Rapid visual detection of Helicobacter pylori and vacA subtypes by Dual-Target RAA-LFD assay.

BACKGROUND: Helicobacter pylori (H. pylori) infects over 50% of the global population and is a significant risk factor for gastric cancer. The pathogenicity of H. pylori is primarily attributed to virulence factors such as vacA. Timely and accurate identification, along with genotyping of H. pylori virulence genes, are essential for effective clinical management and controlling its prevalence. METHODS: In this study, we developed a dual-target RAA-LFD assay for the rapid, visual detection of H. pylori genes (16s rRNA, ureA, vacA m1/m2), using recombinase aided amplification (RAA) combined with lateral flow dipstick (LFD) methods. Both 16s rRNA and ureA were selected as identification genes to ensure reliable detection accuracy. RESULTS: A RAA-LFD assay was developed to achieve dual-target amplification at a stable 37 °C within 20 min, followed by visualization using the lateral flow dipstick (LFD). The whole process, from amplification to results, took less than 30 min. The 95 % limit of detection (LOD) for 16 s rRNA and ureA, vacA m1, vacA m2 were determined as 3.8 × 10-2 ng/µL, 5.8 × 10-2 ng/µL and 1.4 × 10-2 ng/µL, respectively. No cross-reaction was observed in the detection of common pathogens including Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, showing the assay's high specificity. In the evaluation of the clinical performance of the RAA-LFD assay. A total of 44 gastric juice samples were analyzed, immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) were used as reference methods. The RAA-LFD results for the 16s rRNA and ureA genes showed complete agreement with qPCR findings, accurately identifying H. pylori infection as confirmed by IFS in 10 out of the 44 patients. Furthermore, the assay successfully genotyped vacA m1/m2 among the positive samples, showing complete agreement with qPCR results and achieving a kappa (κ) value of 1.00. CONCLUSION: The dual-target RAA-LFD assay developed in this study provides a rapid and reliable method for detecting and genotyping H. pylori within 30 min, minimizing dependency on sophisticated laboratory equipment and specialized personnel. Clinical validation confirms its efficacy as a promising tool for effectively control of its prevalence and aiding in the precise treatment of H. pylori-associated diseases.

Rapid and Sensitive On-site Nucleic Acid Detection of Three Main Fusarium Pathogens of Maize Stalk Rot Based on RPA-CRISPR/Cas12a.

Maize stalk rot is a soil-borne disease that poses a serious threat to maize production worldwide, with the most significant cause being fungal stalk rot. The development of a visual and rapid detection method for the maize stalk rot pathogen is significant for its prompt and accurate identification, enhancing agricultural production efficiency, and implementing timely preventive measures. These measures will help safeguard the maize yield and quality, ultimately reducing agricultural losses. In this study, we aimed to develop an efficient method to detect maize stalk rot pathogens. We focused on three pathogenic fungi commonly found in maize-producing regions worldwide: Fusarium verticillioides, Fusarium proliferatum, and Fusarium graminearum. Based on TEF-1α, we developed a rapid detection technique using RPA-CRISPR/Cas12a, combined with test strips to develop an on-site rapid visual detection test for these pathogens. The method showed detection sensitivity for F. verticillioides, F. proliferatum, and F. graminearum within 20 min at concentrations of 7.8 pg/µL, 0.11 ng/µL, and 0.13 ng/µL, respectively. The sensitivity increased with increasing reaction time. Testing of field disease samples indicated that the method is effective in detecting nucleic acids obtained through crude extraction methods. In conclusion, we developed a visually rapid detection technology that does not rely on complex instruments and equipment for the on-site early detection of F. verticillioides, F. proliferatum, and F. graminearum in the field to implement effective control measures, ensuring stable and high maize yields.

Orthogonal Serine Integrases Enable Scalable Gene Storage Cascades in Bacterial Genome.

Genome integration enables host organisms to stably carry heterologous DNA messages, introducing new genotypes and phenotypes for expanded applications. While several genome integration approaches have been reported, a scalable tool for DNA message storage within site-specific genome landing pads is still lacking. Here, we introduce an iterative genome integration method utilizing orthogonal serine integrases, enabling the stable storage of multiple heterologous genes in the chromosome of Escherichia coli MG1655. By leveraging serine integrases TP901-1, Bxb1, and PhiC31, along with engineered integration vectors, we demonstrate high-efficiency, marker-free integration of DNA fragments up to 13 kb in length. To further simplify the procedure, we then develop a streamlined integration method and showcase the system's versatility by constructing an engineered E. coli strain capable of storing and expressing multiple genes from diverse species. Additionally, we illustrate the potential utility of these engineered strains for synthetic biology applications, including in vivo and in vitro protein expression. Our work extends the application scope of serine integrases for scalable gene integration cascades, with implications for genome manipulation and gene storage applications in synthetic biology.

