MS facilitates the multiplexed analysis of DIM, its ester metabolites and its conjugates in a single, rapid run under gentle conditions to prevent ex vivo degradation. Recent advances such as high-resolution mass spectrometry and microsampling techniques offer new opportunities for sensitive and matrix-adapted analysis. By integrating validation parameters, forensic applicability, and evolving instrumentation, this review provides a practical roadmap for toxicologists and analysts navigating complex biological evidence. Full article

Staphylococcus aureus is a globally crucial foodborne pathogen that can cause diarrhea, vomiting, and bloodstream infection in immunocompromised individuals. S. aureus has three predominant sequence types (STs) (ST7, ST188 and ST398) that are prevalent clones in both food and clinical cases. This study aimed to screen ST-specific targets for S. aureus ST7, ST188 and ST398, and then developed a novel rapid and accurate assay for the detection of these three predominant S. aureus STs in food. A total of 505 Staphylococcus strain genome sequences including 371 sequences of 58 different STs and 134 other non-target S. aureus ST genome sequences were subjected to pan-genome analysis; we successfully screened five novel ST-specific targets (group_10498 and group_10499 target for S. aureus ST7, group_9415 and group_9419 target for S. aureus ST188, group_9911 target for S. aureus ST398). The excellent specificity and sensitivity of all the targets were confirmed by PCR assays. Based on these molecular targets, mPCR and qPCR methods were developed for specifically identifying S. aureus’ three predominant STs without non-target bacterial interference. The limits of detection (LODs) for the mPCR assay in artificially contaminated milk were determined to be 10⁴ CFU/mL for ST7, 10⁵ CFU/mL for ST188, and 10⁴ CFU/mL for ST398, while the LODs achieved by the qPCR method were 8.6 × 10² CFU/mL, 1.2 × 10² CFU/mL, and 6.4 × 10³ CFU/mL, respectively. The testing results for actual food samples suggested that the developed mPCR or qPCR assays could be used as an alternative to standard MLST analysis, for the rapid and reliable identification of S. aureus STs. The novel molecular detection technology established in this study provides an efficient and reliable detection method for the prevention and control of predominant S. aureus ST contamination in food and has important application potential and promotion prospects. Full article

Chicken coccidiosis is a major parasitic disease in poultry that causes substantial economic losses worldwide. The accurate and simultaneous diagnosis of mixed infections with multiple Eimeria species remains challenging using conventional methods. In this study, we developed a multiplex TaqMan-MGB qPCR assay targeting the ITS1 region for simultaneous detection and quantification of four major pathogenic species: E. acervulina, E. necatrix, E. maxima, and E. tenella. The assay exhibited high specificity without cross-reactivity and achieved a sensitivity 100-fold greater than conventional PCR, with detection limits ranging from 10¹ to 10² copies/μL. Standard curves demonstrated strong linearity (R² ≥ 0.95) and amplification efficiency (95–113%). Repeatability was robust, with intra- and inter-assay CVs below 2%. Applied to 165 clinical samples collected from poultry farms in Fujian Province, China (2022–2024), the assay detected an overall Eimeria infection rate of 93.3%, higher than conventional PCR (89.7%). Epidemiological analysis revealed E. tenella (78.8%, [130/165]) and E. necatrix (78.8%, [130/165]) were dominant, while mixed infections were frequent (83.3%, [136/154]), including dual (45.5%, [70/154]), triple (31.2%, [48/154]), and quadruple (11.7%, [18/154]) infections. This multiplex TaqMan-MGB qPCR provides a rapid, sensitive, and quantitative tool for detecting multiple Eimeria species, greatly improving diagnostic efficiency and supporting clinical diagnosis, surveillance, and control strategies. Full article

There have been rising interests in ultra-sensitive biosensing technologies for early diagnosis and prognosis monitoring of infectious diseases, cancers, and neurodegenerative diseases. Digital signal readout strategy represented by digital ELISA or digital PCR, advanced biosensing field enormously, which enables detection of biomolecules under the detection limit of conventional biosensing methods. However, due to the need for compartmentalization and limited multiplex capability, it has been hurdled for utilization in applications requiring hierarchical resolution analysis such as sub-cellular molecules or molecular cargo of single cells or single extracellular vesicles (EVs). Rolling circle amplification (RCA), an isothermal DNA amplification method enabling localization of an amplified signal, can eliminate the need for compartmentalization and increase multiplex capability. It also has potential to expand applications of single molecule counting assay for understanding hierarchy of biological systems. In this review, recent advances in RCA-based single molecule counting assay are overviewed and their applications in single cells and single EVs quantitative analysis are discussed. Furthermore, the limitations and outlook of RCA-based single molecule counting assay are highlighted. Full article

