Search Results (25)

Intensive aquaculture and animal farming along riverbanks have emerged as significant drivers of downstream public health risks, facilitating the transmission of zoonotic pathogens and antimicrobial resistance (AMR) genes from farm effluents into natural water systems. In this study, we conducted a comprehensive 12-week water monitoring program at the Wei River in Shandong, China, using a combination of rapid detection techniques (RPA-LFD) and whole-genome sequencing to trace the origins of detected pathogens. RPA-LFD screening revealed the sequential appearance of Vibrio parahaemolyticus, Aeromonas veronii, norovirus GII, and Brucella spp. in surface water from March onward, coinciding with documented wastewater discharge events from upstream shrimp and fox farms. Subsequent isolation efforts confirmed the presence of V. parahaemolyticus and A. veronii in both river water and shrimp samples, while Brucella abortus was isolated from fox feces and water samples. Whole-genome sequencing of bacterial isolates revealed that V. parahaemolyticus strains from water and shrimp shared identical sequence types (ST150 and ST809) and resistance gene profiles, indicating a clonal relationship. Similarly, B. abortus isolates from water and fox feces differed by fewer than five SNPs, confirming farm-to-water transmission. Norovirus GII.3 and GII.6 sequences from water and fecal samples clustered phylogenetically with regional clinical strains, suggesting local circulation and environmental dissemination. Our findings highlight the critical role of river water monitoring as an early warning system for pathogen spread, emphasizing the need for integrated surveillance systems that monitor both water quality and the health of upstream farms and wildlife populations. The combined use of RPA-LFD and whole-genome sequencing provides a robust framework for real-time detection and source tracing of zoonotic pathogens, offering valuable insights for future environmental monitoring and public health interventions. Full article

Accurate differentiation between Coilia brachygnathus and Coilia nasus is imperative for the effective management of fisheries, the conservation of aquatic ecosystems, and the mitigation of commercial fraud. Current morphological identification remains challenging due to their high morphological similarity—particularly for processed samples—while conventional molecular methods often lack the speed or specificity required for field applications or high-throughput screening. In this study, a novel integrated approach was developed and validated, combining TaqMan quantitative real-time PCR (qPCR). for precise genotyping of C. brachygnathus and C. nasus with Recombinase Polymerase Amplification coupled with Lateral Flow Dipstick (RPA-LFD) for rapid on-site screening. First, species-specific RPA-LFD assays were designed to target the mitochondrial COI gene sequence. This enabled visual detection within 10 min at 37 °C, with a sensitivity of 10² copies/μL, and required no complex equipment. A dual TaqMan MGB qPCR assay was further developed by validating stable differentiating SNPs (chr21:3798155, C/T) between C. brachygnathus and C. nasus, using FAM/VIC dual-labeled MGB probes. Results showed that this assay could distinguish the two species in a single tube: for C. brachygnathus, Ct values in the FAM channel were significantly earlier than those in the VIC channel (ΔCt ≥ 1), with a FAM detection limit of 125 copies/reaction; for C. nasus, only VIC channel amplification was observed, with a detection limit as low as 12.5 copies/reaction. Validation with 171 known tissue samples demonstrated 100% concordance with expected species identities. This integrated approach effectively combines the high accuracy and quantitative capacity of TaqMan qPCR for confirmatory laboratory genotyping with the speed, simplicity, and portability of RPA-LFD for initial field or point-of-need screening. This reliable, efficient, and user-friendly technique provides a powerful tool for resource management, biodiversity monitoring, and ensuring the authenticity of high-quality C. brachygnathus and C. nasus. Full article

Chrysotila dentata (Haptophyta), a harmful algal bloom (HAB) species frequently occurring in coastal waters of China, is one with strong environmental adaptability that poses a serious threat to marine ecosystems and fisheries. Current molecular detection techniques and early warning systems for this species remain limited. To address this, we developed a rapid and highly sensitive detection method for C. dentata. This method integrates recombinase polymerase amplification (RPA) with CRISPR-LbaCas12a and lateral flow dipstick (LFD) technologies, enabling visual readout of results. Key parameters, including the single-stranded DNA (ssDNA) reporter concentration, reaction time, and temperature, were systematically optimized. Field water sample testing demonstrated high specificity and sensitivity, achieving a detection limit of 5 × 10⁻⁶ pg μL⁻¹ for genomic DNA under laboratory conditions and 2.82 × 10¹ cells mL⁻¹ in simulated environmental samples. The entire detection process takes only 1 h (at a constant 39 °C), and results can be directly interpreted via LFD strips. For early warning and prevention of C. dentata outbreaks, this assay provides a powerful, reliable, and field-ready monitoring tool. Full article

