Template Concentration Research Articles

Protein-templated metal nanoclusters for chemical sensing

Metal nanoclusters (MNCs) possess unique optical properties, discrete energy levels, biocompatibility and photostability, making them pivotal photoluminescent probes in chemical sensing. While substantial work has addressed the synthesis, theoretical studies and applications of gold-, copper-, and silver-based MNCs, this review introduces fresh perspectives on how the nature and concentration of templates—particularly protein molecules—affect the optical properties, stability and sensing capabilities of MNCs. We delve into the merits of using protein templates for creating highly stable MNCs with tunable photoluminescence (PL), providing a detailed comparison with non-protein based systems. This review also unveils recent advancements in the photophysical characteristics and chemical sensing applications of protein-templated MNCs, setting it apart from previous reviews by focusing on cutting-edge innovations in template influence. Challenges and future prospects for protein-templated MNCs in chemical sensing are highlighted, marking critical pathways for upcoming research. This work not only integrates current knowledge but also identifies gaps and opportunities not covered extensively in earlier reviews, such as the nuanced effects of template variation on MNCs’ functional properties.

Enhancing efficiency and control in DNA hydrogel synthesis: A dual rolling circle amplification approach and parameter optimization study.

DNA hydrogels are a focus of current research in the realm of cutting-edge functional biomaterials. Rolling circle amplification (RCA) technology has surfaced as a flexible approach for producing functional DNA hydrogels in a one-pot manner, owing to its effectiveness and ease of use. This study introduces a simple dual RCA approach for producing DNA hydrogels. Our research delves into the impacts of different reaction parameters, such as reaction buffer, ethylenediaminetetraacetic acid (EDTA) concentration, base pair types and ratios, circular DNA template concentration, and RCA temperature, on the yield, morphology, and rheological characteristics of the resulting DNA hydrogels. Our findings demonstrate that DNA hydrogels prepared with TE buffer containing 1mM EDTA, higher concentrations of circular DNA templates, and an RCA reaction temperature of 30°C exhibited improved morphological characteristics and favorable mechanical properties. Furthermore, while G-C base pairs significantly enhance the mechanical properties of the hydrogel at lower concentrations, their excessive presence may negatively impact both the formation and mechanical integrity of the hydrogel. The findings from this study are crucial for boosting the efficiency and cost-effectiveness of DNA hydrogel synthesis, enhancing their quality, and broadening their range of applications.

Rational Design of a Novel DNA Polymerase From Clostridium thermocellum to Improve LAMP Detection Performance.

Loop-mediated isothermal amplification (LAMP) is a detection method widely used in pathogen detection and clinical diagnosis. Nevertheless, it is highly constrained by thermal stability, catalytic activity, and resistance to inhibitors of Bst DNA polymerase. In this study, a novel DNA polymerase was characterized from Clostridium thermocellum, exhibiting potential in LAMP detection. Through bioinformatics analysis, the enzyme and the DNA-binding domain (DBD) from Pyrococcus abyssi were mutated for enhanced interaction between proteins and DNA. A chimeric mutant DBDE146K-S738R reaches the detection threshold 13 min earlier than wild-type Cth DNA polymerase in real-time LAMP detection with a template concentration of 1.58 × 105 fg/µL. It also showed the highest enzymatic activity at pH 9.0 and 65°C. The chimeric enzyme DBDE146K-S738R exhibits good thermal stability, capable of performing LAMP reactions after treatment at 73°C or 70°C for 8 h. Moreover, it maintains high activity even under the inhibitory conditions of 50 U/mL heparin, 1.6mM EDTA, 200mM NaCl, 10% ethanol, 1.2M urea, or 0.8% phenol. Notably, it was able to detect 1.58 × 102 ag/µL of the genome and 1.03 CFU/mL of the colony in Salmonella typhimurium detection. The enzyme's performance is superior to commercial Bst 2.0 and comparable to commercial Bst 3.0. The results suggest that DBDE146K-S738R in LAMP exhibits great potential for molecular biological studies and clinical diagnostic analysis.

