Evaluation and comparison of one-step real-time PCR and one-step RT-LAMP methods for detection of SARS-CoV-2.

BACKGROUND: There is an increasing disease trend for SARS-COV-2, so need a quick and affordable diagnostic method. It should be highly accurate and save costs compared to other methods. The purpose of this research is to achieve these goals. METHODS: This study analyzed 342 samples using TaqMan One-Step RT-qPCR and fast One-Step RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification). The One-Step LAMP assay was conducted to assess the sensitivity and specificity. RESULTS: The research reported positive samples using two different methods. In the RT-LAMP method, saliva had 92 positive samples (26.9%) and 250 negative samples (73.09%) and nasopharynx had 94 positive samples (27.4%) and 248 negative samples (72.51%). In the RT-qPCR method, saliva had 86 positive samples (25.1%) and 256 negative samples (74.8%) and nasopharynx had 93 positive samples (27.1%) and 249 negative samples (72.8%). The agreement between the two tests in saliva and nasopharynx samples was 93% and 94% respectively, based on Cohen's kappa coefficient (κ) (P < 0.001). The rate of sensitivity in this technique was reported at a dilution of 1 × 101 and 100% specificity. CONCLUSIONS: Based on the results of the study the One-Step LAMP assay has multiple advantages. These include simplicity, cost-effectiveness, high sensitivity, and specificity. The One-Step LAMP assay shows promise as a diagnostic tool. It can help manage disease outbreaks, ensure prompt treatment, and safeguard public health by providing rapid, easy-to-use testing.

Discrepancies between self-reported medication in adherence and indirect measurement adherence among patients undergoing antiretroviral therapy: a systematic review.

BACKGROUND: Given the critical importance of medication adherence in HIV/AIDS treatment, this study aims to compare medication adherence measured by self-report (SR) and indirect measurement among antiretroviral therapy (ART) patients, exploring the differences of adherence results measured by different tools. METHODS: We systematically searched PubMed, Embase, and the Cochrane Library to identify all relevant literature published up to November 22, 2023, without language restrictions, reporting adherence to ART measured by both SR and indirect measurement methods, while also analyzing individual and group adherence separately. Discrepancies between SR and indirect measurement results were assessed using the Mann-Whitney U test or Wilcoxon signed-rank test, with correlations evaluated using the Pearson correlation coefficient. Following one-to-one comparisons, meta-epidemiological one-step analysis was conducted, and network meta-analysis techniques were applied to compare results obtained through specific adherence assessment tools reported in the identified articles. RESULTS: The analysis encompassed 65 original studies involving 13,667 HIV/AIDS patients, leading to 112 one-to-one comparisons between SR and indirect measurement tools. Statistically significant differences were observed between SR and indirect measurement tools regarding both individual and group adherence (P < 0.05), with Pearson correlation coefficients of 0.843 for individual adherence and 0.684 for group adherence. During meta-epidemiological one-step analysis, SR-measured adherence was determined to be 3.94% (95% CI: -4.48-13.44%) higher for individual adherence and 16.14% (95% CI: 0.81-18.84%) higher for group adherence compared to indirectly measured results. Subgroup analysis indicated that factors such as the year of reporting and geographic region appeared to influence the discrepancies between SR and indirect measurements. Furthermore, network meta-analysis revealed that for both individual and group adherence, the results obtained from most SR and indirect measurement tools were higher than those from electronic monitoring devices, with some demonstrating statistical significance (P < 0.05). CONCLUSIONS: The findings underscored the complexity of accurately measuring medication adherence among ART patients. Significant variability was observed across studies, with self-report methods showing a significant tendency towards overestimation. Year of reporting, geographic region, and adherence measurement tools appeared to influence the differences between SR and indirect measurements. Future research should focus on developing and validating integrated adherence measurements that can combine SR data with indirect measures to achieve a more comprehensive understanding of adherence behaviors.

One-step inverse generation network for sparse-view dual-energy CT reconstruction and material imaging.