This study proposes a region of interest (ROI) setting method to improve the accuracy and efficiency of fluorescence detection in a compact real-time multiplex fluorescence PCR system. Conventional commercial real-time PCR systems are limited in point-of-care (POC) environments due to their high cost and complex optical structures. To address this issue, we developed a low-cost, compact system using an open-platform camera and a Fresnel lens. However, in such a simply structured system, variations between the wells of the polymerase chain reaction (PCR) plate may affect the accuracy of fluorescence detection. In this study, after capturing images with a CMOS camera, we propose two ROI image processing algorithms. The proposed algorithms reliably extract fluorescence signals and compare ROI deviations caused by variations between wells to determine whether physical correction is necessary. To validate the system, we performed comparative analysis of real-time DNA amplification images and fluorescence dye images collected over multiple periods. Based on evaluations using manual detection as a reference, it was confirmed that even a simple algorithm can achieve stable fluorescence detection while minimizing ROI distortion. This study presents an efficient method for enhancing the accuracy of quantitative fluorescence analysis in small PCR systems and is expected to contribute to improving the performance of point-of-care diagnostics, thereby increasing accessibility to on-site diagnostics in the future. Full article

Background/Objectives: While complete loss-of-function (LoF) SPINK1 variants in the simple heterozygous state cause chronic pancreatitis, biallelic complete LoF variants result in a rare pediatric disorder termed severe infantile isolated exocrine pancreatic insufficiency (SIIEPI). To date, only two individuals with a null SPINK1 genotype have been reported—one homozygous for a whole-gene deletion and the other for an Alu insertion in the 3′ untranslated region. Here, we report the genetic basis of a third SIIEPI case, presenting in early infancy with severe exocrine pancreatic insufficiency and diffuse pancreatic lipomatosis. Methods: Targeted next-generation sequencing (NGS) was used to analyze the entire coding region and exon–intron boundaries of the SPINK1 gene. Copy number variant (CNV) analysis was performed with SeqNext, based on normalized amplicon coverage. Results: The proband harbored compound heterozygous complete LoF SPINK1 variants. One was the known NM_001379610.1:c.180_181del (p.(Cys61PhefsTer2)), inherited from the father. The second, initially detected as an exon 2 deletion and confirmed by quantitative fluorescent multiplex PCR (QFM-PCR), was further characterized by long-range PCR as a complex rearrangement comprising a 1185 bp deletion removing exon 2, a 118 bp templated insertion followed by a non-templated nucleotide, and an 8 bp deletion. The mutational signature is consistent with serial replication slippage or template switching involving translesion synthesis. This maternally inherited variant has not been previously reported. Conclusions: This study expands the mutational spectrum of SPINK1-related SIIEPI and suggests that this distinct pediatric disorder may be under recognized in clinical practice. Full article

The accurate identification of mosquito species is critical for effective mosquito surveillance and control, especially when presented with morphologically similar species like Aedes aegypti and Aedes sierrensis. Damaged specimens and morphologically similar life stages such as eggs and larvae make it difficult to distinguish Aedes aegypti from Aedes sierrensis using microscopy and taxonomic keys. To address this, the AegySierr.ID-qPCR assay, a multiplex quantitative PCR assay that utilizes single-nucleotide polymorphisms within the mitochondrial cytochrome oxidase subunit I gene, was developed to distinguish between these two species. The assay was tested on DNA extracted from the eggs, larvae, and adults of both species, as well as from environmental DNA (eDNA) collected from natural mosquito reproduction sites. It demonstrated a high diagnostic accuracy across multiple life stages, with a sensitivity exceeding 95% for most groups and specificity exceeding 90%, except for field-collected adult Ae. sierrensis (75%). For eDNA samples, the assay achieved 100% sensitivity and 94% specificity for samples classified as Ae. sierrensis and 91% sensitivity and 86% specificity for Ae. aegypti. A two-graph receiver operating characteristic analysis was also used as an alternate method with which to establish Ct thresholds for interpreting results from unknown samples. The AegySierr.ID-qPCR assay enables the rapid and sensitive identification of Ae. aegypti and Ae. sierrensis from specimens and eDNA, and may be of use in mosquito surveillance programs. Full article

Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV P72 gene, CSFV 5’UTR region, PRRSV ORF6, PCV2 cap gene, and PRV gB gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control. Full article

Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, play crucial roles in intercellular communication and disease pathology. Their heterogeneous nature, shaped by cellular origin and activation state, requires precise and multiplexed profiling of surface markers for effective characterization. Despite recent advances, current analytical methods remain complex, costly, or inaccessible for routine laboratory use. Here, we present a simple and cost-effective flow cytometry-based assay for the multiplexed analysis of tetraspanin markers (CD63, CD81, CD9) on fluorescently labeled sEVs. Our method combines metabolic labeling with paraformaldehyde fixation and low-speed centrifugation using a benchtop centrifuge, enabling efficient removal of unbound antibodies and minimizing nonspecific signals while preserving vesicle integrity. Using either metabolically labeled exosomes or bulk sEVs stained with carboxyfluorescein succinimidyl ester (CFSE), we demonstrate robust recovery and accurate, semi-quantitative profiling of tetraspanin expression. The assay reveals substantial variability in tetraspanin distribution across different cell lines and does not require ultracentrifugation or immunocapture. Notably, this versatile and reproducible method supports high sEV recovery and is adaptable to additional protein markers. Its compatibility with standard laboratory equipment makes it a practical and scalable alternative to more complex techniques, expanding access to multiplex sEV analysis for both research and clinical applications. Full article

Background/Objectives: Ruptured abdominal aortic aneurysm (RAAA) remains a leading cause of vascular death, with mortality rates approaching 90%. Biomarkers capable of identifying the most at-risk population are urgently needed in the clinic. We aimed to identify potential alterations in the urine proteome that can enable non-invasive detection of abdominal aortic aneurysms (AAA) at high risk of rupture. Methods: We used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MIB/MS) to examine potential biomarkers in urine samples. Quantitative proteomic profiling was conducted using iTRAQ labeling and LC-TEMPO MALDI-TOF/TOF analysis, revealing several dysregulated proteins in the urinary proteome between the two groups. MS and MS/MS data were generated using MALDI TOF/TOF instruments (models 5800 or 4800; AB SCIEX). MS/MS spectra were processed with ProteinPilot™ software version 3.0 (AB SCIEX) and matched against the UniProt/Swiss-Prot database for identification of proteins with an Unused ProtScore >1.3. Statistical tests were performed using R/Bioconductor software and bioinformatics analysis using open-source software. Results: We quantitatively measured activity over 130 kinases from various kinase families using MIB/MS with a threshold of 1.5-fold change in expression. Statistical analysis assigned significance to EPHB6, AXL, EPHB4, DDR1, EPHA2 and EPHB3. All were tyrosine kinases, and the Ephrin receptor type was dominant. The reduced expression of specific kinases identified by MIB/MS analysis was validated by Western blot. Conclusions: This pilot study presents a promising breakthrough in the diagnosis and surveillance of AAA. We identified six dysregulated tyrosine kinases in the urine proteome of patients with RAAAs, suggesting their potential as urinary biomarkers for early detection of AAA at high risk of rupture. However, these preliminary findings require confirmation in larger, prospective cohorts to validate their diagnostic utility and generalizability. Full article

The Vibrio genus represents a critical group of bacterial pathogens in the marine environment globally, leading to massive mortality in the aquaculture industry. Diagnosing vibriosis, an infection caused by Vibrio species, in clinical samples poses challenges due to its non-specific clinical manifestations. In this study, we developed a TaqMan probe-based multiplex real-time PCR method for the simultaneous detection and quantification of four Vibrio pathogens: Vibrio anguillarum (Va), Vibrio alginolyticus (Val), Vibrio harveyi (Vh), and Vibrio scophthalmi (Vsc). The assay targets conserved intra-species regions and specific inter-species regions using specific primers and TaqMan probes to ensure specificity. Sensitivity analysis demonstrated that the multiplex real-time PCR assay could simultaneously detect the four different bacteria, with detection limits of 26–60 copies per reaction, making it 100 times more sensitive than conventional PCR assays. Additionally, the assay exhibited high reproducibility, with intra- and inter-group coefficients of variation below 1.4%. A total of 63 clinical samples was analyzed using this established assay, which successfully detected both single and mixed infections. These results demonstrate that the multiplex quantitative PCR assay is a rapid, specific, and sensitive diagnostic tool for the detection of Va, Val, Vh, and Vsc, making it suitable for monitoring these bacteria in both single- and co-infected clinical samples. Full article