Search Results (118)

The rapid emergence of SARS-CoV-2 variants underscores the need for accurate, rapid, and affordable diagnostic tools, particularly in resource-limited settings. An isothermal amplification-based assay was developed integrating reverse-transcriptase recombinase polymerase amplification (RT-RPA), T7 transcription, and duplex-specific nuclease (DSN)-mediated detection for variant discrimination. The assay targets three genomic regions: a conserved region within ORF1a and two variant regions, ORF1a (Δ3675–3677) and the S gene (Δ69–70), enabling differentiation between the Wuhan-Hu-1 reference isolate and the B.1.1.7 variant. The method demonstrated high specificity and a limit of detection of 200 copies per sample using low-cost instrumentation. DSN-mediated cleavage improved discrimination between matched and mismatched RNA targets while enabling signal amplification through target recycling. The assay requires minimal laboratory infrastructure, relying on a heat block and fluorescent plate reader. These results demonstrate a scalable and cost-effective strategy for SARS-CoV-2 variant screening with potential as a future strategy for pathogen screening and variant surveillance. Full article

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) is a major pathogen causing severe growth retardation in shrimp, leading to substantial economic losses in global aquaculture. To address the urgent need for accurate, rapid, and field-deployable diagnostic tools for EHP, this study developed a novel one-pot detection platform by integrating asymmetric Enzymatic Recombinase Amplification (aERA) with a PAM-independent CRISPR/Cas12a system (AYERA-Cas12a) based on ssDNA activation. This design circumvents the compatibility challenge between isothermal amplification and CRISPR activity in a single tube by generating single-stranded DNA amplicons that activate Cas12a without requiring a PAM sequence. The assay operates at a constant temperature of 46 °C and completes detection within 15 min. It achieves a sensitivity of 10 copies/μL, equivalent to qPCR, and shows no cross-reactivity with six other prevalent shrimp pathogens. Validation using 56 clinical shrimp (Litopenaeus vannamei, L. vannamei) samples demonstrated complete agreement with qPCR results. With its simple procedure, isothermal conditions, and clear endpoint fluorescence readout under blue light, the AYERA-Cas12a platform is suitable for point-of-care testing (POCT). This work provides a user-friendly tool for the on-site surveillance and early diagnosis of EHP, offering significant potential for improving disease management in shrimp farming. Full article

Vector-borne protozoal infections—including babesiosis, theileriosis, hepatozoonosis, trypanosomosis, and leishmaniosis—impose a substantial burden on livestock and companion animal health worldwide and carry important zoonotic and public health implications. Accurate diagnosis is essential yet challenging, given the diversity of parasite genera, their markedly different tissue tropisms, and the uneven distribution of diagnostic resources across veterinary settings. This review provides an integrated overview of the principal diagnostic approaches available, structured around the biological logic that guides test selection in practice. Microscopic examination remains the first-line method; its strengths and limitations are discussed for intraerythrocytic parasites (Plasmodium spp., Babesia spp., Theileria spp., Cytauxzoon spp.—the latter two with additional extra-erythrocytic schizont stages in leukocytes and tissue macrophages, respectively), leukocyte-associated forms (Hepatozoon spp.), extracellular trypanosomes, and tissue-stage parasites, including emerging applications of artificial intelligence. Serological methods—enzyme-linked immunosorbent assay (ELISA), indirect fluorescence antibody test (IFAT), and point-of-care lateral flow assays—are evaluated for their role in exposure detection, population screening, and international trade certification, with attention to cross-reactivity and the active-versus-past-infection distinction. Molecular diagnostics, encompassing conventional PCR, qPCR, droplet digital PCR, isothermal amplification, and next-generation sequencing, are reviewed with respect to target selection, sensitivity, and point-of-care applicability. Finally, diagnostic challenges are contextualised within a One Health framework, highlighting the fragmentation of veterinary surveillance and the need for integrated, cross-sector approaches to detect emerging threats. Full article

