Search Results (67)

In this study, a multienzyme isothermal and lateral flow dipstick combination assay for PPRV detection was established, the designed primers and probes targeting the N gene were screened and optimized, and analytical sensitivity, specificity, and repeatability of developed method were systematically evaluated. The experimental results demonstrated that this method is easy to operate, can complete detection within 30 min at 42 °C, and is capable of detecting all lineages of peste des petits ruminants virus (PPRV) without cross-reactivity with other viruses. The limit of detection could reach 10 copies/μL. Repeatability validation showed that the coefficients of variation (CV) for both intra-assay and inter-assay experiments were below 3.0%. The positive detection rate for clinical samples could reach 100%. The test results are visually interpretable via fluorescence and lateral flow strips. In conclusion, this method exhibits high analytical sensitivity, specificity, and excellent repeatability, enabling rapid diagnosis of peste des petits ruminants (PPR). Full article

As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve a Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum, and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI), and in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37–39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in a wheat blast hotspot district, Meherpur, Bangladesh. Training 52 diverse stakeholders validated the kit’s field readiness, with 88% of trainees endorsing its user-friendly design. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site genomic surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as an early warning modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers. Full article

Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease in ruminants. PTB is difficult to diagnose, control, and eradicate, leading to substantial economic losses. Thus, sensitive and specific detection methods are urgently required. crRNA and primers targeting the MAP ATPase FtsK gene were designed for recombinase polymerase amplification (RPA) and nested PCR. Fecal DNA was amplified using RPA or nested PCR, purified with Tris-saturated phenol-chloroform-isoamyl alcohol, and detected via CRISPR-Cas12a. Moreover, signals were read using a qPCR instrument, fluorescence reader, or lateral flow strips. RPA–CRISPR-Cas12a and nested PCR–CRISPR-Cas12a assays were optimized and validated on 50 clinical samples and 7 MAP cultures. The limits of detection were 1 × 10⁻¹⁰ μg/μL for RPA–CRISPR-Cas12a and 1 × 10⁻¹⁴ μg/μL for nested PCR–CRISPR-Cas12a. Efficient cleavage of the ssDNA reporter occurred at DNA concentrations of ≥1 × 10⁻⁴ μg/μL, producing a strong fluorescent signal. All three detection methods showed perfect agreement with reference assays across both sample sets. This study presents the first integration of RPA or nested PCR with CRISPR-Cas12a for MAP detection, enabling rapid, specific, and highly sensitive diagnosis. Flexible detection options allow adaptation to available resources and bacterial loads, supporting practical use in PTB control. Full article

Cardiac troponin I (cTnI) is a critical biomarker for the diagnosis of acute myocardial infarction (AMI), but conventional detection methods are often time-consuming and require specialized laboratory equipment. To meet the need for rapid and feasible detection, there is an urgent demand for methods that are fast, specific, and easy to use. In this study, two aptamers (Tro4 and Tro6), which specifically bind to different epitopes of cTnI, were employed to construct a dual-aptamer sandwich system on a lateral flow assay (LFA) strip. The test strip can deliver results within 10 min and shows a detection limit of 11.70 ng·mL⁻¹. It also exhibited excellent stability after storage at room temperature for up to four months. The assay demonstrated high analytical accuracy, as evidenced by recovery rates from spiked serum samples ranging from 95.11% to 103.17%. These results suggest that the proposed aptamer-based LFA is highly suitable for rapid screening of cTnI, especially in point-of-care settings and resource-limited environments. From a diagnostic perspective, this method holds great promise for improving the timely detection and management of AMI and other myocardial injuries. Full article

