Search Results (84)

PEDV poses a significant threat to the global swine industry, necessitating rapid and accurate diagnostic methods for effective disease management. In this study, we developed a foldable, easy-to-use paper-based microfluidic analytical device (μPAD) for on-site detection of PEDV. The device seamlessly integrates paper-based nucleic acid enrichment, LAMP reaction, and visual lateral flow detection into a single platform. Key parameters, including nucleic acid extraction protocols, chromatographic channel configurations, colorimetric indicators, and reaction temperature and duration, were systematically optimized. The resulting LAMP-μPAD assay detects PEDV within 30 min at 60 °C, achieving a limit of detection of 4.82 × 10² copies/μL with no cross-reactivity against other viruses. When evaluated against RT-PCR using clinical specimens, the assay demonstrated a specificity of 100%, a sensitivity of 95.3%, and an overall concordance of 98.5%. This paper-based sensor offers a promising alternative for the rapid, on-site detection of PEDV and other highly transmissible pathogens. 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

Bacillus cereus is a widespread foodborne pathogen that can cause food poisoning when present in food at certain levels. Ingesting contaminated food may lead to symptoms such as abdominal pain, diarrhea, and, in severe cases, life-threatening conditions. In this study, a simple and super-fast method for detecting B. cereus was developed, which combines cellulose filter paper-based DNA extraction, multienzyme isothermal rapid amplification (MIRA), and lateral flow dipstick (LFD) technology. Initially, PCR was adopted to evaluate the DNA extraction efficiency of the filter paper, followed by the optimization of the lysis formula and extraction conditions. With the above optimization, DNA that can be used for subsequent nucleic acid amplification can be obtained within 3 min. Then, the isothermal amplification of MIRA–LFD was established and optimized to evaluate the detection specificity and sensitivity. Finally, the developed method was applied to detect B. cereus in cooked rice samples. The results indicated that the entire amplification procedure of MIRA-LFD only takes 15 min at 39 °C. The whole super-fast detection system could be completed in less than 20 min, from DNA extraction to result interpretation, which achieved a detection limit of 12 fg/μL of DNA concentration, corresponding to approximately 115 CFU/mL in actual samples. Full article

MicroRNA122 (miR-122) is a microRNA that is highly expressed in hepatocytes and has been identified as a prospective therapeutic target and biomarker for liver injury. An expanding body of research has demonstrated that miR-122 is a critical regulator in both the initiation and progression of a wide range of liver diseases. Traditional methods for detecting miR-122 mainly include Northern blotting and qRT-PCR, but they are technically complex and cumbersome, requiring expensive instruments and high technical requirements. In this paper, we present a novel rapid testing method utilizing a lateral flow nucleic acid biosensor (LFNAB) for the sensitive and time-efficient detection of miR-122. This approach offers several advantages, including a high specificity for miR-122, the ability to detect low concentrations of the target molecule, and a significantly reduced testing time compared to conventional detection methods. In this study, a thiol-modified single-stranded detection DNA probe (Det-DNA), a biotinylated single-stranded capture DNA probe (Cap-DNA), and a biotinylated single-stranded control DNA probe (Con-DNA) are used to construct the LFNAB. A gold nanoparticle (AuNP) is a colored tag, which is used to label the Det-DNA probe. The principle of detecting miR-122 is based on dual DNA-miRNA hybridization reactions on the LFNAB to form sandwich-type AuNP-Det-DNA-miR-122-Cap-DNA complexes, which are captured on the test area of LFNAB for visualization and quantification. After systematic optimization of conditions of experiment, the response of LFNAB was highly linear within the scope of 0 pM-100 pM miR-122, and the detection limit in 15 min was 3.90 pM. The use of LFNAB to detect miR-122 in serum and fingertip blood has yielded satisfactory results. This successful application indicates the effectiveness of LFNAB in detecting miR-122 in both serum and fingertip blood samples, showcasing its potential utility in clinical and research settings for assessing miR-122 levels in different biological samples. Full article

