Ultraspecific One-Pot CRISPR-Based "Green-Yellow-Red" Multiplex Detection Strategy Integrated with Portable Cartridge for Point-of-Care Diagnosis.

Versatile, informative, sensitive, and specific nucleic acid detection plays a crucial role in point-of-care pathogen testing, genotyping, and disease monitoring. In this study, we present a novel one-pot Cas12b-based method coupled with the "Green-Yellow-Red" strategy for multiplex detection. By integrating RT-LAMP amplification and Cas12b cleavage in a single tube, the entire detection process can be completed within 1 h. Our proposed method exhibits high specificity, enabling the discrimination of single-base mutations with detection sensitivity approaching single molecule levels. Additionally, the fluorescent results can be directly observed by the naked eye or automatically analyzed using our custom-designed software Result Analyzer. To realize point-of-care detection, we developed a portable cartridge capable of both heating and fluorescence excitation. In a clinical evaluation involving 20 potentially SARS-CoV-2-infected samples, our method achieved a 100% positive detection rate when compared to standard RT-PCR. Furthermore, the identification of SARS-CoV-2 variants using our method yielded results that were consistent with the sequencing results. Notably, our proposed method demonstrates excellent transferability, allowing for the simultaneous detection of various pathogens and the identification of mutations as low as 0.5% amidst a high background interference. These findings highlight the tremendous potential of our developed method for molecular diagnostics.

Sustainable poultry practices: integrating green light interventions to control pecking in chicken.

BACKGROUND: The present study aimed to investigate the impact of the light-emitting diode (LED) green light alone or in combination with melatonin on pecking-related hormone regulation during incubation under normal and under hormonal stress conditions in breeder eggs. This study was divided into 2 experiments: In the first experiment effect of LED green light incubation on pecking-related hormones under normal conditions, on Hy-line brown (low pecking phenotype) and Roman pink (high pecking phenotype) eggs were tested. The 296 eggs of each strain were divided into two groups: LED green light incubation and dark incubation (control), each containing four replicates (37 eggs/replicate). The second experiment was conducted to evaluate the effect of LED green light incubation alone or in combination with melatonin under hormonal stress conditions on Roman pink eggs. A total of 704 Roman pink eggs were taken and divided into four groups, each consisting of 176 eggs. Each group was further divided into 2 subgroups, LED green light-regulated incubation and dark incubation with 88 eggs per subgroup, having 4 replicates of 22 eggs each. The groups were as follows: corticosterone solution injection (CI), corticosterone + melatonin mixed solution injection (CMI), Phosphate buffer solution injection (PI), and no injection (UI). RESULTS: Results of the first experiment revealed a higher level of serotonin hormone and lower corticosterone hormone in Hy-Line brown embryos compared to Roman pink embryos during dark incubation. The LED green light incubation significantly (P < 0.05) increased the level of 5-HT while decreasing the CORT level in Roman pink embryos indicating its regulatory effect on pecking-related hormones. Results of the second experiment showed that LED green light incubation significantly (P < 0.05) alleviated the CORT-induced hyperactivity of plasma 5-HT in Roman pink embryos. Furthermore, Melatonin (MLT) injection and LED green light together significantly (P < 0.05) reduced the hormonal stress caused by corticosterone injection in the eggs. CONCLUSIONS: Overall, the LED green light regulatory incubation demonstrated a regulatory effect on hormones that influence pecking habits. Additionally, when coupled with MLT injection, it synergistically mitigated hormonal stress in the embryos. So, LED green light incubation emerged as a novel method to reduce the damaging pecking habits of poultry birds.

Magnetically separable and recyclable bamboo-like carbon nanotube-based FRET assay for sensitive and selective detection of Hg2.

The global occurrence of toxic hazards in aquatic ecosystems has aroused concern about the potential impacts on the ecological environment and human health in recent decades. Mercury(II) ions that originate from widespread sources including the mining industry, fossil fuel consumption, and industrial wastes are now well known as a highly toxic pollutant. Despite various detection methods which have been reported to sense Hg2+, it still poses a great challenge for us to develop a new effective sensing platform to replenish current fluorescent detection techniques. Here, we report a novel fluorescent biosensor using bamboo-like magnetic carbon nanotubes (BMCNTs) and FAM-labeled T-rich ssDNA for efficient detection of Hg2+ in aqueous solution. The proposed biosensor shows a good response toward Hg2+ detection over a linear response range of 0.05~1 µM (R2 = 0.98) with a detection limit of 20 nM. It also exhibits the capability to discriminate Hg2+ ions with negligible response to other metal ions, such as Ca2+, Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+. Interestingly, the BMCNTs could be separated and recycled easily by using an external magnet, which means a much more cost-effective, easy-to-operate, and eco-friendly method for Hg2+ ion detection.