Objectives: The purpose of this study is to develop and verify a sandwich-type lateral flow immunoassay (LFA) integrated with a smartphone, enabling semi-quantitative 25-hydroxyvitamin D [25(OH)D] measurement including automated image analysis function, thereby establishing a reliable and accessible vitamin D evaluation system for point-of-care (POCT). Methods: A smartphone-based sandwich-type LFA was constructed, and 25(OH)D was measured semi-quantitatively. The system combined a customized test strip with an automatic image acquisition, calibration, and classification module integrated into an application dedicated to a smartphone. Analysis performance, reproducibility, and equivalence between sample types were comprehensively evaluated. Results: The developed analysis achieved a detection range of 5–100 ng/mL, and there were little interference and cross-reactivity for endogenous substances or structurally similar vitamin D derivatives. The image processing algorithm accurately classified the samples into three clinically important categories: deficiency (<20 ng/mL), insufficient (20–30 ng/mL), and sufficient (>30 ng/mL). Cross-platform testing between Android and iOS devices showed excellent reproducibility (r = 0.99, R² = 0.9967). Comparative analysis with the Atellica IM 1600 analyzer showed a high degree of agreement between 97.0% category consensus and κ = 0.951 (r = 0.99, R² ≥ 0.98). Comparative tests between serum and capillary samples also confirmed a 100% classification agreement rate and an overall diagnostic accuracy of 95.5%. Conclusions: This next-generation smartphone integration platform enables rapid, accurate, and semi-quantitative detection of 25(OH)D from fingerstick and serum specimens. By combining the sandwich-type LFA design with computational-based imaging analysis, the system effectively overcomes the major limitations of small-molecule immunoassay and has the potential to be applied to field diagnosis (POCT), decentralized diagnostics, and vitamin D screening in large populations. Full article

Pneumonia caused by Pneumocystis jirovecii poses a serious threat, particularly to immunocompromised patients such as those with HIV/AIDS, transplant recipients, or individuals undergoing chemotherapy. Its diagnosis is challenging because current methods, such as microscopy and certain molecular tests, have limitations in sensitivity and specificity, and require specialized equipment, which delays treatment initiation. In this context, CRISPR-Cas12-based methods offer a promising alternative: they are rapid, highly specific, sensitive, and low-cost, enabling more timely and accessible detection, even in resource-limited settings. We developed a simple and rapid detection platform based on the CRISPR-Cas12 coupled with lateral flow strips. A guide RNA was designed against DHPS, β-tubulin, and mtLSU rRNA genes. The guide corresponding to β-tubulin showed high sensitivity in the detection of P. jirovecii to produce a detectable fluorescence signal within the first 20–30 min. In addition, it demonstrated high specificity for P. jirovecii when DNA from other microorganisms was used. When coupled with lateral flow strips, high sensitivity and specificity were also observed for detecting positive samples, without the need for genetic amplification. CRISPR-Cas12 successfully detected P. jirovecii infection in an initial diagnostic application, demonstrating the potential of this method for integration into public health diagnostic systems, particularly in field, due to its adaptability, speed, and ease of use. Full article

Citrus alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is one of the most severe fungal diseases affecting citrus crops. Currently, there is a critical need for rapid and visual detection techniques to identify the tangerine pathotype of A. alternata. In this study, a novel detection system was developed by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology, targeting the ACTT3 gene specific to the tangerine pathotype of A. alternata. Through optimization of reaction time and component concentrations, the assay demonstrated a detection sensitivity of 1 pg μL⁻¹ within 40 min at a constant temperature of 37 °C. The results can be visually interpreted using nucleic acid test strips, offering advantages in specificity, sensitivity, and speed. This system has been successfully validated for the rapid detection of the pathogen within plant tissues, including leaves and fruits, providing an efficient and practical solution for real-time field detection of the tangerine pathotype of A. alternata. Full article

Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription–recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks. Full article

A conventional PCR (cPCR) remains an effective molecular technique for the diagnosis of canine monocytic ehrlichiosis. However, agarose gel electrophoresis requires additional time after thermal cycling. In the present study, we developed a PCR-based lateral flow biosensor (PCR-LFB) to detect Ehrlichia canis (E. canis). Lateral flow strips allow for the simple and rapid detection of PCR products and provide an alternative to gel electrophoresis. The sensitivity, specificity, and detection limit of PCR-LFB were compared to those of TaqMan probe-based real-time PCRs (qPCRs). The PCR-LFB was performed with 5′ 6-FITC and biotin-labeled primers specific to E. canis, targeting the dsb gene. The detection limit of the PCR-LFB assay was 10⁻⁶ for the target DNA sequence in a 10-fold dilution of the recombinant plasmid, which is 10 times lower than that of qPCR. Among the confirmed qPCR results in the 30 dog samples, false-positive results were not detected by the PCR-LFB. Compared to qPCR, the sensitivity and specificity of PCR-LFB were 63.6% (95% CI; 42.9–80.2%) and 100% (95% CI; 67.5–100%), respectively. The Kappa value of the PCR-LFB is in moderate agreement with the qPCR (κ = 0.483). Perfect agreement (κ = 1) was observed between cPCR and PCR-LFB. Lower cost and shorter time consumption were demonstrated using PCR-LFB. Full article