OBJECTIVE: Sparse-view dual-energy spectral computed tomography (DECT) imaging is a challenging inverse problem. Due to the incompleteness of the collected data, the presence of streak artifacts can result in the degradation of reconstructed spectral images. The subsequent material decomposition task in DECT can further lead to the amplification of artifacts and noise. APPROACH: To address this problem, we propose a novel one-step inverse generation network (OIGN) for sparse-view dual- energy CT imaging, which can achieve simultaneous imaging of spectral images and materials. The entire OIGN consists of five sub-networks that form four modules, including the pre-reconstruction module, the pre-decomposition module, and the following residual filtering module and residual decomposition module. The residual feedback mechanism is introduced to synchronize the optimization of spectral CT images and materials. MAIN RESULTS: Numerical simulation experiments show that the OIGN has better performance on both reconstruction and material decomposition than other state-of-the-art spectral CT imaging algorithms. OIGN also demonstrates high imaging efficiency by completing two high-quality imaging tasks in just 50 seconds. Additionally, anti-noise testing is conducted to evaluate the robustness of OIGN. Significance. These findings have great potential in high-quality multi-task spectral CT imaging in clinical diagnosis.

A Facile One-Step Self-Assembly Strategy for Novel Carbon Dots Supramolecular Crystals with Ultralong Phosphorescence Controlled by NH4.

A new methodological design is proposed for carbon dots (CDs)-based crystallization-induced phosphorescence (CIP) materials via one-step self-assembled packaging controlled by NH4 +. O-phenylenediamine (o-PD) as a nitrogen/carbon source and the ammonium salts as oxidants are used to obtain CDs supramolecular crystals with a well-defined staircase-like morphology, pink fluorescence and ultralong green room-temperature phosphorescence (RTP) (733.56 ms) that is the first highest value for CDs-based CIP materials using pure nitrogen/carbon source by one-step packaging. Wherein, NH4 + and o-PD-derived oxidative polymers are prerequisites for self-assembled crystallization so as to receive the ultralong RTP. Density functional theory calculation indicates that NH4 + tends to anchor to the dimer on the surface state of CDs and guides CDs to cross-arrange in an X-type stacking mode, leading to the spatially separated frontier orbitals and the through-space charge transfer (TSCT) excited state in turn. Such a self-assembled mode contributes to both the small singlet-triplet energy gap (ΔEST) and the fast inter-system crossing (ISC) process that is directly related to ultralong RTP. This work not only proposes a new strategy to prepare CDs-based CIP materials in one step but also reveals the potential for the self-assembled behavior controlled by NH4 +.

Acoustofluidic one-step production of plasmonic Ag nanoparticles for portable paper-based ultrasensitive SERS detection of bactericides.

SERS measurements for monitoring bactericides in dairy products are highly desired for food safety problems. However, the complicated preparation process of SERS substrates greatly impedes the promotion of SERS. Here, we propose acoustofluidic one-step synthesis of Ag nanoparticles on paper substrates for SERS detection. Our method is economical, fast, simple, and eco-friendly. We adopted laser cutting to cut out appropriate paper shapes, and aldehydes were simultaneously produced at the cutting edge in the pyrolysis of cellulose by laser which were leveraged as the reducing reagent. In the synthesis, only 5 µL of Ag precursor was added to complete the reaction, and no reducing agent was used. Our recently developed acoustofluidic device was employed to intensely mix Ag+ ions and aldehydes and spread the reduced Ag nanoparticles over the substrate. The SERS substrate was fabricated in 1 step and 3 min. The standard R6G solution measurement demonstrated the excellent signal and prominent uniformity of the fabricated SERS substrates. SERS detection of the safe concentration of three bactericides, including tetracycline hydrochloride, thiabendazole, and malachite green, from food samples can be achieved using fabricated substrates. We take the least cost, time, reagents, and steps to fabricate the SERS substrate with satisfying performance. Our work has an extraodinary meaning for the green preparation and large-scale application of SERS.

A Single-Centre Retrospective Analysis of Pregnancies with Placenta Accreta Spectrum (PAS): From One-Step Surgery towards Two-Step Surgical Approach.