Tilapia Lake Virus (TiLV) is well known as a highly contagious viral infection in aquaculture, particularly affecting Tilapia worldwide. Until recently, various TiLV diagnostic methods have been used for rapid and accurate diagnostic procedures that are crucial for timely disease detection and reducing losses. In this study, we developed an alternative method for investigating TiLV diagnosis using Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA) assay combined with a lateral flow dipstick (LFD). The test was generated by specific anti-FITC and anti-Biotin capture antibodies that are compatible with the TiLV-specific primers tagged with FITC and Biotin. The test was conducted by the reverse transcriptase of target TiLV RNA and RPA amplification at 39 °C for 20 min. The products were then determined by a positive band signal via LFD. The RT-RPA-LFD assay detected the plasmid of TiLV (pTiLV) with a Limit of Detection (LOD) of 3.19 copies/µL, while the RT-PCR-LFD assay detected it with an LOD of 319 copies/µL. Our findings demonstrate that RT-RPA-LFD represents a possible alternative to RT-PCR for the rapid and sensitive detection of TiLV, especially in areas with limited infrastructure. Full article

PoRV is a significant etiological agent of neonatal diarrhea in piglets, resulting in substantial economic losses within the global swine industry due to elevated mortality rates and reduced productivity. To address the urgent need for accessible and rapid diagnostics in resource-limited settings, we have developed a CRISPR/Cas12a-based assay integrated with recombinase polymerase amplification (RPA) for the visual detection of PoRV. This platform specifically targets the conserved VP6 gene using optimized RPA primers and crRNA, harnessing Cas12a’s collateral cleavage activity to enable dual-readout via fluorescence or lateral flow dipsticks (LFDs). The assay demonstrates a detection limit of 10² copies/μL within 1 h, exhibiting no cross-reactivity with phylogenetically related pathogens such as Transmissible Gastroenteritis Virus (TGEV). By eliminating reliance on thermal cyclers or specialized equipment, this method is fully deployable in swine farms, veterinary clinics, or field environments. The lateral flow format provides immediate colorimetric results that require minimal technical expertise, while the fluorescence mode allows for semi-quantitative analysis. This study presents a robust and cost-effective platform for decentralized PoRV surveillance in swine populations, addressing the critical need for portable diagnostics in resource-limited settings and enhancing veterinary health management. Full article

Phytophthora pini, a globally dispersed plant pathogen, poses a significant threat to natural ecosystems and cultivated horticultural crops. Early and precise detection of P. pini is essential for effective disease management. This study focused on developing specific, rapid, and sensitive molecular diagnostic techniques to identify the pathogenic oomycete P. pini. We employed recombinase polymerase amplification with lateral flow device (RPA-LFD) and RPA combined with CRISPR/Cas12a. The RPA-LFD method can identify P. pini at concentrations as low as 10 pg/μL in 30 min, while the RPA-CRISPR/Cas12a approach can detect the pathogen at 1 pg/μL in approximately 50 min. These methods are highly effective in identifying disease caused by P. pini and provide a basis for future field detection, which may reduce the economic losses associated with this devastating disease. Full article

Pigs are susceptible to the deadly infectious disease known as African swine fever (ASF), which is brought on by the African swine fever virus (ASFV). As such, prompt and precise disease detection is essential. Deletion of the virulence-related genes MGF-505/360 and EP402R generated from the virulent genotype II virus significantly reduces its virulence, and animal tests using one of the recombinant viruses show great lethality and transmissibility in pigs. The isothermal technique known as recombinase polymerase amplification (RPA) is perfect for rapid in-field detection. To accurately identify ASFV MGF-505R gene-deleted mutants and assess the complex infection situation of ASF, RPA assays in conjunction with real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA-LFD assay) were created. These innovative methods allow for the direct detection of ASFV from pigs, offering in-field pathogen detection, timely disease management, and satisfying animal quarantine requirements. The specific primers and probes were designed against conserved regions of ASFV B646L and MGF-505R genes. Using recombinant plasmid DNA containing ASFV MGF-505R gene-deleted mutants as a template, the sensitivity of both ASF real-time RPA and ASF RPA-LFD assays were demonstrated to be 10 copies per reaction within 20 min at 37 °C. Neither assay had cross-reactions with CSFV, PRRSV, PPV, PRV, ot PCV2, common viruses seen in pigs, indicating that these methods were highly specific for ASFV. The evaluation of the performance of ASFV real-time RPA and ASFV RPA-LFD assays with clinical samples (n = 453) demonstrated their ability to specifically detect ASFV or MGF-505R gene-deleted mutants in samples of pig feces, ham, fresh pork, and blood. Both assays exhibited the same diagnostic rate as the WOAH-recommended real-time fluorescence PCR, highlighting their reliability and validity. These assays offer a simple, cost-effective, rapid, and sensitive method for on-site identification of ASFV MGF-505R gene-deleted mutants. As a promising alternative to real-time PCR, they have the potential to significantly enhance the prevention and control of ASF in field settings. Full article