SAM-dPCR: Accurate and Generalist Nuclei Acid Quantification Leveraging the Zero-Shot Segment Anything Model.

Digital PCR (dPCR) has transformed nucleic acid diagnostics by enabling the absolute quantification of rare mutations and target sequences. However, traditional dPCR detection methods, such as those involving flow cytometry and fluorescence imaging, may face challenges due to high costs, complexity, limited accuracy, and slow processing speeds. In this study, SAM-dPCR is introduced, a training-free open-source bioanalysis paradigm that offers swift and precise absolute quantification of biological samples. SAM-dPCR leverages the robustness of the zero-shot Segment Anything Model (SAM) to achieve rapid processing times (<4 seconds) with an accuracy exceeding 97.10%. This method has been extensively validated across diverse samples and reactor morphologies, demonstrating its broad applicability. Utilizing standard laboratory fluorescence microscopes, SAM-dPCR can measure nucleic acid template concentrations ranging from 0.154 copies µL-1 to 1.295 × 103 copies µL-1 for droplet dPCR and 0.160 × 103 to 3.629 × 103 copies µL-1 for microwell dPCR. Experimental validation shows a strong linear relationship (r2 > 0.96) between expected and determined sample concentrations. SAM-dPCR offers high accuracy, accessibility, and the ability to address bioanalytical needs in resource-limited settings, as it does not rely on hand-crafted "ground truth" data.

Characterization and Application of Hydroxyapatite From Chicken Egg Shell with Green Template as a Potential Drug Delivery System

Hydroxyapatite (HAp) is a calcium phosphate biomaterial widely studied in the medical field because of its good biocompatibility properties. This study aims to identify the effect of increasing variations in banana peel template concentration on the characteristics of hydroxyapatite so that it has the potential as a drug delivery system. This study includes the characterization and application of the potential of the hydroxyapatite drug delivery system with the addition of variations in the concentration of green template. Then, hydroxyapatite was characterized using X-ray diffraction (XRD) and a surface area analyzer (SAA). This study's results indicate that adding a banana peel template affects the characteristics of hydroxyapatite. The higher the template concentration, the smaller the crystallite size, pore size, pore volume, and surface area, but the degree of crystallinity is greater. The results showed that the characterization of hydroxyapatite K15% with a crystal size of 34.73 nm, a degree of crystallinity of 89%, a pore size of 7.2178 nm, a surface area of 30.111 m2/g, and a pore volume of 0.1145 cc/g. The results of the study of the potential of the drug delivery system obtained a loading efficiency value of 67% for ibuprofen. However, the ibuprofen release process results at time intervals tend to be unstable.

Optimization of loop mediated isothermal amplification assay (LAMP) for detection of chloroquine resistance in P. vivax malaria

Chloroquine is still used as a first-line treatment for uncomplicated Plasmodium vivax malaria in India and resistance to this therapy can act as a major hurdle for malaria elimination. It is difficult to monitor drug-efficacy and drug resistance through in vivo and in vitro studies in case of Plasmodium vivax so analysis of molecular markers serves as an important tool to track resistance. Molecular methods that are currently in use for detecting single nucleotide polymorphisms in resistant genes including Polymerase chain reaction (PCR), Realtime-Polymerase chain reaction require highly sophisticated labs and are time consuming. So, with this background the study has been designed to optimize Loop Mediated Isothermal Amplification Assay to detect single nucleotide polymorphisms in chloroquine resistance gene of Plasmodium vivax in field settings. Eighty-eight Plasmodium vivax positive samples were collected. Pvmdr1 gene was amplified for all the samples and sequenced. Obtained sequences were analyzed for the presence of single nucleotide polymorphisms in the target gene. Further Loop Mediated Isothermal Amplification Assay primer sets were designed for the target mutants and the assay was optimized. Clinical as well as analytical sensitivity and specificity for the assay was calculated. Double mutants with variations at T958M and F1076L were detected in 100% of the Plasmodium vivax clinical isolates with haplotype M958 Y976 Y1028 L1076. Designed primers for Loop Mediated Isothermal Amplification Assay successfully detected both the mutants (T958M and F1076L) in 100% of the isolates and do not show cross-reactivity with other strains. So, the assay was 100% sensitive and specific for detecting single nucleotide polymorphisms in the target Pvmdr1 gene. Limit of detection was found to be 0.9 copies/µl and lowest DNA template concentration detected by designed assay was 1.5 ng/µL. Observed prevalence of single nucleotide polymorphisms in Pvmdr 1 gene is indicating a beginning of trend towards chloroquine resistance in Plasmodium vivax. The present study optimized LAMP for detecting single nucleotide polymorphisms in Plasmodium vivax cases in field settings, thus would help in finding significant hubs of emerging chloroquine drug resistance and ultimately helping in the management of suitable antimalarial drug policy.