Background: Placenta accreta spectrum (PAS) can be the cause of major morbidity and its optimal management is still controversial. The aim of this study was to compare the traditional one-step surgery with a two-step surgical approach in which the placenta is left in situ and the second final operation is delayed to minimise blood loss. Methods: We conducted a single-centre retrospective cohort study including all patients managed for PAS between 2007 and 2023. The number of units of red blood cells (RBCs) needed during surgery was the primary outcome used to compare these two approaches. Results: A total of 43 cases were included in this analysis. Twenty of these were managed with the delayed two-step surgical approach, whereas 23 received one-step surgery. The median estimated blood loss during surgery was 2000 mL and 2800 mL for two-step and one-step surgery, respectively (p = 0.095). In the two-step surgical approach, the median number of RBC units transfused during surgery was significantly lower (p = 0.049) and the odds ratio for needing more than four units of RBCs was 0.28 (95%-CI: 0.08-0.98, p = 0.043). A longer interval between the caesarean section and the second operation showed a trend toward lower blood loss (p = 0.065) and was associated with a significantly lower number of RBC units needed during surgery (p = 0.019). Conclusions: Two-step surgery for the treatment of PAS was safe in our cohort and could lead to a reduction in blood transfusion. Leaving the placenta in situ and delaying the final operation represents a possible alternative to traditional caesarean hysterectomy.

Polyelectrolyte-coated liposomes microfluidically assembled in one-step for enhancing cell endocytosis and in-vivo immune responses.

To enhance the cellular uptake of liposomes, we prepared conventional liposomes with targeting molecules and surface-charged liposomes and evaluated their potential as nano-carriers and vaccine adjuvants by comparing their endocytosis efficiencies using immune cells. Surface-charged liposomes were synthesized via a one-step microfluidic method, which provided a novel, simple, fast, and highly reproducible method for preparing liposomes. Flow cytometry revealed that cationic polyelectrolyte-coated liposomes exhibited higher endocytosis efficiencies (of up to a factor of 100) in A774A.1 cells and JAWs II cells compared with uncoated liposomes or those coated with anionic polyelectrolytes. Positively charged liposomes exhibited some cytotoxicity at quaternary-chitosan coating concentrations higher than 6 mg/mL; however, significantly lower cytotoxicities (by a factor of almost ten) were obtained by protein mixing. Furthermore, BALB/c mice vaccinated with a mixture of Anthrax vaccine adsorbed (AVA) and quaternary chitosan-coated liposomes showed faster and stronger anti-PA IgG inductions compared to those vaccinated with AVA alone, with titers positively correlating with the amount of cationic liposome used. This finding clearly reveals that quaternary chitosan-coated liposomes act as both nano-carriers and vaccine adjuvants that significantly enhance in-vivo immune responses to vaccines with low immunogenicities.

Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics.

We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass spectrometry data acquisition. The Tecan Uno Single Cell Dispenser provides rapid cell isolation and nanoliter-volume reagent dispensing within 384-well PCR plates. A newly developed sample processing workflow achieves cell lysis, protein denaturation, and digestion in 1 h with a single reagent dispensing step. Low-flow liquid chromatography coupled with wide-window data-dependent acquisition results in the quantification of nearly 3000 proteins per cell using an Orbitrap Exploris 480 mass spectrometer. This approach greatly broadens accessibility to sensitive single-cell proteome profiling for nonspecialist laboratories.

Simple Solution Plasma Synthesis of Ni@NiO as High-Performance Anode Material For Lithium-ion Batteries Application.

The pursuit of straightforward and cost-effective methods for synthesizing high-performance anode materials for lithium-ion batteries is a topic of significant interest. This study elucidates a one-step synthesis approach for a conversion composite using glow discharge in a nickel formate solution, yielding a composite precursor comprising metallic nickel, nickel hydroxide, and basic nickel salts. Subsequent annealing of the precursor facilitated the formation of the Ni@NiO composite, exhibiting exceptional electrochemical properties as anode material in Li-ion batteries: a capacity of approximately 1000 mAh·g-1, cyclic stability exceeding 100 cycles, and favorable rate performance (200 mAh·g-1 at 10 A·g-1). Comparative analysis across various methods for synthesizing NiO-based materials underscored the superiority of the Ni@NiO composite. Furthermore, an assessment of resource costs demonstrated the cost-effectiveness and scalability of the approach in terms of resource consumption per Ah. Lastly, the integration of a Ni@NiO anode with an NMC532 cathode in a full battery highlights Ni@NiO's potential for conversion anodes, achieving a practical gravimetric energy density of 92 Wh kg-1.

Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs.

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics.

One-step spraying of protein-anchored chitosan oligosaccharide antimicrobial coating for food preservation.

The persistent global issues of unsafe food and food waste continue to exist. Microbial contamination stands out as a major cause of losses in perishable foods like vegetables and fruits. Herein, we report a self-assembling coating based on disulfide bond cleavage-induced bovine serum albumin (BSA), where the antimicrobial activity of chitosan oligosaccharide (COS) is stably anchored in the coating by electrostatic interactions during the unfolding-aggregation phase of BSA. The intrinsic antimicrobial activity of COS, combined with the positively charged and hydrophobic regions enriched on the BSA coating, significantly disrupts the integrity of bacterial structures. Furthermore, the BSA@COS coating can easily adhere in situ to the grooves on the surface of strawberries through a simple one-step spraying process, extending the shelf life of strawberries and bananas by nearly three times. This makes it a potential economic alternative to current commercial antimicrobial coatings, offering a solution to the rampant global issue of food waste.

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 3 - Scouting optimization strategies for separation of monoclonal antibodies by dual simultaneous independent gradients of pH & salt on a weak cation exchange stationary phase.

In previous publications we have described the pISep dual simultaneous, independent gradients (DSIGs) liquid chromatography (LC) for uncoupling gradients of non-buffering solute (NaCl, urea or acetonitrile) from externally generated pH gradients. In DSIGs the shape and slope of the [salute] gradient does not depend on the shape and slope of the pH gradient. The technique allows in a single run true simultaneous two dimensional LC separation of complex protein mixtures on various stationary phases including anion, cation exchangers (AEX, CEX), reversed phase (RP), mixed mode and mixed bed. Using a humanized IgG1 (HIgG1) monoclonal antibody (MAb) and a variety of pH & [NaCl] DSIGs, we show that most of MAb isoforms can be successfully separated from each other. These experimental observations are supported by an initial theoretical argument presented here predicting an overall improvement of all MAb isoforms separation by DSIGs of pH & [NaCl]. Theoretical calculations predict that, in general, there exists an optimal non-zero isocratic salt concentration in a pH gradient separation that will resolve isoforms close in binding energy, but a wide range of salt concentrations will be required for acceptable resolution of all isoforms. Theory also predicts better separation of weaker rather than stronger binding isoforms. Experimentally, we have found that no one set of DSIGs LC conditions could optimally baseline resolve all identifiable MAb isoforms in a single run of reasonable duration. The versatility and simplicity of the pH & [NaCl] pISep DSIGs LC allows fast, automated scouting of protein separations over any range of pH from 2.4 to 10.8 and [NaCl] from 0 to 1 M without changing the chemistry of the buffering system. Due to the universal applicability of the pISep buffering system in IEX LC, the researcher is given a powerful tool to easily develop pH & [NaCl] DSIGs protocols that vary mobile phase compositions to achieve high resolution separations of targeted proteins.

Purification of Recombinant N-terminal Histidine-Tagged Arabidopsis thaliana Phosphoglycolate Phosphatase 1, Glycolate Oxidase 1 and 2, and Hydroxypyruvate Reductase 1.

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni2+-NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD.

High methoxyl pectin grafted onto gallic acid by one- and two-pot redox-pair procedures.