Strawberry crown and root rot diseases are caused by soil-borne pathogens including Macrophomina phaseolina (Mp) and Verticillium dahliae (Vd). The symptoms caused by these pathogens are very similar and difficult to distinguish, and traditional culture-based detection methods are laborious, time-consuming, and slow in providing results. In this work, we developed a duplex PCR-NALFIA assay using two pairs of species-specific primers labeled at the 5′ end with different molecules for the simultaneous identification of Mp and Vd. For the NALFIA assay, a lateral flow device (LFD) for the detection of two analytes was used. The method was developed by single and duplex PCR (Mp, Vd, Mp + Vd) using increasingly complex biological systems: (i) DNA from pure cultures of the pathogens; (ii) DNA from artificially inoculated cut melon stems; and (iii) DNA from artificially inoculated strawberry plants cv. Aromas. The duplex PCR protocol was effective in detecting the two pathogens within melon tissues and provided good results with strawberry crown tissues only when the DNA samples were purified by removing the PCR inhibitors. The amplicons were used for both agarose gel electrophoresis (AGE) and NALFIA assays and demonstrated the greater sensitivity of the NALFIA assay (10 pg) for simultaneous detection of the two pathogens. Full article

Due to the price and demand of Ophiocordyceps sinensis having increased dramatically, adulteration with other fungi is a common problem. Thus, a reliable method of authentic O. sinensis identification is essential. In the present work, a rapid DNA extraction and double-tailed recombinase polymerase amplification (RPA) coupled with nucleic acid hybridization lateral flow strip (NAH-LFS) was developed to distinguish authentic O. sinensis ingredients from other fungi substitutes. In the presence of O. sinensis, the RPA amplicons with two ssDNA tails in the opposite ends, which could simultaneously bind with the SH-probes on gold nanoparticles (AuNPs) and capture the probe on the test line, formed visible red bands. RPA combined with NAH-LFS can efficiently detect O. sinensis DNA down to 1.4 ng/μL; meanwhile, the specificity test validated no cross reaction with common adulterants, including Cordyceps gunnii, Cordyceps cicadae, Cordyceps militaris, yungui Cordyceps, and Ophiocordyceps nutans. The whole RPA-NAH-LFS could be completed within 16 min. The RPA-NAH-LFS results in detecting 20 commercial O. sinensis samples are consistent with PCR-AGE and RT-PCR, confirming the feasibility of the RPA-NAH-LFS method. In conclusion, these results are expected to facilitate the application of RPA-NAH-LFS in the authentication detection of O. sinensis materials, providing a convenient and efficient method for O. sinensis quality control. Full article

Feline calicivirus (FCV) is one of the most common viral pathogens in domestic cats worldwide, which mainly causes upper respiratory tract infections in felines and seriously threatens the health of felines. Consequently, it is crucial to establish a rapid detection method to efficiently take control and prevent the spread of FCV. To construct the Cas13a-RAA-LFD reaction system, this study specifically designed recombinase-aided amplification (RAA) primers added with a T7 promoter and CRISPR RNA (crRNA), which were both based on the FCV relatively conserved sequence. The Cas13a protein cleaved the reporting probes only when crRNA recognized the target sequence. The results could be directly observed by lateral flow dipsticks (LFDs). To evaluate this system, factors such as RAA amplification time, Cas13a protein concentration, crRNA concentration, and CRISPR reaction time were optimized. Then, a comparison of the coincidence rate for clinical samples between this method and the polymerase chain reaction (PCR) agarose electrophoresis method was performed to evaluate the reliability of the method. Eventually, the results indicated that the target gene could be effectively amplified by the Cas13a-RAA-LFD method, and the results could be visually observed by LFD. The method could detect FCV specifically, whilst having no cross-reaction with other common viruses which infect felines, such as feline parvovirus (FPV), feline coronavirus (FCoV) and feline herpesvirus (FHV). This method is extremely sensitive and has been validated to detect viral nucleic acids down to 10⁰ copies/μL. The good reproducibility and stability of the method were also verified by this study. Testing of clinical samples proved that the coincidence rate of clinical detection reached 96.39%. In summary, this study established a simplistic, efficient, accurate, and visualized FCV detection method, which can be utilized for early prevention and control of FCV. Full article