Design and Elementary Evaluation of a Highly-Automated Fluorescence-Based Instrument System for On-Site Detection of Food-Borne Pathogens.

A simple, highly-automated instrument system used for on-site detection of foodborne pathogens based on fluorescence was designed, fabricated, and preliminarily tested in this paper. A corresponding method has been proved effective in our previous studies. This system utilizes a light-emitting diode (LED) to excite fluorescent labels and a spectrometer to record the fluorescence signal from samples. A rotation stage for positioning and switching samples was innovatively designed for high-throughput detection, ten at most in one single run. We also developed software based on LabVIEW for data receiving, processing, and the control of the whole system. In the test of using a pure quantum dot (QD) solution as a standard sample, detection results from this home-made system were highly-relevant with that from a well-commercialized product and even slightly better reproducibility was found. And in the test of three typical kinds of food-borne pathogens, fluorescence signals recorded by this system are highly proportional to the variation of the sample concentration, with a satisfied limit of detection (LOD) (nearly 10²-10³ CFU·mL-1 in food samples). Additionally, this instrument system is low-cost and easy-to-use, showing a promising potential for on-site rapid detection of food-borne pathogens.

Beyond the Spectrum: Unleashing the Potential of Infrared Radiation in Poultry Industry Advancements.

The poultry industry is dynamically advancing production by focusing on nutrition, management practices, and technology to enhance productivity by improving feed conversion ratios, disease control, lighting management, and exploring antibiotic alternatives. Infrared (IR) radiation is utilized to improve the well-being of humans, animals, and poultry through various operations. IR radiation occurs via electromagnetic waves with wavelengths ranging from 760 to 10,000 nm. The biological applications of IR radiation are gaining significant attention and its utilization is expanding rapidly across multiple sectors. Various IR applications, such as IR heating, IR spectroscopy, IR thermography, IR beak trimming, and IR in computer vision, have proven to be beneficial in enhancing the well-being of humans, animals, and birds within mechanical systems. IR radiation offers a wide array of health benefits, including improved skin health, therapeutic effects, anticancer properties, wound healing capabilities, enhanced digestive and endothelial function, and improved mitochondrial function and gene expression. In the realm of poultry production, IR radiation has demonstrated numerous positive impacts, including enhanced growth performance, gut health, blood profiles, immunological response, food safety measures, economic advantages, the mitigation of hazardous gases, and improved heating systems. Despite the exceptional benefits of IR radiation, its applications in poultry production are still limited. This comprehensive review provides compelling evidence supporting the advantages of IR radiation and advocates for its wider adoption in poultry production practices.

New trends in impedimetric biosensors for the detection of foodborne pathogenic bacteria.

The development of a rapid, sensitive, specific method for the foodborne pathogenic bacteria detection is of great importance to ensure food safety and security. In recent years impedimetric biosensors which integrate biological recognition technology and impedance have gained widespread application in the field of bacteria detection. This paper presents an overview on the progress and application of impedimetric biosensors for detection of foodborne pathogenic bacteria, particularly the new trends in the past few years, including the new specific bio-recognition elements such as bacteriophage and lectin, the use of nanomaterials and microfluidics techniques. The applications of these new materials or techniques have provided unprecedented opportunities for the development of high-performance impedance bacteria biosensors. The significant developments of impedimetric biosensors for bacteria detection in the last five years have been reviewed according to the classification of with or without specific bio-recognition element. In addition, some microfluidics systems, which were used in the construction of impedimetric biosensors to improve analytical performance, are introduced in this review.

TriD-LAMP: A pump-free microfluidic chip for duplex droplet digital loop-mediated isothermal amplification analysis.