Development and optimization of the qPCR-based rapid detection method for Chinese species of Sanghuangporus

Development and optimization of the qPCR-based rapid detection method for Chinese species of Sanghuangporus

Establishment of a Sensitive and Reliable Droplet Digital PCR Assay for the Detection of Bursaphelenchus xylophilus.

Pine wilt disease (PWD), which poses a significant risk to pine plantations across the globe, is caused by the pathogenic agent Bursaphelenchus xylophilus, also referred to as the pine wood nematode (PWN). A droplet digital PCR (ddPCR) assay was developed for the quick identification of the PWN in order to improve detection sensitivity. The research findings indicate that the ddPCR assay demonstrated significantly higher analysis sensitivity and detection sensitivity in comparison to traditional quantitative PCR (qPCR). However, it had a more limited dynamic range. High specificity was shown by both the ddPCR and qPCR techniques in the diagnosis of the PWN. Assessments of reproducibility revealed that ddPCR had lower coefficients of variation at every template concentration. Inhibition tests showed that ddPCR was less susceptible to inhibitors. There was a strong linear association between standard template measurements obtained using ddPCR and qPCR (Pearson correlation = 0.9317; p < 0.001). Likewise, there was strong agreement (Pearson correlation = 0.9348; p < 0.001) between ddPCR and qPCR measurements in the evaluation of pine wood samples. Additionally, wood samples from symptomatic (100% versus 86.67%) and asymptomatic (31.43% versus 2.9%) pine trees were diagnosed with greater detection rates using ddPCR. This study's conclusions highlight the advantages of the ddPCR assay over qPCR for the quantitative detection of the PWN. This method has a lot of potential for ecological research on PWD and use in quarantines.

Quantitative Characterization of RCA-Based DNA Hydrogels - Towards Rational Materials Design.

DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.

Selective solid phase extraction of folic acid from tomato as a biological source with a magnetic molecularly imprinted polymer measured by fluorescence spectroscopy

AbstractA novel magnetic molecular-imprinted polymer (MMIP) was used to selectively extract folic acid directly from real samples. Folic acid was used as template molecule, Fe3O4/SiO2-3-triethoxysilyl-propyl-acrylamide as functional monomer, azobisisobutyronitrile as initiation, ethylene glycol dimethacrylate as crosslinker agent, and acrylamide as the secondary monomer in a mixed ethanol-water solvent. The effect of different parameters on the extraction efficiency was studied, and the optimum conditions were established as follows: the concentrations of crosslinking and template were fixed at 0.05 and 0.06 g, absorption percentage was 96.5, pH was adjusted to 8, and extraction time was 8 h with a temperature of 25 °C. By examining the effect of pH, we tried to investigate the effect of the amide groups that present in MMIP and its intermolecular hydrogen interaction with folic acid. After optimising the effective parameters in polymer synthesis and adsorption rate, a magnetic imprinting dispersive solid-phase extraction method combined with fluorescence spectrophotometry at λem = 367 nm (MMIP-DSPE-FL) was constructed for sensitive determination of folic acid in tomato samples. The limit of quantification (LOQ) and limit of detection (LOD) values were 30.00 ± 0.01 μg L−1 and 10.00 ± 0.03 μg L−1, respectively, after the MMIP-DSPE preconcentration. Three tomato samples were analysed to give recoveries in the range of 80.2–81.6%, with relative standard deviation values below 0.6% (n = 3). The prepared MMIP-DSPE showed high selectivity toward folic acid, which could be used six times without changing adsorption capacity. The adsorption isotherm of the folic acid-imprinted polymer pursued the Langmuir model (RL = 0.029), and the kinetics model followed pseudo-first-order (R2 = 0.9974).