Diagnostics often require specialized equipment and trained personnel in laboratory settings, creating a growing need for point-of-care tests (POCTs). Among the genetic testing methods available, Loop-mediated Isothermal Amplification (LAMP) offers a viable solution for developing genetic POCT due to its compatibility with simplified devices. This study aimed to create a genetic test that integrates all steps from sample processing to analyzing results while minimizing the complexity, handling, equipment, and time required. Several challenges were addressed to achieve this goal: (1) the development of a buffer for bacterial DNA extraction that is compatible with both LAMP and immunochromatographic tests; (2) the adaption of the LAMP protocol for use with the SPID device; and (3) the optimization of the detection protocol for specific test conditions, with a lateral flow immunoassay format selected for its POCT compatibility. Following these developments, the test was validated using Escherichia coli (E. coli) and non-E. coli strains. A portable heating station was also developed to enable amplification without costly equipment. The resulting genetic POCT achieved 100% sensitivity and 85% specificity, with results available in 60 to 75 min. This study demonstrated that our POCT efficiently performs DNA extraction, amplification, and detection for bacterial identification. The test’s simplicity and cost-effectiveness will support its implementation in various settings. Full article

We developed a rapid and sensitive diagnostic platform that integrates isothermal viral gene amplification with a nucleic acid lateral flow assay (NALFA) to detect SARS-CoV-2 RNA. Isothermal gene amplification was performed by combining reverse transcription of viral RNA with recombinase polymerase amplification (RPA). In our diagnostic platform, DNA primers for the RPA reaction were modified by appending DNA tails, enabling the synthesis of tailed amplicon DNAs. These tailed amplicon DNAs were subsequently annealed to the complementary capture DNA probe affixed to the lateral flow strip during the NALFA of the reaction samples. The other side of each amplicon DNA tail was annealed to the reporter probe DNA conjugated with gold nanoparticles to visually detect the test line in the strip. This diagnostic platform reduces the time required to obtain readouts to within 1 h and can detect viral RNA concentrations as low as 3.1 cp/μL. Furthermore, when applied to nasopharyngeal clinical samples, our NALFA diagnostic platform yielded highly reliable molecular diagnostic readouts that were 100% consistent with the results of conventional RT-qPCR. Full article

Urinary tract infection (UTI) is the most prevalent kind of pathogenic bacteria infection, and the midstream urine culture is regarded as the gold standard in UTI diagnosis. Recently, even with modern media and techniques such as polymerase chain reaction (PCR), urinary cultures still create a considerable workload for hospital laboratories. Other UTI-detecting methods, such as flow cytometry and lateral flow immunoassay, suffer from various drawbacks like long time consumption and low sensitivity. Therefore, looking for reliable biomarkers in UTI is urgently needed. In this review, the current definitions of UTI can be basically divided into two main categories: uncomplicated UTI and complicated UTI. In light of anatomical sites, it can be classified as either lower UTI or upper UTI. We take the classification of UTI as a clue and review the reported extensive literature to classify the existing studied markers into the following three categories: Biomarkers used clinically; Promising biomarkers; and Controversial biomarkers. Particularly, the nucleic acid-associated, metabolomic, and lipidomic biomarkers are highlighted. At the end, we discuss the challenges and prospects of biomarkers in UTI, hoping to further inspire the diagnosis of UTI. Full article

In the last decade, pulmonary fungal infections such as invasive pulmonary aspergillosis (IPA) have increased in incidence due to the increased number of immunocompromised individuals. This increase is especially problematic when considering mortality rates associated with IPA are upwards of 70%. This high mortality rate is due to, in part, the length of time it takes to diagnose a patient with IPA. When diagnosed early, mortality rates of IPA decrease by as much as 30%. In this review, we discuss current technologies employed in both medical and research laboratories to diagnose IPA, including culture, imaging, polymerase chain reaction, peptide nucleic acid–fluorescence in situ hybridization, enzyme-linked immunosorbent assay, lateral flow assay, and liquid chromatography mass spectrometry. For each technique, we discuss both promising results and potential areas for improvement that would lead to decreased diagnosis time for patients suspected of contracting IPA. Further study into methods that offer increased speed and both analytical and clinical sensitivity to decrease diagnosis time for IPA is warranted. Full article