Bitter gourd (Momordica charantia L.) is an important vegetable and medicinal crop in tropical/subtropical regions, but suffers severe yield losses (even total failure) from Fusarium wilt caused by Fusarium oxysporum f. sp. momordicae (Fom). There is no specific detection system available to detect this pathogen, and the methods used for other pathogens exhibit cross-reactivity and require specialized equipment. Therefore, this study developed a loop-mediated isothermal amplification (LAMP) assay for early Fom diagnosis. Initially, five sets of LAMP primers targeting the conserved regions of Fom, located within the region amplified by the FOMM-SPF/SPR PCR primers, were tested for specificity and sensitivity. In this experiment, FoM-1-2 showed optimal specificity, identifying 44 Fom strains without cross-reactivity with 10 other non-Fom species after a 60 min incubation at 64 °C. A visual readout based on a fluorescent dye (green for positive, pale orange for negative) eliminated the need for gel electrophoresis and specialized instruments. The LAMP assay was 100-fold more sensitive than conventional PCR (detection limit: 5.6 pg/μL vs. 560 pg/μL). In inoculated seedlings, LAMP detected Fom in basal stems at four days post-inoculation and top leaves at six days, whereas conventional PCR yielded faint bands in the basal stem after eight days. Moreover, LAMP enabled non-destructive detection. Thus, the present study developed a rapid, specific, and sensitive visual LAMP assay, supporting early diagnosis of bitter gourd Fusarium wilt. Full article

Cervical cancer is associated with persistent human papillomavirus (HPV) infections. The early detection of HPV is one of the key strategies for the effective treatment of cervical cancer. Current HPV molecular detection methods use enzyme-based nucleic acid amplification strategies that, although specific and sensitive, involve extensive workflows. Enzyme-free isothermal amplification detection strategies with the potential to adapt to low-resource settings for HPV oncogenic transcripts remain limited. This study aimed to validate a fluorescence-based branched hybridization chain reaction (bHCR) assay for the targeted detection of HPV 16/35 E6 oncogenic transcripts. Analytical performance was evaluated using a synthetic target and a negative clinical matrix, whereas the diagnostic performance of the bHCR assay was evaluated using clinically characterized samples (n = 67). The study demonstrated assay linearity over an analyte concentration range of 0.625–40 µM, with a statistically significant correlation between the fluorescence signal and target concentration (r² = 0.928, p < 0.0001). Analytical accuracy was assessed by pre-extraction spike recovery; achieved recoveries ranged from 70% to 86%, indicating potential RNA loss during the assay workflow. Analytical sensitivity determined the background signal threshold limit of blank (LoB) as 16,251.6 RFU, with detection and quantification at concentrations of 0.0625 µM (≈2.6 × 10¹¹ copies per reaction, limit of detection (LoD) and 0.125 µM (≈5.3 × 10¹¹ copies per reaction, limit of quantification (LoQ). The assay exhibited high diagnostic performance, with a diagnostic cut-off of 16,481 RFU and an area under the curve (AUC) of 0.9194. Specificity and sensitivity of the assay were 94% and 86%, respectively, with a Negative Predictive Value (NPV) of 85% and a Positive Predictive Value (PPV) of 94%. These findings demonstrate a reliable analytical assay with excellent diagnostic discrimination and warrant further optimization and expanded clinical validation. Full article

Background/Objectives: Rapid identification of foodborne pathogens is of high practical significance because it enables prompt epidemiological response, timely patient management, and effective sanitary control of food products. In this study, we developed an integrated molecular platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology for rapid, sensitive, and specific detection of Salmonella enterica. Methods: Four virulence genes (sirA, stn, siiD, and pagN) were selected as targets to ensure reliable pathogen identification. Reaction conditions were optimized using the Moraxella bovoculi Cas12a (MbCas12a) nuclease. The study focused on integrating isothermal amplification with a custom-engineered hardware solution for visual fluorescence detection. Results: The developed method demonstrated sensitive and specific detection, with no cross-reactivity to non-target microorganisms. Optimization allowed for a substantially reduced assay time of approximately 30 min. As a result, a portable fluorescence visualization approach was developed, featuring a 3D-printed housing and an integrated ultraviolet light source for direct visual fluorescence detection. This allows rapid differentiation of samples without specialized laboratory equipment, making it suitable for field applications. Conclusions: The combination of isothermal amplification, MbCas12a-based detection, and the portable fluorescence visualization approach provides a versatile platform for rapid diagnostics and food safety monitoring. This approach has strong potential to improve public health outcomes and enhance the resilience of food supply chains by enabling accessible, field-deployable pathogen detection. Full article