Digital nucleic acid amplification techniques are powerful and attractive approaches for providing sensitive and absolute quantification in biology. Among these, digital loop-mediated isothermal amplification (dLAMP) shows the potential for field detection, since its robustness and independence from thermal cycling. The key of dLAMP is to generate a large number of individual droplets or microwells. However, an auxiliary precision pump is always required for sample digitalization. In addition, current systems for droplet dLAMP usually need to transfer the droplets after digitalization or amplification. Herein, we developed and evaluated a pump-free microfluidic chip for duplex droplet dLAMP (TriD-LAMP) detection. This chip was designed based on step emulsification and contains a droplet generation zone and a droplet storage zone. Droplets are formed through the step due to the difference in Laplace pressure. There are 64 parallel nozzles that could generate tens of thousands of uniform droplets manually (variation <5%). The storage zone for droplets collection was previously filled with oil containing fluorosurfactant that keeps the droplets from fusing and evaporation during the amplification. Therefore, this custom chip is able to perform all stages of the dLAMP process without transferring droplets. Combined with the optimized fluorescent probe method, the chip achieves accurate quantification of the E. coli DNA down to 19.8 copies/µL. As a proof of concept, the simultaneous quantification of two targets was successfully realized on this custom chip. Conclusively, this integrated, pump-free TriD-LAMP chip can provide a promising tool for multiple targets detection in clinical diagnostics and point-of-care applications.

Subtractive inhibition assay for the detection of E. coli O157:H7 using surface plasmon resonance.

A surface plasmon resonance (SPR) immunosensor was developed for the detection of E. coli O157:H7 by means of a new subtractive inhibition assay. In the subtractive inhibition assay, E. coli O157:H7 cells and goat polyclonal antibodies for E. coli O157:H7 were incubated for a short of time, and then the E. coli O157:H7 cells which bound antibodies were removed by a stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using a BIAcore 3000 biosensor. The results showed that the signal was inversely correlated with the concentration of E. coli O157:H7 cells in a range from 3.0 × 10(4) to 3.0 × 10(8) cfu/mL with a detection limit of 3.0 × 10(4) cfu/mL. Compared with direct SPR by immobilizing antibodies on the chip surface to capture the bacterial cells and ELISA for E. coli O157:H7 (detection limit: both 3.0 × 10(5) cfu/mL in this paper), the detection limit of subtractive inhibition assay method was reduced by one order of magnitude. The method simplifies bacterial cell detection to protein-protein interaction, which has the potential for providing a practical alternative for the monitoring of E. coli O157:H7 and other pathogens.

[Rapid detection of Escherichia coli O157 : H7 using surface plasmon resonance (SPR) biosensor].

A biosensor based on surface plasmon resonance (SPR) was developed for rapid detection of Escherichia coli O157 : H7. BIACORE 3000 SPR instrument and a dextran-modified sensor chip (CM5) were used. After activation with EDC/NHS, anti-E. coli O157 : H7 antibody was immobilized on the gold surface of the SPR sensor, and then following ethanolamine was injected, and the chip was ready for E. coli O157 : H7. Regeneration was achieved using NaOH in order to detect several samples. The limit of detection was found to be 3 x 10(5) CFU x mL(-1) for E. coli O157 : H7, and the change of RU was linearly correlated with the concentration of E. coli O157 : H7 cells (R2 = 0.99). The detection time ranged from 5 to 7 min, and the result of regeneration was effective which allowed the chip to be reused for more than 50 samples. This method is convenient, and stable, and shows potentials for applications in food areas.

Two-dimensional nanocomposite-based electrochemical sensor for rapid determination of trans-resveratrol.

A two-dimensional nanocomposite-based disposable electrochemical sensor was fabricated for the rapid analysis of trans-resveratrol (TRA) in red wine. The sensor was prepared by modifying graphene-molybdenum disulfide (Gr-MoS2) nanocomposite on the surface of screen-printed electrode (SPE). Results show that the Gr-MoS2 nanocomposite with sheet-on-sheet structure can accelerate the oxidation reaction kinetics of TRA due to its large effective electrochemical surface area and high electron transfer rate. As a result, the Gr-MoS2 nanocomposite appears the synergistic effects, making the highly sensitive detection of TRA come true. The prepared sensor showed a linear response in TRA concentration from 1.0 to 200 µmol L-1 (with a limit of detection of 0.45 µmol L-1). After validating the accuracy with high performance liquid chromatography (HPLC), this nanocomposite-based electrochemical sensor can be applied for the detection of TRA in real red wine samples.

End-point dual specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor.

A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready@). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc.

Rapid on-site detection of genetically modified soybean products by real-time loop-mediated isothermal amplification coupled with a designed portable amplifier.

Herein, we developed a rapid method for detection of genetically modified soybean (GTS 40-3-2) products using loop-mediated isothermal amplification (LAMP). A crude 5-minute extraction method was established for DNA extraction from soybeans and soybean products. LAMP reaction for CaMV35S promoter was optimized and the fastest threshold time (Tt) was 14 min with 4 mM magnesium ions at 63 °C. A portable instrument was designed to perform real-time LAMP in the field. As little as 0.1% GM soybean, specifically, or 0.5% GM ingredients in Chinese traditional tofu could be detected in 30 min from sampling to results. We used this method to further assess other five soybean products to determine whether they contained transgenic ingredients and compared the results with those obtained using PCR, which suggested the proposed method was applicable for rapid detection of genetically modified soybean in food products.