In this study, a novel rapid immunochromatographic (IC) test for African swine fever virus (ASFV) antibodies is presented. An immunochromatographic test (IC) is a detection technique that combines membrane chromatography with immunolabeling. This approach saves time for antibody preparation, resulting in a shorter production cycle. p54 is an important structural protein of African swine fever, and an ideal protein for serotype diagnosis. Gold nanoparticles are attached to the ASFV p54-Fc fusion protein, and the ASFV-specific antigen p54 and Staphylococcus aureus protein A (SPA) are labeled on a nitrocellulose membrane, at positions T and C, respectively. We developed a SPA double sandwich IC test strip, and assessed its feasibility using ASFV p54 and p54-Fc fusion proteins as antigens. ASFV p54 and p54-Fc fusion proteins were expressed and purified. A sandwich cross-flow detection method for p54, which is the primary structural protein of ASFV, was established, using colloidal gold conjugation. Our method can detect ASFV antibodies in field serum samples in about 15 min using a portable colloidal gold detector, demonstrating high specificity and sensitivity (1:320), and the coincidence rate was 98% using a commercial ELISA kit. The dilution of the serum sample can be determined by substituting the absorbance (T-line) interpreted by portable devices into the calibration curve function formula of an African swine fever virus standard serum. In summary, our method is rapid, cost-effective, precise, and highly selective. Additionally, it introduces a new approach for constructing IC test strips using SPA protein without antibody preparation, making it a reliable on-site antibody test for ASFV. Full article

Toxoplasma gondii (T. gondii) is an important zoonotic pathogen which induces both acute and chronic toxoplasmosis. Timely diagnosis of T. gondii is crucial for effective disease management. Here, we present a pioneering approach using europium (III)-chelated nanoparticles (EuNPs) in a rapid lateral flow immunochromatographic test strip (ICTS) for detecting T. gondii antibodies in serum samples. By conjugating EuNPs with Staphylococcus aureus protein A, we efficiently captured T. gondii-specific antibodies, which bound to T. gondii antigens on the test line (T-line), generating a distinct fluorescent signal. Employing this novel method, we conducted an extensive epidemiological investigation of T. gondii infections among dogs and cats in Shanghai, China. This innovative ICTS allows for rapid results within 25 min, which include a qualitative result through naked-eye observation under an ultraviolet lamp and a quantitative one derived using a strip reader. With a detection limit of 1:6400 for dog positive serum and no cross-reactivity with other canine and feline pathogens, the EuNPs-ICTS demonstrated excellent consistency with standard enzyme-linked immunosorbent assay results for dogs (κ = 0.91) and cats (κ = 0.92). In addition, 20.38% of 996 dog serum samples and 14.18% of 416 cat serum samples revealed T. gondii antibodies, highlighting the efficacy of this approach. Our study presents a rapid, sensitive, specific, and reproducible EuNPs-ICTS, serving as a promising tool for on-the-spot diagnosis of T. gondii infections in dogs and cats. Full article

Brucellosis is a global problem, with the causative agent being the genus Brucella. B. canis can cause undulant fever in dogs, which is a zoonotic disease that can spread not only among dogs but also to humans. This poses a public health threat to society. In this study, a rapid and straightforward immune colloidal gold test strip was developed for the diagnosis of canine brucellosis through the detection of anti-LPS antibodies in serum samples. Rabbit anti-canine IgG conjugated with colloidal gold was employed as the colloidal gold-labeled antibody. The extracted high-purity R-LPS was employed as the capture antigen in the test line (T-line), while goat anti-rabbit IgG was utilized as the capture antibody in the control line (C-line). The colloidal gold strip exhibited high specificity in the detection of brucellosis, with no cross-reaction observed with the common clinical canine diseases caused by Canine coronavirus (CCV), Canine distemper virus (CDV), and Canine parvovirus (CPV). In comparison to the commercial iELISA kit, the sensitivity and specificity of the colloidal gold test strip were found to be 95.23% and 98.76%, respectively. The diagnostic coincidence rate was 98.47%. The findings of this study indicate that colloidal gold test strips may be employed as a straightforward, expeditious, sensitive, and specific diagnostic instrument for the identification of canine brucellosis, particularly in resource-limited regions. Full article