The aim of this research was to graft gallic acid (GA) onto high methoxyl pectin (HMP) through the redox-pair of ascorbic acid (Aa) and hydrogen peroxide (H2O2) with one- and two-pot procedures. The effectiveness of the both procedures and the chemical, physical and antioxidant properties of the obtained HMP-GA were evaluated. HMP-GAone-pot (23.3 ± 0.21 mg GA Equivalent (GAE)/g) and HMP-GAtwo-pot (32.3 ± 0.52 mg GAE/g) were best obtained at H2O2/Aa molar ratio-HMP/GA weight ratio of 9.0-0.5 and 16.0-0.5, respectively. The UV-Vis and FT-IR spectra and along with their derivative and thermal gravimetric analyses, revealed differences between HMP-GAone-pot and HMP-GAtwo-pot. The latter exhibited a greater antioxidant capacity than the former in single electron transfer (ET), hydrogen atom transfer (HAT), and ET-HAT mixed assays. The chemical differences can be attributed to side reactions that may have interfered with the grafting reaction. Consequently, HMP-GA, possessing unique antioxidant and prebiotic properties, can be synthesized through redox-pair procedures.

One-Pot Synthesis of Alkyl Functionalized Reduced Graphene Oxide Nanocomposites as the Lubrication Additive Enabling Enhanced Tribological Performance.

Recently, aiming for the enhanced dispersibility of graphene-based nanomaterials in lubricating oil matrices to serve as highly efficient lubricant additives, numerous modification approaches have been extensively studied. However, these previous modification routes usually involve a tedious multistep modification process or multitudinous toxic reagents, restricting their extensive practical application. In this work, novel graphene oxide (GO) nanoadditives (RGO-g-BO) featuring excellent durable dispersion capability and remarkable tribological performance were successfully prepared via an environmentally friendly one-step approach consisting of surface grafting of long-chain bromooctadecane (BO) and in situ chemical reduction. Benefiting from the greatly improved lipophilicity (resulting from the introduction of hydrophobic long-chain alkane groups and chemical reduction), along with the miniaturization effect, RGO-g-BO exhibits superior long-term dispersion stability in the finished oil. Moreover, the tribological properties results demonstrated that the finished oil filled with RGO-g-BO nanolubricants achieved an outstanding friction-reducing and antiwear performance. Particularly, under the optimum content of RGO-g-BO (as low as 0.005 wt%), the friction coefficient as well as the wear volume of the composite finished oil were greatly reduced by 13% and 53%, respectively, as compared with nascent finished oil. Therefore, in view of the advantages of low-cost, one-step facile synthesis, desirable dispersion capability, and remarkable tribological performance, RGO-g-BO holds great prospects as a highly efficient lubrication additive in the tribology field.

Validation of one-step reverse transcription digital PCR assays for Norovirus GI.

Regular monitoring of Norovirus presence in environmental and food samples is crucial due to its high transmission rates and outbreak potential. For detecting Norovirus GI, reverse transcription qPCR method is commonly used, but its sensitivity can be affected by assay performance. This study shows significantly reduced assay performance in digital PCR or qPCR when using primers targeting Norovirus GI genome 5291-5319 (NC_001959), located on the hairpin of the predicted RNA structure. It is highly recommended to avoid this region in commercial kit development or diagnosis to minimizing potential risk of false negatives.

[Rapid determination of 15 N-nitrosamines in air-dried yak meat using one-step QuEChERS-gas chromatography-triple quadrupole mass spectrometry].