Conventional Escherichia coli (E. coli) detection methods are often time-consuming, while molecular diagnostics typically rely on expensive thermocycling equipment. To address these limitations, this study developed a rapid nucleic acid detection method for E. coli by integrating multienzyme isothermal rapid amplification (MIRA) with Pyrococcus furiosus Argonaute (PfAgo)-mediated targeted cleavage. The conserved housekeeping gene phoA was selected as the target, and specific MIRA primers and 5′-phosphorylated guide DNAs (gDNAs) were designed accordingly. After exponential amplification at 39 °C, the amplicons were specifically recognized by PfAgo at 95 °C, leading to molecular beacon cleavage and generation of a detectable FAM fluorescence signal. Among the tested guides, gDNA6 showed the highest cleavage efficiency. Optimal performance was achieved with 1 μM PfAgo, 0.5 μM gDNA, and 5 mM MnCl₂. The optimized MIRA-PfAgo assay demonstrated a limit of detection of 10⁰ copies/μL, comparable to qPCR, and exhibited high specificity with no cross-reactivity against common enteric pathogens. In 28 clinical and environmental samples, the assay results were fully consistent with those of qPCR. Overall, the MIRA-PfAgo platform provides a rapid, sensitive, and specific approach for E. coli detection, demonstrating strong potential to reduce reliance on precision thermal cyclers for resource-limited applications. Full article

The development of high-throughput, sensitive and portable strategies for detecting foodborne pathogens is urgently needed in food safety, especially during an outbreak. Herein, an ultrasensitive suspension array was constructed by designing photonic crystal microsphere (PCM)-assisted loop-mediated isothermal amplification (LAMP) for Staphylococcus aureus detection. The PCM-LAMP suspension array integrated the optical signal enhancement capability of the biomimetic microporous three-dimensional PCM surface with the thousand-fold signal amplification of LAMP. The biomimetic PCMs displayed a periodic dielectric nanostructure and enhanced the fluorescence intensity of the LAMP reaction, leading to high sensitivity. The PCM-LAMP suspension array allowed sensitive detection of the target DNA of S. aureus without long-term culture. Under optimal conditions, the limit of detection for S. aureus genomic DNA reached as low as 0.18 fM, and the assay exhibited excellent specificity against other bacteria. Furthermore, trace target DNA in food samples was accurately quantified, demonstrating its potential for practical applications. Therefore, the developed PCM-LAMP suspension array holds great promise for ultrasensitive and rapid detection of foodborne pathogens. Full article

Abies georgei var. smithii (Viguie & Gaussen) is a dominant conifer along the southeastern margin of the Qinghai–Tibet Plateau, where heart rot often develops covertly, complicating forest health monitoring and disease management. Fomitopsis subpinicola B.K. Cui, M.L. Han & Shun Liu is an important causal agent of heart rot affecting A. georgei var. smithii in this region, yet rapid, field-deployable molecular diagnostics of this pathogen remain limited. Here, we developed and evaluated two TEF1α-based isothermal platforms for specific detection of F. subpinicola: RAA and LAMP. To reduce potential cross-reactivity, TEF1α sequences from representative taxa within the F. pinicola species complex and closely related non-complex species were aligned for primer/probe design. Candidate RAA primers were screened by gel electrophoresis to select an optimal pair, and two LAMP primer sets were compared by specificity testing to identify the best-performing set. Both assays specifically detected F. subpinicola with no cross-amplification in the tested non-target fungi. Limits of detection were 9.97 copies/μL for fluorescent RAA (25 min), 9.97 × 10² copies/μL for RAA-LFD (15 min), and 9.97 × 10³ copies/μL for LAMP (35 min). In 30 increment core samples from A. georgei var. smithii, all methods consistently detected samples with obvious decay, while fluorescent RAA additionally yielded positives in some apparently asymptomatic samples, indicating promise for early or low-abundance screening. Together, these assays constitute a tiered and application-oriented detection system, enabling flexible selection of diagnostic approaches according to sensitivity requirements, operational conditions, and field surveillance needs for heart rot of A. georgei var. smithii. Full article