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration.

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson's disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how and if it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any dopaminergic neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute dopaminergic neurodegeneration.

Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa.

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/µL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

Rapid and equipment-free identification of papaya mealybug Paracoccus marginatus based on RPA-CRISPR/Cas12a.

BACKGROUND: Paracoccus marginatus has invaded many countries, spreading rapidly and causing significant economic losses to crops. Accurate detection during the monitoring process is critical to prevent its expansion into new areas, therefore it is necessary to develop efficient and reliable detection methods. Traditional detection methods are time-consuming and instrument-dependent owing to the morphological similarities and small sizes of P. marginatus and other mealybugs, therefore establishing an efficient, rapid, and sensitive method for field detection in resource-limited settings is critical. RESULTS: A sensitive and rapid detection system was developed to detect P. marginatus using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. The RPA-CRISPR/Cas12a assay distinguished P. marginatus from 10 other mealybugs. The entire process can be completed in approximately an hour, and the identification results can be determined by the naked eye using lateral flow strips or a portable mini-UV torch. A method was developed to extract DNA from P. marginatus within 5 min. This method was combined with the RPA-CRISPR/Cas12a assay to achieve rapid and simple detection. In addition, two portable thermos cups with temperature displays were used to maintain the reagents and assay reactions in the field. CONCLUSION: This assay represents the first application of portable and easily available items (mini-UV torch and thermos cup) based on the combination of RPA and CRISPR/Cas12a for rapid pest detection. This method is rapid, highly specific, and instrument-flexible, allowing for the early monitoring of P. marginatus in the field. This study provides guidance for the development of suitable management strategies. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Rapid detection of Impatiens necrotic spot virus from thrips vectors using reverse transcription-recombinase polymerase amplification.

The plant virus, Impatiens necrotic spot virus (INSV), is an economically important pathogen of vegetables, fruits, and ornamental crops. INSV is vectored by the western flower thrips, Frankliniella occidentalis, a small insect pest that is globally distributed. In recent years, INSV outbreaks have reached epidemic levels in the Salinas Valley of California-an agriculturally rich region where most of the lettuce (Lactuca sativa) is produced in the United States. Due to the obligate nature in which virus transmission occurs, new tools that could rapidly detect INSV from thrips vectors would enhance our ability to predict where virus outbreaks may occur. Here, we report on the development of a reverse transcription-recombinase polymerase amplification (RT-RPA) assay that can detect INSV from individual thrips. The assay uses crude extraction methods, is performed at a single temperature of 42 °C, can be completed in 25 min, and provides sensitivity levels that are comparable to other available detection methods. When the assay was used on field populations of thrips, INSV was successfully identified and quantified from individual larvae and adults. The work provides a new cost-effective surveillance tool that can rapidly detect INSV from its insect vector and from plants.

Rapid one-pot human single nucleotide polymorphism genotyping platform with Cas13a nuclease.

Single nucleotide polymorphism (SNP), as one of the key components of the genetic factors, is important for disease detection and early screening of hereditary diseases. Current SNP genotyping methods require laboratory instruments or long operating times. To facilitate the diagnosis of hereditary diseases, we developed a new method referred to as the LwaCas13a-based SNP genotyping platform (Cas13a platform), which is useful for detecting disease-related SNPs. We report a CRISPR/Cas13a-based SNP genotyping platform that couples recombinase-aided amplification (RAA), T7 transcription, and Leptotrichia wadei Cas13a (LwaCas13a) detection for simple and fast genotyping of human disease-related SNPs. We used this Cas13a platform to identify 17 disease-related SNPs, demonstrating that position 2 in gRNA is suitable for the introduction of additional mismatches to achieve high discrimination in genotyping across a wide range of SNP targets. The discrimination specificity of 17 SNPs was improved 3.0-35.1-fold after introducing additional mismatches at position 2 from the 5'-end. We developed a method, which has a lower risk of cross-contamination and operational complexity, for genotyping SNPs using human saliva samples in an one-pot testing that delivers results within 60 min. Compared to TaqMan probe qPCR, RFLP, AS-PCR and other SNP genotyping methods, the Cas13a platform is simple, rapid and reliable, expanding the applications of the CRISPR/Cas system in nucleic acid detection and SNP genotyping.