A method based on gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) coupled with one-step QuEChERS technique was developed for the simultaneous determination of 15 N-nitrosamines in air-dried yak meat. The hydration volume, extraction solvent, extracting salt, and cleaning material were optimized according to the characteristics of the N-nitrosamines and sample matrix. The optimized conditions were as follows: 10 mL of purified water for sample hydration, acetonitrile as the extraction solvent for the sample after hydration, 4.0 g of anhydrous MgSO4 and 1.0 g of NaCl as extracting salts, 500 mg of MgSO4+25 mg of C18+50 mg of PSA as cleaning materials. Favorable recoveries of the 15 N-nitrosamines were obtained when the extraction solution was incompletely dried. Thus, the final extract was dried to below 0.5 mL under a mild nitrogen stream and then redissolved to 0.5 mL with acetonitrile. After filtration, 200 µL of the sample was transferred to an autosampler vial for GC-MS/MS analysis. The 15 N-nitrosamines were determined using GC-MS/MS on a DB-HeavyWAX column (30 m×0.25 mm×0.25 µm) with an electron impact ion source in multiple-reaction monitoring (MRM) mode, and quantified using an external standard method. Under the optimized experimental conditions, the results showed that the calibration curves exhibited good linearities for the 15 N-nitrosamines, with correlation coefficients (r2) greater than 0.9990. The limits of detection (LODs) and the limits of quantification (LOQs) ranged from 0.05 to 0.20 µg/kg and from 0.10 to 0.50 µg/kg, respectively. At spiked levels of 1LOQ, 2LOQ, and 10LOQ, the average recoveries were 79.4%-102.1%, 80.6%-109.5%, and 83.0%-110.6%, respectively, and the relative standard deviations were in the range of 0.8%-16.0%. The low matrix effects of the 15 N-nitrosamines indicated the high sensitivity of the proposed method. The method was applied to detect representative commercial air-dried yak meat samples obtained using different processing techniques. Seven N-nitrosamines, including N-nitrosodimethylamine, N-nitrosodiisobutylamine, N-nitrosodibutylamine, N-methyl-N-phenylnitrous amide, N-ethyl-N-nitrosoaniline, N-nitrosopyrrolidine, and N-nitrosodiphenylamine were detected in all samples. The average contents of the seven N-nitrosamines was 0.08-20.18 µg/kg. The detection rates and average contents of the N-nitrosamines in cooked air-dried yak meat samples were higher than those in traditional raw air-dried yak meat samples. Compared with the manual QuEChERS method, the one-step QuEChERS method developed integrated the extraction and clean-up procedures into one single run, and the detection efficiency was considerably improved. The developed method is simple, rapid, highly sensitive, and insusceptible to human errors. Thus, it is useful for the determination of N-nitrosamines in air-dried yak meat and can be extended to the qualitative and quantitative analysis of N-nitrosamines in other meat products. It also provides method support and a data reference for the general determination of N-nitrosamines, which is of great significance for food safety.

One-step growth of Cu-doped carbon dots in amino-modified carbon nanotube-modified electrodes for sensitive electrochemical detection of BPA.

Copper-doped carbon dots and aminated carbon nanotubes (Cu-CDs/NH2-CNTs) nanocomposites were synthesized by a one-step growth method, and the composites were characterized for their performance. An electrochemical sensor for sensitive detection of bisphenol A (BPA) was developed for using Cu-CDs/NH2-CNTs nanocomposites modified with glassy carbon electrodes (GCE). The sensor exhibited an excellent electrochemical response to BPA in 0.2 M PBS (pH 7.0) under optimally selected conditions. The linear range of the sensor for BPA detection was 0.5-160 µM, and the detection limit (S/N = 3) was 0.13 µM. Moreover, the sensor has good interference immunity, stability and reproducibility. In addition, the feasibility of the practical application of the sensor was demonstrated by the detection of BPA in bottled drinking water and Liu Yang River water.

Optimized Antibiotic Resistance Genes Monitoring Scenarios Promote Sustainability of Urban Water Cycle.

Dissemination of antibiotic resistance genes (ARGs) in urban water bodies has become a significant environmental and health concern. Many approaches based on real-time quantitative PCR (qPCR) have been developed to offer rapid and highly specific detection of ARGs in water environments, but the complicated and time-consuming procedures have hindered their widespread use. Herein, we developed a facile one-step approach for rapid detection of ARGs by leveraging the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA). This efficient method matches the sensitivity and specificity of qPCR and requires no complex equipment. The results show a strong correlation between the prevalence of four ARG markers (ARGs: sul1, qnrA-1, mcr-1, and class 1 integrons: intl1) in tap water, human urine, farm wastewater, hospital wastewater, municipal wastewater treatment plants (WWTPs), and proximate natural aquatic ecosystems, indicating the circulation of ARGs within the urban water cycle. Through monitoring the ARG markers in 18 WWTPs in 9 cities across China during both peak and declining stages of the COVID epidemic, we found an increased detection frequency of mcr-1 and qnrA-1 in wastewater during peak periods. The ARG detection method developed in this work may offer a useful tool for promoting a sustainable urban water cycle.

The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission.

Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites' viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.