Evaluation of Genetic Diversity in Sorghum Genotypes via Simple Sequence Repeat (SSR) Markers

This study aimed to evaluate the genetic diversity of 20 sorghum [Sorghum bicolor (L.) Moench] genotypes via simple sequence repeat (SSR) markers, with a focus on identifying drought-tolerant varieties. Leaf samples were collected from three-week-old seedlings, and genomic DNA was extracted via the CTAB method, a widely recognized protocol for obtaining high-quality DNA. The DNA samples were quantified to ensure consistent template concentrations for amplification via 25 SSR primers. PCR amplification was performed, followed by agarose gel electrophoresis to separate and visualize the SSR band patterns, which were then recorded in a binary matrix format on the basis of the presence or absence of bands. Among the SSR primers used, nine were polymorphic, generating 13 scorable markers, highlighting the genetic variability among the genotypes. The polymorphic information content (PIC) values ranged across the SSR loci, with the Xtxp145 locus exhibiting the highest PIC value of 0.998, indicating its high discriminatory power and informativeness in differentiating between genotypes. Genetic similarity indices were calculated via Jaccard's similarity coefficient [1], and the data were subjected to cluster analysis via the unweighted pair group method with arithmetic mean (UPGMA) method. The resulting dendrogram grouped the genotypes into seven main clusters at a 50% similarity threshold, underscoring the genetic diversity present within the sorghum genotypes studied. Cluster I contained a single genotype, SVD-1272R, whereas, Cluster II included seven genotypes with subcluster formation. Cluster III comprised one ungrouped genotype, SPV-486. Cluster IV included eight genotypes, whereas Clusters V, VI, and VII each contained a single ungrouped genotype. This dendrogram illustrates the genetic diversity and relationships among the sorghum genotypes on the basis of similarity indices. The findings of this study confirmed the efficacy of SSR markers in assessing genetic diversity and emphasized their potential utility in breeding programs aimed at improving drought tolerance.

Selective detection of ketamine in human urine samples by electrochemiluminescence sensor based on conductive molecular imprinted polypyrrole: A hydrogen peroxide‐free electrochemiluminescence assay

AbstractA new sensor was fabricated for the determination of ketamine (Ket) in urine samples based on enhanced electrochemiluminescence (EECL) behavior of luminol on a carbon paste electrode (CPE) modified with the conductive molecularly imprinted polymer (CMIP) film. The FESEM technique was applied to demonstrate the rod shape morphology of CMIP. Unlike the common luminol system, EECL signal of luminol no need to addition of hydrogen peroxide and it might find application in efforts to avoid instability of hydrogen peroxide or its interference with the analytes of interest. The EECL emission has been optimized by an investigation of the applied potentials, electrochemical polymerization conditions, template and modifier concentrations, and the pH solution. Under the optimum conditions, the EECL signal depended linearly on the Ket concentration within a range from 0.1 to 1.25 μM with a limit of detection (LOD) of 0.08 μM. The sensor exhibited very good recovery percentages ranging from 92% to 101% in urine samples, while precisions were ≤3%. These results suggested that the proposed sensor would be applicable for analysis Ket in complex matrixes without interfering of other drugs (anesthetics) and thus has the potential to be used in clinical application.