Rapid detection of avian leukemia virus using CRISPR/Cas13a based lateral flow dipstick.

Avian leukemia virus (ALV) is one of the main pathogens of poultry tumor diseases, and has caused significant economic losses to the poultry industry since its discovery. Therefore, establishing a rapid detection method is essential to effectively prevent and control the spread of ALV. In this study, specific CRISPR RNA (crRNA) and recombinase-aided amplification (RAA) primers with T7 promoter were designed based on the relatively conserved sequence of avian leukemia virus. When crRNA recognized the target sequence, Cas13a protein was activated to cut the reporting probes, and then the detection results were read by using lateral flow dipstick (LFD). The RAA-CRISPR/Cas13a-LFD reaction system was constructed. The RAA amplification time, Cas13a protein concentration, crRNA concentration and CRISPR reaction time were optimized to evaluate the specificity, sensitivity and reproducibility of the system. Finally, RAA-CRISPR/Cas13a-LFD method was compared with Polymerase chain reaction (PCR)-Agarose electrophoresis method and qPCR method in the detection of clinical samples, and the reliability of RAA-CRISPR/Cas13a-LFD method was evaluated. The results showed that the RAA-CRISPR/Cas13a-LFD method could effectively amplify the target gene at 37°C for 40 min, and the test results could be determined by LFD visual observation. The method had good specificity and no cross-reaction with Marek's disease virus (MDV), Fowl adenovirus (FAdV), Infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), Infectious laryngotracheitis virus (ILTV), and Infectious bronchitis virus (IBV). The minimum detection limit of the method was 100 copies/µL, and it had good repeatability and stability. The coincidence rate of clinical detection reached 97.69% and 99.23%. In summary, this study established a simple, efficient, accurate and visualized ALV detection method, which can be used for the prevention and rapid clinical diagnosis of avian leukosis (AL).

Optogenetic control of early embryos labeling using photoactivatable Cre recombinase 3.0.

Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light-mediated optical regulation of Cre-loxP recombination using PA-Cre3.0 transgenic early mouse pre-implantation embryos. We found that inducing PA-Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA-Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA-Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.

Multiplex reverse transcription recombinase polymerase amplification combined with lateral flow biosensor for simultaneous detection of three viral pathogens in cattle.

Bovine viral diarrhea virus (BVDV), bovine epidemic fever virus (BEFV), and bovine respiratory syncytial virus (BRSV) cause respiratory symptoms in cattle. The absence of rapid, precise, and easily accessible diagnostic methods poses difficulties for herders and veterinary epidemiologists during outbreaks of major infectious animal diseases. Considering the mixed infection of viruses, a multiple-detection method, reverse transcription recombinase polymerase amplification (mRT-RPA) combined with a lateral flow biosensor (LFB), was established to simultaneously detect the three pathogens. This technique is based on the specific binding of three differently labeled RT-RPA products (DNA sequences) to antibodies on the three test lines of the LFB, achieving multiplex detection through the presence or absence of coloration on the LFB test lines. The fluorescence values of the LFB test lines are recorded by a test strip reader. The mRT-RPA-LFB assay completes detection at a constant temperature of 41 °C within 33 min. The limits of detection (LODs) for BVDV, BEFV and BRSV were 2.62 × 101, 2.42 × 101 and 2.56 × 101 copies/µL, respectively. No cross-reactivity was observed with the other six bovine viruses. The developed method showed satisfactory intra- and inter-assay precision, and the average coefficients of variation were ranged from 2.92 % to 3.99 %. The diagnostic sensitivity and specificity were 98.11 % and 100 %, respectively, which were highly consistent with the RT-qPCR assay, and the kappa value was 0.988 (95 % confidence interval, CI). In general, the mRT-RPA-LFB assay has the potential to become a powerful tool for rapid screening of cattle diseases because of its advantages such as fast detection speed, convenient operation, strong specificity, and high sensitivity.

Rapid and visual detection of Mycoplasma genitalium using recombinase polymerase amplification combined with lateral flow strips.