Estimating the postmortem interval (PMI) is critical in the field of forensic science, and necrophagous insects play a significant role in this process. Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) is a common necrophagous insect species, making its rapid and accurate identification essential. However, commonly used molecular biology methods, such as DNA barcode, still have some limitations in identifying necrophagous insects as they are often complex, time-consuming, and reliant on laboratory instruments. Therefore, in this study, we have developed an innovative detection system for the rapid and accurate identification of C. megacephala based on the Cytochrome b gene using recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) in combination. The developed RPA-LFD detection system achieved complete amplification in just 15 min at 37 °C with good sensitivity and specificity. Only 7.8 × 10⁻⁴ ng or more of target DNA fragments were required, and a positive detection rate of 100% was achieved in 18 C. megacephala samples from actual cases. In addition, the ability of the developed RPA-LFD detection system in combination with rapid DNA extraction methods to enable on-site detection was preliminarily explored. The results suggested that when the RPA-LFD detection system was combined with the grinding ddH₂O extraction method (a rapid DNA extraction method), the process from species acquisition to visualization of detection results could be completed in less than 20 min. In conclusion, this innovative RPA-LFD detection system outperforms commonly used molecular biology methods for C. megacephala identification in terms of speed, sensitivity and convenience, making it suitable for direct application at crime scenes, promising to provide important assistance in estimating PMI and expanding the impact of forensic entomological evidence. Full article

Mycobacterium bovis (M. bovis), the microorganism responsible for bovine tuberculosis (bTB), is transferred to people by the ingestion of unpasteurized milk and unprocessed fermented milk products obtained from animals with the infection. The identification of M. bovis in milk samples is of the utmost importance to successfully prevent zoonotic diseases and maintain food safety. This study presents a comprehensive description of a highly efficient molecular test utilizing recombinase-aided amplification (RPA)–clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) 13a–lateral flow detection (LFD) for M. bovis detection. In contrast to ELISA, RPA–CRISPR–Cas13a–LFD exhibited greater accuracy and sensitivity in the detection of M. bovis in milk, presenting a detection limit of 2 × 10⁰ copies/μL within a 2 h time frame. The two tests exhibited a moderate level of agreement, as shown by a kappa value of 0.452 (95%CI: 0.287–0.617, p < 0.001). RPA–CRISPR–Cas13a–LFD holds significant potential as a robust platform for pathogen detection in complex samples, thereby enabling the more dependable regulation of food safety examination, epidemiology research, and medical diagnosis. Full article

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis. Full article

The Chinese giant salamander (Andrias davidianus), listed as an endangered species under “secondary protection” in China, faces significant threats due to ecological deterioration and the expansion of human activity. Extensive field investigations are crucial to ascertain the current status in the wild and to implement effective habitat protection measures to safeguard this species and support its population development. Traditional survey methods often fall short due to the elusive nature of the A. davidianus, presenting challenges that are time-consuming and generally ineffective. To overcome these obstacles, this study developed a real-time monitoring method that uses environmental DNA (eDNA) coupled with recombinase polymerase amplification and lateral flow strip (RPA-LFD). We designed five sets of species-specific primers and probes based on mitochondrial genome sequence alignments of A. davidianus and its close relatives. Our results indicated that four of these primer/probe sets accurately identified A. davidianus, distinguishing it from other tested caudata species using both extracted DNA samples and water samples from a tank housing an individual. This method enables the specific detection of A. davidianus genomic DNA at concentrations as low as 0.1 ng/mL within 50 min, without requiring extensive laboratory equipment. Applied in a field survey across four sites in Huangshan City, Anhui Province, where A. davidianus is known to be distributed, the method successfully detected the species at three of the four sites. The development of these primer/probe sets offers a practical tool for field surveying and monitoring, facilitating efforts in population recovery and resource conservation for A. davidianus. Full article

The measles virus is highly contagious, and efforts to simplify its diagnosis are essential. A reverse transcriptase/recombinase polymerase amplification assay coupled with CRISPR/Cas12a and an immunochromatographic lateral flow detection (RT-RPA-CRISPR-LFD) was developed for the simple visual detection of measles virus. The assay was performed in less than 1 h at an optimal temperature of 42 °C. The detection limit of the assay was 31 copies of an RNA standard in the reaction tube. The diagnostic performances were evaluated on a panel of 27 measles virus RT-PCR-positive samples alongside 29 measles virus negative saliva samples. The sensitivity and specificity were 96% (95% CI, 81–99%) and 100% (95% CI, 88–100%), respectively, corresponding to an accuracy of 98% (95% CI, 94–100%; p < 0.0001). This method will open new perspectives in the development of the point-of-care testing diagnosis of measles. Full article