To address the issues of operational complexity, long duration association, and reliance on specialized equipment with existing detection methods for Vibrio parahaemolyticus, this study established a rapid detection method for V. parahaemolyticus in exported aquatic products based on the domestically developed Enzymatic Recombinase Amplification (ERA) technology. To target the thermolabile hemolysin gene (tlh) and the iron-regulated virulence regulatory protein gene (irgB) of V. parahaemolyticus, highly specific ERA primers and probes were designed and screened. Two detection platforms, a colorimetric method and a fluorescent method, were developed. Method validation results showed that this detection system achieved specific amplification for all 30 tested V. parahaemolyticus strains, with no cross-reactivity observed with 30 other common foodborne pathogenic bacteria. The detection sensitivity for both the fluorescent and colorimetric methods reached 10⁻¹ ng/μL, with a minimum detection limit of 10 CFU/25 g for artificially contaminated samples. The entire detection process, including sample preparation, requires only approximately 20 min—significantly faster than traditional culture (24–72 h) or even conventional PCR methods. Collaborative validation across five independent laboratories confirmed excellent reproducibility, with inter-laboratory agreement yielding a Kappa coefficient of 0.98. The ERA method operates at a low, constant temperature (37–39 °C), eliminating the need for thermal cyclers. When combined with portable isothermal amplification devices and visual (colorimetric) readout, it offers a distinct advantage in terms of speed, cost-effectiveness, and suitability for resource-limited or field settings compared to existing PCR-based or culture-based platforms. This method is simple to operate, rapid, sensitive, and highly suitable for on-site application, providing a reliable and practical technical solution for the rapid screening and risk monitoring of V. parahaemolyticus in exported aquatic products. Full article

CRISPR-Cas12a enables rapid and specific detection of PCR/LAMP (loop-mediated isothermal amplification) reaction products; however, this approach often requires open-tube manipulation, rendering it prone to cross-contamination. Here, we developed a novel one-pot reaction system that eliminated carryover contamination and facilitated endpoint detection using a CRISPR/Cas12a-based system. We leveraged the dependence of the CRISPR-Cas12a cleavage system on the protospacer-adjacent motif (PAM) to design PCR/LAMP primers that incorporated the PAM site (TTT) into amplified DNA. Pre-incubation of Cas12a with crRNA1 and crRNA2 using PCR/LAMP resulted in efficient cleavage of cross-contaminating DNA, while the target gene remained intact due to the lack of PAM sites. Furthermore, a Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and the ssDNA probe) pre-stored on the lid was introduced to mix with the PCR/LAMP amplicons, which triggered the non-specific cleavage of fluorescent probes for direct visual detection under a blue LED instrument. This method effectively degraded up to 10⁶ copies of carryover contaminants within one hour, demonstrating the potential of one-pot detection methods in complex samples. Full article

In order to establish a rapid and sensitive LAMP visual detection method for Cherry Virus A on-site, this study used the conserved fragment of the CVA coat protein (CP) sequence as a template for primer design. The rapid visual LAMP detection method for Cherry Virus A was successfully established by optimizing the reaction system components (concentration ratio of internal and external primers, and concentrations of loop primers, Bst DNA, Mg²⁺, dNTPs and betaine) and reaction conditions (temperature and time). This method enables specific detection of Cherry Virus A and facilitates visual inspection of crude nucleic acid extracts within 40 min, significantly reducing the diagnostic turnaround time. The limit of detection is 67.54 pg μL⁻¹ (cDNA), which is 100 times more sensitive than PCR. Analysis of 70 field sweet cherry samples revealed an RT-LAMP positivity rate of 91.42%, significantly surpassing the 71.42% achieved by RT-PCR. This method is suitable for the rapid on-site detection of Cherry Virus, and can also provide a theoretical reference for the early diagnosis of cherry viral diseases. Full article