Effect of DNA Template Concentration on Standard Polymerase Chain Reaction

The polymerase chain reaction (PCR) was an essential fundamental procedure widely used in molecular biology to amplify specific deoxyribonucleic acid (DNA) sequences. To investigate the efficiency and specificity of PCR using DNA extracted from human blood cells as sample, several concentrations of DNA templates were used to obtain accurate and reproducible results during this optimisation process. The principal objective was to identify which concentration was the best for amplifying the DNA target sequence and study the effects of the scale of concentrations to standard PCR. For this study, a series of PCR were conducted using various concentrations of DNA template ranging from 1 ng/μL to 100 ng/μL per reaction under optimized conditions of primer concentrations, annealing temperature and extension time. The results demonstrated that DNA template concentration significantly influenced the efficiency and specificity of PCR as the intensity of the target gene amplification band was increased on the agarose gel electrophoresis under ultraviolet light indicating the PCR product yield. Formation of primer-dimers became more prominent as the DNA template concentration reduced resulting in non-specific amplification. The concentrations ranging from 10 ng/μL to 70 ng/μL were the most optimal reactions profiting a remarkable feat of target gene amplification while concentrations less than 1 ng/μL produced none and non-specific outcomes retaining little amount of target gene amplification. In conclusion, our study highlights the pivotal role of DNA template concentration optimises PCR to increase reliability and reproducibility, extending our understanding of genetic analysis, diagnostic, and forensic sciences.

A phosphorylated guanidine chitosan and UiO-66-NH2 modified magnetic nanoparticle platform for enrichment and detection of multiple bacteria

Wastewater-based epidemiology (WBE) is a powerful tool for early warning of infectious disease outbreaks. Hence, a rapid and portable pathogen monitoring system is urgent needed for on-site detection. In this work, we first reported synthesis of an artificial modulated wide-spectrum bacteria capture nanoparticle (Arg-CSP@UiO@Fe3O4). Arginine-modified phosphorylated chitosan (Arg-CSP) coating could provide strongly positive charged guanidinium group for pathogen interaction by electrostatic attraction, and UiO-66-NH2 layer could help Arg-CSP graft onto Fe3O4 magnetic particles. The capture efficiency of Arg-CSP@UiO@Fe3O4 reached 92.2 % and 97.3 % for Escherichia coli (E.coli) and Staphylococcus epidermidis (S.epidermidis)within 40min, in 10mL sample. To prevent pathogen degradation in sewage, a portable nucleic acid extraction-free method was also developed. UiO-66-NH2 could disintegrate in buffer with high concentration of PO43- for bacterium desorption, and then nucleic acid of the bacteria was released by heating. The DNA template concentration obtained by this method was 779.28 times higher than that of the direct thermal lysis product and 8.63 times higher than that of the commercial kit. Afterwards, multiple detection of bacteria was realized by loop-mediated isothermal amplification (LAMP). Artificial regulated pathogen desorption could prevent non-specific adsorption of nucleic acid by nanoparticles. The detection limit of Arg-CSP@UiO@Fe3O4-LAMP method was 80cfu/mL for E.coli and 300cfu/mL for S.epidermidis. The accuracy and reliability of the method was validated by spiked sewage samples. In conclusion, this bio-monitoring system was able to detect multiple bacteria in environment conveniently and have good potential to become an alternative solution for rapid on-site pathogen detection.