The harmful algal bloom (HAB) species Pseudo-nitzschia multiseries is widely distributed worldwide and is known to produce the neurotoxin domoic acid, which harms marine wildlife and humans. Early detection and preventative measures are more critical than late management. However, the major challenge related to early detection is the accurate and sensitive detection of microalgae present in low abundance. Therefore, developing a sensitive and specific method that can rapidly detect P. multiseries is critical for expediting the monitoring and prediction of HABs. In this study, a novel assay method, recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD), is first developed for the detection of P. multiseries. To obtain the best test results, several important factors that affected the amplification effect were optimized. The internal transcribed spacer sequence of the nuclear ribosomal DNA from P. multiseries was selected as the target region. The results showed that the optimal amplification temperature and time for the recombinase polymerase amplification (RPA) of P. multiseries were 37 °C and 15 min. The RPA products could be visualized directly using the lateral flow dipstick after only 3 min. The RPA-LFD assay sensitivity for detection of recombinant plasmid DNA (1.9 × 10⁰ pg/μL) was 100 times more sensitive than that of RPA, and the RPA-LFD assay sensitivity for detection of genomic DNA (2.0 × 10² pg/μL) was 10 times more sensitive than that of RPA. Its feasibility in the detection of environmental samples was also verified. In conclusion, these results indicated that the RPA-LFD detection of P. multiseries that was established in this study has high efficiency, sensitivity, specificity, and practicability. Management measures made based on information gained from early detection methods may be able to prevent certain blooms. The use of a highly sensitive approach for early warning detection of P. multiseries is essential to alleviate the harmful impacts of HABs on the environment, aquaculture, and human health. Full article

Pentatrichomonas hominis is a trichomonad protozoan that infects the cecum and colon of humans and other mammals. It is a zoonotic pathogen that causes diarrhea in both animals and humans. As companion animals, dogs infected with P. hominis pose a risk of transmitting it to humans. Current methods, such as direct smears and polymerase chain reaction (PCR), used for P. hominis detection have limitations, including low detection rates and the need for specialized equipment. Therefore, there is an urgent need to develop rapid, sensitive, and simple detection methods for clinical application. Recombinase polymerase amplification (RPA) has emerged as a technology for rapid pathogen detection. In this study, we developed a lateral flow dipstick (LFD)-RPA method based on the highly conserved SPO11-1 gene for detecting P. hominis infection by optimizing the primers, probes, and reaction conditions, and evaluating cross-reactivity with genomes of Giardia duodenalis and other parasites. The LFD-RPA method was then used to test 128 dog fecal samples collected from Changchun. The results confirmed the high specificity of the method with no cross-reactivity with the five other parasites. The lowest detection limit of the method was 10² copies/µL, and its sensitivity was 10⁰ times higher than that of the conventional PCR method. Consistent with the positivity rate observed using nested PCR, 12 samples (out of 128) tested positive using this method (positivity rate, 9.38%). In conclusion, the LFD-RPA method developed in this study represents a simple and sensitive assay that allows for the rapid detection of P. hominis infection in dogs, especially in this field. Full article

Stewart’s vascular wilt and leaf blight of sweet corn is caused by the Gram-negative enteric bacterium Pantoea stewartii subsp. stewartii. Stewart’s wilt results in substantial yield losses worldwide warranting rapid and accurate disease diagnosis. Recombinase polymerase amplification (RPA) is an isothermal technique that is tolerant to host plant-derived inhibitors and is, therefore, ideally suited for rapid in-field detection vis-à-vis traditional polymerase chain reaction-based molecular assays. An RPA assay coupled with a Lateral Flow Device (LFD) was developed for rapid, accurate, and sensitive real-time detection of P. stewartii subsp. stewartii directly from the infected host offering in-field pathogen detection, timely disease management, and satisfying quarantine and phytosanitary requirements. Twelve novel primer sets were designed against conserved genomic regions of P. stewartii subsp. Stewartii; however, only the primers for amplification of the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB were discernibly unique and adequate for unambiguous identification of P. stewartii subsp. stewartii. The P. stewartii subsp. stewartii-specific primers were further validated in a simplex RPA assay for specificity against twenty-six bacterial species representing several Pantoea and other closely related bacterial species/subspecies/strains found in the same niche, and naturally or artificially infected plant samples. The integrated RPA/LFD assay was also optimized for rapid and sensitive on-site detection of P. stewartii subsp. stewartii with an empirical detection limit of 0.0005 pg μL⁻¹ bacterial DNA and 1 × 10² CFU mL⁻¹ (app. two bacterial cells used per RPA reaction) in minimally processed samples for accurate, low-cost, and point-of-need diagnosis of the quarantine pathogen P. stewartii subsp. stewartii. Full article