Forensic DNA profiling commonly relies on polymerase chain reaction (PCR) amplification followed by capillary electrophoresis (CE) or massively parallel sequencing (MPS), which requires expensive, laboratory-based equipment that depends on a stable power supply and is unsuitable for field applications. Here, we present a proof-of-concept assay that uses recombinase polymerase amplification (RPA) combined with exo probe detection for rapid, isothermal genotyping of insertion–deletion (InDel) markers. To the best of our knowledge, this study represents the first demonstration of forensic DNA typing using RPA coupled with exo probes. The reaction proceeds at 39 °C and combines amplification and detection in a single 20 min step. Thirteen DNA samples were genotyped in triplicate across eight InDel loci using allele-specific fluorescent probes. Genotypes were derived from differential endpoint fluorescence between matched and mismatched probes. Compared with benchmark genotyping, 97.07% of genotypes (n = 307) were correct at 1 ng DNA input. Accurate profiles were reliably obtained for DNA inputs as low as 250 pg, and partial profiles were still detectable at 31 pg. The results demonstrate that RPA-based InDel genotyping is fast, sensitive, and reproducible. With further optimization, such as refined probe design and selection of robust loci, the assay has clear potential to achieve complete accuracy and to be integrated into portable lab-on-a-chip platforms for rapid, field-deployable forensic identification. Full article

Duck plague virus (DPV), a highly contagious α-herpesvirus in the livestock and poultry environment, poses a significant threat to the healthy growth of ducks, potentially causing substantial economic losses. Effective control of DPV requires the development of specific diagnostic tools. A new fluorescent biosensor (R-C-CHA) was developed to detect virulent strains of DPV. It combined recombinase polymerase amplification (RPA), a CRISPR/Cas12a system, and catalytic hairpin assembly (CHA) for signal enhancement. The RPA primers were specifically designed to target the conserved DPV-CHv UL2 gene region, allowing for the rapid, efficient amplification of the target nucleic acids in isothermal conditions. The CRISPR/Cas12a system was used for sequence-specific recognition, activating its lateral cleavage activity. Furthermore, the CHA cascade reaction was utilized for enzyme-free fluorescent signal amplification. The results showed that the R-C-CHA biosensor completed the detection process in 40 min with a detection limit of 0.02 fg/μL, which was an approximate five-fold improvement compared to traditional RPA-CRISPR/Cas12a biosensors. The R-C-CHA biosensor also demonstrated perfect consistency with clinical detection and polymerase chain reaction (PCR) diagnosis, highlighting its strong potential for rapid detection in livestock and poultry farming settings. Full article

The primary objective of this study was to develop and optimize a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for porcine abortion-associated pestivirus (PAAPeV)—an emerging pathogen that causes severe reproductive disorders in swine, for which no effective treatments or vaccines are currently available. In this study, four sets of LAMP primers were designed and screened for the colorimetric RT-LAMP assay, targeting the highly conserved 5′ untranslated region (5′UTR) of PAAPeV. Three reaction parameters, including reaction temperature, reaction duration, and inner-to-outer primer ratio, were then optimized based on cycle threshold (Ct) values, fluorescence intensity, and color changes of the endpoint products. Subsequently, the specificity and sensitivity of the optimized colordetect RT-LAMP assay were systematically validated, and its diagnostic performance was compared with that of the gold-standard reverse transcription quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that the optimized assay achieved a detection limit of 2 copies/μL under the conditions of 65 °C incubation for 25 min and an inner-to-outer primer ratio of 8:1, with results amenable to naked-eye interpretation. Furthermore, this assay exhibited high specificity, showing no cross-reactivity with other known pestiviruses or prevalent swine pathogens. Clinical sample testing results showed 100% concordance between colordetect RT-LAMP and RT-qPCR. Collectively, this colordetect RT-LAMP assay represents a rapid, sensitive, and specific tool for PAAPeV RNA detection in both clinical laboratories and field settings. Full article