Mycoplasma genitalium (MG) is an important sexually transmitted pathogen that can cause urethritis in males and pelvic inflammatory disease in females. Due to its complex growth requirements and lengthy incubation times, culturing MG in clinical laboratories is impractical. Here we describe a rapid and visual assay combining recombinase polymerase amplification (RPA) with lateral flow (LF) strips to detect MG (MG-RPA-LF). The limit of detection (LoD) of this method was 33.6 genome equivalents (GE) per reaction, using a dilution series of purified genomic DNA. Clinical performance was evaluated by testing 100 urogenital swabs. Compared to the Simultaneous Amplification and Testing assay, our MG-RPA-LF assay showed a sensitivity of 94 % (95 % CI, 82 %-98 %) and a specificity of 100 % (95 % CI, 91 %-100 %). The overall concordance between the two methods was 97 % (95 % CI, 91 %-99 %) with a κ coefficient of 0.94 (P < 0.001). Without cumbersome and expensive instruments, this method is anticipated to be a promising alternative to diagnose MG infection, especially in resource-poor settings.

Recent development in CRISPR-Cas systems for human protozoan diseases.

Protozoan parasitic diseases pose a substantial global health burden. Understanding the pathogenesis of these diseases is crucial for developing intervention strategies in the form of vaccine and drugs. Manipulating the parasite's genome is essential for gaining insights into its fundamental biology. Traditional genomic manipulation methods rely on stochastic homologous recombination events, which necessitates months of maintaining the cultured parasites under drug pressure to generate desired transgenics. The introduction of mega-nucleases (MNs), zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) greatly reduced the time required for obtaining a desired modification. However, there is a complexity associated with the design of these nucleases. CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) is the latest gene editing tool that provides an efficient and convenient method for precise genomic manipulations in protozoan parasites. In this chapter, we have elaborated various strategies that have been adopted for the use of CRISPR-Cas9 system in Plasmodium, Leishmania and Trypanosoma. We have also discussed various applications of CRISPR-Cas9 pertaining to understanding of the parasite biology, development of drug resistance mechanism, gene drive and diagnosis of the infection.

BEAM: A combinatorial recombinase toolbox for binary gene expression and mosaic genetic analysis.

We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.

An efficient AAV vector system of Rec2 serotype for intravenous injection to study metabolism in brown adipocytes in vivo.

OBJECTIVE: Recombinant adeno-associated virus (rAAV) vectors are powerful tools for the sustained expression of proteins in vivo and have been successfully used for mechanistic studies in mice. A major challenge associated with this method is to obtain tissue specificity and high expression levels without need of local virus administration. METHODS: To achieve this goal for brown adipose tissue (BAT), we developed a rAAV vector for intravenous bolus injection, which includes an expression cassette comprising an uncoupling protein-1 enhancer-promoter for transcription in brown adipocytes and miR122 target sequences for suppression of expression in the liver, combined with packaging in serotype Rec2 capsid protein. To test tissue specificity, we used a version of this vector expressing Cre recombinase to transduce mice with floxed alleles to knock out MLXIPL (ChREBP) or tdTomato-Cre reporter mice. RESULTS: We demonstrated efficient Cre-dependent recombination in interscapular BAT and variable effects in minor BAT depots, but little or no efficacy in white adipose tissues, liver and other organs. Direct overexpression of glucose transporter SLC2A1 (GLUT1) using the rAAV vector in wild type mice resulted in increased glucose uptake and glucose-dependent gene expression in BAT, indicating usefulness of this vector to increase the function even of abundant proteins. CONCLUSION: Taken together, we describe a novel brown adipocyte-specific rAAV method to express proteins for loss-of-function and gain-of-function metabolic studies. The approach will enable researchers to access brown fat swiftly, reduce animal breeding time and costs, as well as enable the creation of new transgenic mouse models combining multiple transgenes.

Establishment and methodological evaluation of a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes based on CRISPR-Cas12a technology.

PURPOSE: The opportunistic pathogens causing Cryptococcal meningitis are Cryptococcus neoformans and Cryptococcus gattii species complexes. At present, clinical detection methods for this condition include culture, ink staining, and cryptococcal antigen detection. In addition, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR) can be applied for the detection of Cryptococcus. Nevertheless, these methods cannot achieve point-of-care detection (POCT); thus, there is a pressing need to establish a fast, sensitive, and effective detection method. METHODS: Recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR) techniques are effective tools for achieving rapid POCT. In this study, RPA was combined with CRISPR-Cas12a to establish a fast, sensitive, and specific detection method for cryptococcal meningitis. RESULTS: This study included RPA-Cas12a fluorescence detection and RPA-Cas12a immunochromatographic detection, which can be performed within 50 min. Moreover, the detection limit was as low as 102 copies/µL. Interestingly, the developed method demonstrated satisfactory specificity and no cross-reactivity with other fungi and bacteria. 36 clinical samples were tested, and the consistency between the test results and those obtained using the commonly used clinical culture method was 100 %. CONCLUSION: In this study, a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes was developed based on CRISPR-Cas12a technology, characterized by its high sensitivity and specificity, ease of use, and cost-effectiveness, making it suitable for on-site detection.

Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.

A compact microfluidic platform for rapid multiplex detection of respiratory viruses via centrifugal polar-absorbance spectroscopy.

Nucleic acid detection technology has become a crucial tool in cutting-edge research within the life sciences and clinical diagnosis domains. Its significance is particularly highlighted during the respiratory virus pandemic, where nucleic acid testing plays a pivotal role in accurately detecting the virus. Isothermal amplification technologies have been developed and offer advantages such as rapidity, mild reaction conditions and excellent stability. Among these methods, recombinase polymerase amplification (RPA) has gained significant attention due to its simple primer design and resistance to multiple reaction inhibitors. However, the detection of RPA amplicons hinders the widespread adoption of this technology, leading to a research focus on cost-effective and convenient detection methods for RPA nucleic acid testing. In this study, we propose a novel computational absorption spectrum approach that utilizes the polar GelRed dye to efficiently detect RPA amplicons. By exploiting the asymmetry of GelRed molecules upon binding with DNA, polar electric dipoles are formed, leading to precipitate formation through centrifugal vibration and electrostatic interaction. The quantification of amplicon content is achieved by measuring the residual GelRed concentration in the supernatant. Our proposed portable and integrated microfluidic device successfully detected five respiratory virus genes simultaneously. The optimized linear detection was achieved and the sensitivity for all the targets reached 100 copies/µL. The total experiment could be finished in 27 min. The clinical experiments demonstrated the practicality and accuracy. This cost-effective and convenient detection scheme presents a promising biosensor for rapid virus detection, contributing to the advancement of RPA technology.

Development and application of a novel recombinase polymerase amplification-Pyrococcus furiosus argonaute system for rapid detection of goose parvovirus.

Rapid and accurate detection of goose parvovirus (GPV) is crucial for controlling outbreaks and mitigating their economic impact on the poultry industry. This study introduces recombinase polymerase amplification combined with the Pyrococcus furiosus argonaute (RPA-PfAgo) system, a novel diagnostic platform designed to address the limitations of traditional GPV detection methods. Capitalizing on the rapid DNA amplification of RPA and stringent nucleic acid cleavage by the PfAgo protein, the RPA-PfAgo system offers high specificity and sensitivity in detecting GPV. Our optimization efforts included primer and probe configurations, reaction parameters, and guided DNA selection, culminating in a detection threshold of 102 GPV DNA copies per microlitre. The specificity of the proposed method was rigorously validated against a spectrum of avian pathogens. Clinical application to lung tissues from GPV-infected geese yielded a detection concordance of 100%, surpassing that of qPCR and PCR in both rapidity and operational simplicity. The RPA-PfAgo system has emerged as a revolutionary diagnostic modality for managing this disease, as it is a promising rapid, economical, and onsite GPV detection method amenable to integration into broad-scale disease surveillance frameworks. Future explorations will extend the applicability of this method to diverse avian diseases and assess its field utility across various epidemiological landscapes.

RT-RPA Assay Combined with a Lateral Flow Strip to Detect Soybean Mosaic Virus.

Soybean (Glycine max L.) is one of the most widely planted and used legumes in the world, being used for food, animal feed products, and industrial production. The soybean mosaic virus (SMV) is the most prevalent virus infecting soybean plants. This study developed a diagnostic method for the rapid and sensitive detection of SMV using a reverse transcription-recombinase polymerase amplification (RT-RPA) technique combined with a lateral flow strip (LFS). The RT-RPA and RT-RPA-LFS conditions to detect the SMV were optimized using the selected primer set that amplified part of the VPg protein gene. The optimized reaction temperature for the RT-RPA primer and RT-RPA-LFS primer used in this study was 38℃ for both, and the minimum reaction time was 10 min and 5 min, respectively. The RT-RPA-LFS was as sensitive as RT-PCR to detect SMV with 10 pg/µl of total RNA. The reliability of the developed RT-RPA-LFS assay was evaluated using leaves collected from soybean fields. The RT-RPA-LFS diagnostic method developed in this study will be useful as a diagnostic method that can quickly and precisely detect SMV in the epidemiological investigation of SMV, in the selection process of SMV-resistant varieties, on local farms with limited resources.