Recent development of nano-materials used in DNA biosensors.

As knowledge of the structure and function of nucleic acid molecules has increased, sequence-specific DNA detection has gained increased importance. DNA biosensors based on nucleic acid hybridization have been actively developed because of their specificity, speed, portability, and low cost. Recently, there has been considerable interest in using nano-materials for DNA biosensors. Because of their high surface-to-volume ratios and excellent biological compatibilities, nano-materials could be used to increase the amount of DNA immobilization; moreover, DNA bound to nano-materials can maintain its biological activity. Alternatively, signal amplification by labeling a targeted analyte with nano-materials has also been reported for DNA biosensors in many papers. This review summarizes the applications of various nano-materials for DNA biosensors during past five years. We found that nano-materials of small sizes were advantageous as substrates for DNA attachment or as labels for signal amplification; and use of two or more types of nano-materials in the biosensors could improve their overall quality and to overcome the deficiencies of the individual nano-components. Most current DNA biosensors require the use of polymerase chain reaction (PCR) in their protocols. However, further development of nano-materials with smaller size and/or with improved biological and chemical properties would substantially enhance the accuracy, selectivity and sensitivity of DNA biosensors. Thus, DNA biosensors without PCR amplification may become a reality in the foreseeable future.

[Application of immunomagnetic separation to E. coli O157 : H7 detection].

In the present paper, the capture efficiencies of four different proportions of E. coli O157 : H7 and immunomagnetic beads (IMB) (1 : 6.34, 1 : 12.69, 1 : 25.38 and 1 : 30.07) and three different proportions of E. coli O157 : H7 and Bacillus subtilis (0.3 mL : 0.1 mL, 0.2 mL : 0.2 mL and 0.1 mL : 0.3 mL) were compared, with the total volume and capacity of IMB remaining constant. It was revealed that the capture efficiency could reach 100% when the proportion of E. coli O157 : H7 and IMB was 1 : 30, meaning that almost all targets E. coli O157 : H7 could be captured at 1 : 30, and that the capture efficiency decreased and then increased with the number of Bacillus subtilis increasing. Moreover, the combination of ATP bioluminescence and IMS was correlated well with conventional culture methods, R2 = 0.9882, and the detection limit was as low as 10(2) CFU x mL(-1).

[Application of near-infrared spectroscopy to detection of pesticide phoxim residues].

Near-infrared (NIR) spectroscopy technique was applied directly to the detection of pesticide phoxim residues. A sample pretreatment method was introduced. Samples were mixed with silica gel. Silica gel as a sorbent was employed to extract and enrich the low-concentration samples. Subsequently, diffuse reflection spectrum was measured on silica gel. Calibration models were developed using partial least square regression (PLSR) algorithm. Leave-one-out cross-validation was used to evaluate and compare the models. Two experiments were carried out, and the results show that 21 samples with the concentration gradient of 0.5 mg x L(-1) exhibited a high correlation coefficient of cross-validation of 0. 958, and a root mean square error of cross validation (RMSECV) of 0.872 mg x L(-1), while 41 samples with the concentration gradient of 0.25 mg x L(-1) gave a correlation coefficient of cross-validation of 0.924 and RMSECV of 1.15 mg x L(-1). It is indicated that with the reduction in concentration gradient, the prediction capacity of models dropped, but there still existed a high correlation coefficient with the concentration of phoxim in the samples. The experiments proved that the sample pretreatment method with the introduction of silica gel as an absorber to enrich low concentration analyte was effective. The method was able to lower the detection limit of NIR. The developed technique has a potential application in low-concentration sample detection by NIR spectroscopy, such as pesticide residues.

[Studies on detection of E. coli O157:H7 by ATP bioluminescence].

In the present paper, a quantitative linear model betweena series of concentrations of E. coliO157 : H7 and counts by BPCL ultra weak luminescence analyzer was built up. And the influences of four different buffers with the same pH (pH = 7.4), Tris-HCl, PBS, KH2PO4-NaOH and Na2 HPO4-C6H8O7, and five different chemical substances with the same mass concentration (10 g x L), NaCl, KCl, NaOH, MgCl2 and NaH2PO4 on ATP bioluminescence were compared. The results showed that Tris-HCl was a suitable buffer for dilution, since it could distinguish well between different concentrations and had the lowest background signals. And MgCl2 could intensify luminescence distinctly, while the other four chemical substances decreased luminescence, of which NaOH decreased luminescence most obviously. Moreover, ATP bioluminescence was correlated well with conventional culture methods (r = 0.96), and the detection limit was 10(3) cells x mL(-1).