The prevalence and impact of Getah virus (GETV) are significant concerns in China. GETV can infect a wide range of animals, including horses, pigs, sheep, cattle, birds, and humans, resulting in substantial losses in the livestock and agricultural industries. GETV infection can cause the development of ulcers and inflammation in the mouth and gums of horses, which result in pain and discomfort and lead to symptoms such as reduced appetite, drooling, and difficulty chewing. As a result, there is a pressing need for efficient and rapid disease diagnosis methods. However, the currently available diagnostic methods have limitations in terms of operational time, equipment, and the experience of the individuals using them. In this study, a rapid, specific, and sensitive detection method was developed using a colloidal gold-based immunochromatographic strip (ICS) for the detection of antibodies against GETV in horses. To prepare the ICS, the antigen domain of the E2 glycoprotein of GETV was expressed using the Escherichia coli expression system after analysis with DNAstar v7.1 software. The nitrocellulose membrane was coated with rE2 protein or SPA to form the test line and control line, respectively. After optimizing the reaction conditions, the sensitivity, specificity, and repeatability of the strip were verified. The results showed that the test strip had a detection limit of up to 1:320 dilutions for GETV-positive serum, with no cross-reactivity observed with other equine-susceptible pathogens such as equine arteritis virus (EAV), equine herpesvirus-1 (EHV-I), equine infectious anemia virus (EIAV), equine influenza virus (EIV), African horse sickness virus (AHSV), and Japanese encephalitis virus (JEV). Furthermore, the ICS exhibited a concordance rate of 94.0% when testing 182 clinical serum samples compared to the virus neutralization test. Overall, this ICS diagnosis method will be an effective tool for the rapid detection of GETV in the field. Full article

Groupers are valuable economic fish in the southern sea area of China, but the threat of disease is becoming more and more serious. Vibrio harveyi, V. parahaemolyticus, and Singapore grouper iridovirus (SGIV) are three important pathogens that cause disease in groupers, and infection with either a single one or a mix of these pathogens poses a serious threat to the healthy development of grouper culture. To enhance the rapid diagnosis and screening in the early stages, it is necessary to develop rapid detection methods of these pathogens. To simultaneously and rapidly detect the three pathogens, in this study, we utilized the TolC of V. harveyi, DNAJ of V. parahaemolyticus, and RAD2 of SGIV as the target genes and established a triple visual loop-mediated isothermal amplification (LAMP) method. This LAMP method showed a detection time as fast as 30 min and a high sensitivity of 100 fg/μL. Moreover, this method exhibited strong specificity and no cross-reaction with seven types of Vibrio and Staphylococcus aureus, as well as five common viruses in aquatic animals. Then, the LAMP products were enzymically cut, and three characteristic strips were used to identify the pathogen species. The results of the clinical trials demonstrated that the method could accurately and specifically detect V. harveyi, V. parahaemolyticus, and SGIV in grouper tissues. In summary, this study successfully established a triple visual LAMP rapid detection method for V. harveyi, V. parahaemolyticus, and SGIV. The method offers several advantages including simple equipment, easy operation, rapid reaction, high specificity, high sensitivity, and visual results. It is suitable for the early and rapid diagnosis of groupers infected with V. harveyi, V. parahaemolyticus, and SGIV, thereby providing useful technical support for further application in the large-scale disease surveillance of aquaculture animals. Full article

Lateral flow immunoassays (LFIAs) are recognized for their practicality in homecare and point-of-care testing, owing to their simplicity, cost-efficiency, and rapid visual readouts. Despite these advantages, LFIAs typically fall short in sensitivity, particularly in detecting viruses such as SARS-CoV-2, thus limiting their broader application. In response to this challenge, we have innovated an approach to substantially enhance LFIA sensitivity. This involves the integration of a water-soluble dextran–methacrylate polymer wall with a 15% grafting degree positioned between the test and control lines on the LFIA strip. This novel modification significantly improved the sensitivity of the assay, achieving detection limits as low as 50 pg mL⁻¹ and enhancing the sensitivity by 5–20-fold relative to existing LFIA kits available on the market. Furthermore, our developed LFIA kit (WSPW-LFIA) demonstrated exceptional specificity for SARS-CoV-2. Coupled with a straightforward fabrication process and robust stability, the WSPW-LFIA represents a promising advancement for real-time in vitro diagnosis across a spectrum of diseases. Full article