Development of a CRISPR/Cas12a-based fluorescent detection method of Senecavirus A

BackgroundSenecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread.MethodsIn this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system.ResultsThe method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours.ConclusionsImportantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

Synthesizing hierarchically porous SAPO-11 with polyvinyl alcohol phosphates as mesopore template: effects of phosphorus content and concentration of templates

Synthesizing hierarchically porous SAPO-11 with polyvinyl alcohol phosphates as mesopore template: effects of phosphorus content and concentration of templates

Effects of synthesis conditions on particle size and pore size of spherical mesoporous silica

The particle size and pore size of spherical mesoporous silica materials play significant roles in their application. However, relatively limited systematic research has been conducted on how preparation conditions influence these properties. In particular, the effects of some important factors have not been adequately studied, including reaction time, reaction temperature, and organic solvent type. In this work, octane and water were used as solvents, and tetraethyl orthosilicate was used as the silicon source to systematically study the effects of reaction time, reaction temperature, different organic solvents, octane/water mass ratio, styrene template concentration, and surfactant (cetyltrimethylammonium bromide, CTAB)/H2O mass ratio on the particle morphology, particle size, and pore size of silica. The results suggest that the above-mentioned neglected factors exert a substantial influence on both particle size and pore size. In the experimental temperature range, the pore diameter decreases and the particle size increases with increasing temperature. The maximum particle size and pore size are achieved after a reaction time of 3 h, and a further increase in reaction time leads to a smaller particle size and pore size. As the number of carbon atoms in the organic solvent decreases, the pore size also gradually increases. Styrene and organic solvents that dissolve in CTAB micelles are crucial factors in pore formation, while the aggregation of the swollen CTAB micelles influences the particle size. The changes in the pore structure stability and hydroxyl density of the synthesized samples in water were also studied. After undergoing water treatment at temperatures ranging from 20 to 60 °C for 72 h, both the pore structure and morphology remain relatively unchanged. When the temperature increases, the surface hydroxyl density exhibits a more pronounced increase in the presence of water. After water treatment for 5 h, the surface hydroxyl density reaches saturation.

Digital droplet RT‐LAMP increases speed of SARS‐CoV‐2 viral RNA detection

AbstractNucleic acid amplification testing (NAAT) remains one of the most reliable methods for pathogen identification. However, conventional bulk NAATs may not be sufficiently fast or sensitive enough for the detection of clinically‐relevant pathogens in point‐of‐care testing. Here, we have developed a digital droplet RT‐LAMP (ddRT‐LAMP) assay that rapidly and quantitatively detects the SARS‐CoV‐2 viral E gene in microfluidic drops. Droplet partitioning using ddRT‐LAMP significantly accelerates detection times across a wide range of template concentrations compared to bulk RT‐LAMP assays. We discover that a reduction in droplet diameter decreases assay times up to a certain size, upon which surface adsorption of the RT‐LAMP polymerase reduces reaction efficiency. Optimization of drop size and polymerase concentration enables rapid, sensitive, and quantitative detection of the SARS‐CoV‐2 E gene in only 8 min. These results highlight the potential of ddRT‐LAMP assays as an excellent platform for quantitative point‐of‐care testing.

Development of Real-time PCR Method for Detection of Xanthomonas campestris pv. juglandis Causing Walnut Bacterial Blight

Walnut is one of the important economic tree species in the world. For the past few years, with the continuous expansion of walnut planting area, walnut bacterial blight caused by Xanthomonas campestrispv. juglandis has critically affected walnut production and caused economic losses. In this research, by comparing the gene sequences of X. campestrispv. juglandis and its closely related bacterial species, the 16S rDNA gene sequence was selected, and the specific primers were evaluated using PCR. Subsequently, the real-time fluorescence PCR specific detection method of X. campestrispv. juglandis was established and optimized. The specificity of real-time PCR was verified for 23 strains isolated from walnut and its surrounding soil, including eight target pathogens. The specific amplification was possible even when the cDNA template concentration was 7.0×10-5 µg/mL, hence, the detection sensitivity of the standard method is 7.0×10-5 µg/mL. The reliability of the established real-time PCR detection method in field detection was verified by the identification of target bacteria in 8 randomly collected healthy leaves and leaves of suspected walnut blight. This method laid a foundation for the early diagnosis of X. campestrispv. juglandis.

Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus

Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/μL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.