Immunosensors for detection of pesticide residues.

Immunosensors are biosensors that use antibodies or antigens as the specific sensing element and provide concentration-dependent signals. There is great potential in the applications of immunosensing technologies for rapid detection of pesticide residues in food and environment. This paper presents an overview of various transduction systems, such as electrochemical, optical, piezoelectric, and nanomechanics methods, which have been reported in the literature in the design and fabrication of immunosensors for pesticide detection. Various immobilization protocols used for formation of a biorecognition interface are also discussed. In addition, techniques of regeneration, signal amplification, miniaturization, and antibodies are evaluated for the development and applications of these immunosensors. It can be concluded that despite some limitations of the immunosensing technologies, these immuosensors for pesticide monitoring are becoming more and more relevant in environmental and food analysis.

Field Detection of Citrus Huanglongbing Associated with ' Candidatus Liberibacter Asiaticus' by Recombinese Polymerase Amplification within 15 min.

' Candidatus Liberibacter asiaticus' (Las) is the most prevalent bacterium associated with huanglongbing, which is one of the most destructive diseases of citrus. In this paper, an extremely rapid and simple method for field detection of Las from leaf samples, based on recombinase polymerase amplification (RPA), is described. Three RPA primer pairs were designed and evaluated. RPA amplification was optimized so that it could be accomplished within 10 min. In combination with DNA crude extraction by a 50-fold dilution after 1 min of grinding in 0.5 M sodium hydroxide and visual detection via fluorescent DNA dye (positive samples display obvious green fluorescence while negative samples remain colorless), the whole detection process can be accomplished within 15 min. The sensitivity and specificity of this RPA-based method were evaluated and were proven to be equal to those of real-time PCR. The reliability of this method was also verified by analyzing field samples.

An anti-passivation ink for the preparation of electrodes for use in electrochemical immunoassays.

p-Nitrophenylphosphate (PNPP) is usually employed as the substrate for enzyme-linked immunosorbent assays. p-Nitrophenol (PNP), the product of PNPP, with the catalyst alkaline phosphatase (ALP), will passivate an electrode, which limits applications in electrochemical analysis. A novel anti-passivation ink used in the preparation of a graphene/ionic liquid/chitosan composited (rGO/IL/Chi) electrode is proposed to solve the problem. The anti-passivation electrode was fabricated by directly writing the graphene-ionic liquid-chitosan composite on a single-side conductive gold strip. A glassy carbon electrode, a screen-printed electrode, and a graphene-chitosan composite-modified screen-printed electrode were investigated for comparison. Scanning electron microscopy was used to characterize the surface structure of the four different electrodes and cyclic voltammetry was carried out to compare their performance. The results showed that the rGO/IL/Chi electrode had the best performance according to its low peak potential and large peak current. Amperometric responses of the different electrodes to PNP proved that only the rGO/IL/Chi electrode was capable of anti-passivation. The detection of cardiac troponin I was used as a test example for electrochemical immunoassay. Differential pulse voltammetry was performed to detect cardiac troponin I and obtain a calibration curve. The limit of detection was 0.05 ng/ml.

A fast and visual method for duplex shrimp pathogens detection with high specificity using rapid PCR and molecular beacon.

Molecular diagnosis of genome is one of the major methods for pathogens detection. The commonly used PCR method can realize an exponential amplification of the target gene but is time-consuming. In this work, we proposed a duplex and visual method using rapid PCR combined with molecular beacons to specifically detect two kinds of shrimp pathogens in one reaction tube. We only need to observe the fluorescence change of the reaction tube with naked eye to determine the result. A home-made automatic transfer equipment allows reaction tubes shuttling quickly between two water baths to achieve rapid PCR amplification. A simple device was also designed to present the detection results easily determined with naked eye. This duplex and visual detection method is fast, low-cost and of high specificity. From DNA extraction to results judgment, only 15 min was enough. Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Vibrio parahaemolyticus (VP) are two common shrimp pathogens which were chosen as our detection objects. This method may give a possibility to conduct end-point visual duplex detection, which may make a positive influence on the pathogen prevention.