Pigeon Newcastle disease, caused by pigeon paramyxovirus type 1 (PPMV-1), is a significant infectious disease in pigeons that can result in substantial mortality and poses a severe threat to the pigeon industry. The rapid and accurate onsite diagnosis of pigeon disease is crucial for timely diagnosis and the implementation of effective prevention and control measures. In this study, we established a rapid detection method for PPMV-1 based on recombinase-aided amplification (RAA) and CRISPR/Cas12a. The RAA primers target the conserved regions of the L gene for preamplification in clinical nucleic acid samples, followed by CRISPR/Cas12a detection of the target gene. Visualization could be achieved by combination with a lateral flow dipstick (LFD). This method demonstrated high specificity, showing no cross-reactivity with non-PPMV-1 samples. The sensitivity of the method assessed by fluorescence analysis reached 10⁰ copies/µL, and when it was combined with an LFD, the sensitivity was 10³ copies/µL. The constructed RAA-CRISPR/Cas12a-LFD visual detection method was applied to clinical sample testing and was found to enable the rapid and accurate detection of swab samples and tissue specimens. Its sensitivity was consistent with the current gold standard, quantitative real-time PCR results. The RAA-CRISPR/Cas12a-LFD detection method we developed provides a novel approach for the rapid, simple, precise, and specific onsite diagnosis of pigeon Newcastle disease. Full article

Mercury ions (Hg²⁺) are toxic heavy metals present in the environment that pose significant health risks. An advanced detection system could allow for a prompt response and alleviate serious damage to humans. In this study, we developed a cost-effective, on-site detection method for Hg²⁺ using a multicomponent nucleic acid enzyme (MNAzyme)-assisted nucleic acid lateral flow assay (NALFA). The MNAzyme, which was engineered to contain thymine–thymine mismatches, is responsive only to the presence of Hg²⁺ and exerts efficient cleavage activity on substrates that can be captured by the NALFA strip, and thus the proposed system enables the visual detection of Hg²⁺ in the NALFA strip. Our assay demonstrated sufficient detection sensitivity and specificity to meet the WHO standards, offering a good practical alternative for rapid environmental and public health monitoring. Full article

A promising and sought-after class of nanozymes for various applications is Pt-containing nanozymes, primarily Au@Pt, due to their easy preparation and remarkable catalytic properties. This study aimed to explore the freeze–thaw method for functionalizing Pt-containing nanozymes with oligonucleotides featuring a polyadenine anchor. Spherical gold nanoparticles ([Au]NPs) were synthesized and subsequently used as seeds to produce urchin-like Au@Pt nanoparticles ([Au@Pt]NPs) with an average diameter of 29.8 nm. The nanoparticles were conjugated with a series of non-thiolated DNA oligonucleotides, each consisting of three parts: a 5′-polyadenine anchor (A_n, with n = 3, 5, 7, 10; triple-branched A₃, or triple-branched A₅), a random sequence of 23 nucleotides, and a linear polyT block consisting of seven deoxythymine residues. The resulting conjugates were characterized using transmission electron microscopy, spectroscopy, dynamic light scattering, and emission detection of the fluorescent label at the 3′-end of each oligonucleotide. The stability of the conjugates was found to depend on the type of oligonucleotide, with decreased stability in the row of [Au@Pt]NP conjugates with A₇ > A₅ > 3A₃ > 3A₅ > A₁₀ > A₃ anchors. These [Au@Pt]NP–oligonucleotide conjugates were further evaluated using lateral flow test strips to assess fluorescein-specific binding and peroxidase-like catalytic activity. Conjugates with A₃, A₅, A₇, and 3A₃ anchors showed the highest levels of signals of bound labels on test strips, exceeding conjugates in sensitivity by up to nine times. These findings hold significant potential for broad application in bioanalytical systems. Full article

This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. A protein-free nucleic acid-based lateral flow assay (NALFA) with a limit of detection of 1 pmol of DNA is shown for the first time. The total production duration of such an assay was successfully reduced from the currently known several days to just a few hours. The simplification and acceleration of the protocol make the method more accessible and practical for various applications. The developed method supports multiplexing, enabling the simultaneous detection of up to six DNA targets. This multiplexing capability is a significant improvement over traditional line tests and offers more comprehensive diagnostic potential in a single assay. The approach significantly reduces the run time compared to traditional line tests, which enhances the efficiency of diagnostic procedures. The protein-free aspect of this assay minimizes the prevalent complications of cross-reactivity in immunoassays especially in cases of multiplexing. It is also demonstrated that the NALFA developed in this study is amplification-free and hence does not rely on specialized technicians, nor does it involve labour-intensive steps like DNA extraction and PCR processes. Overall, this study presents a robust, efficient, and highly sensitive platform for DNA or RNA detection, addressing several limitations of current methods documented in the literature. The advancements in sensitivity, cost reduction, production time, and multiplexing capabilities mark a substantial improvement, holding great potential for various applications in diagnostics, forensics, and molecular biology. Full article