Based on the differences in drug resistance characteristics between Candida glabrata and Candida krusei and the clinical need for rapid discrimination, this study established a duplex recombinase polymerase amplification-lateral flow strip (RPA-LFS) detection system using dual-labeled probes. By optimizing ITS2-targeted primers and probes (5′-end labeled with FITC/DIG, 3′-end labeled with biotin) and integrating dual test lines on the strip (streptavidin-T line for directional capture), simultaneous visual detection of both targets was achieved. Performance validation demonstrated: a detection limit of 10 copies/mL and 100 copies/mL, matching qPCR sensitivity; 100% detection rate for 30 target strains (including 12 reference and clinical isolates) with strict discrimination from 8 closely related pathogens (0% cross-reactivity); high concordance with qPCR in 328 clinical samples (sensitivity, specificity, and total concordance all 100%). The system delivers “sample-to-result” output within 30 min without complex instrumentation, providing technical support for precise point-of-care discrimination of drug-resistant Candida infections and rational antifungal drug use, particularly in primary healthcare settings and outbreak scenarios.

Candida tropicalis and Candida parapsilosis are two common non-albicans Candida species implicated in invasive candidiasis. They differ markedly in antifungal susceptibility—C. tropicalis often resists fluconazole, whereas C. parapsilosis shows reduced echinocandin susceptibility—highlighting the need for rapid, species-level identification to guide therapy. Traditional culture methods are slow, and molecular techniques usually require complex instrumentation, limiting their use in resource-limited settings. In this study, we established a duplex recombinase polymerase amplification combined with lateral flow strip (RPA-LFS) assay for the simultaneous visual detection of these two pathogens. Species-specific primers and probes were designed against the ITS2 region of C. tropicalis and the FKS2 gene of C. parapsilosis, with probes labeled at the 5′ end with FITC or DIG and reverse primers biotinylated. After optimizing reaction conditions (39 °C, 15 min), the assay achieved a limit of detection of 10 genomic equivalents/reaction for C. tropicalis and 100 genomic equivalents/reaction for C. parapsilosis. It showed 100% inclusivity across 30 target strains and 100% specificity against 14 non-target pathogens, including related Candida species and common bacteria. Clinical evaluation with 374 clinical samples (predominantly respiratory specimens) showed perfect agreement with quantitative real-time PCR, yielding 100% sensitivity (95% CI 97.8–100%), 100% specificity (95% CI 98.2–100%), and a Cohen’s kappa of 0.99 (95% CI 0.99–1.00). The entire procedure takes less than 30 min and requires no specialized equipment. This duplex RPA-LFS assay offers a rapid, accurate, diagnostic tool for the simultaneous detection of C. tropicalis and C. parapsilosis, well-suited for point-of-care testing in resource-limited environments and for supporting timely antifungal stewardship.

Carbapenem-Resistant Acinetobacter baumannii (CRAB) poses a major public health threat due to its multidrug resistance and high mortality rates, necessitating rapid and accurate diagnostic tools for timely intervention and infection control. This study developed and evaluated a novel detection method combining duplex recombinase polymerase amplification (DRPA) with a lateral flow strip (LFS) for the rapid visual identification of CRAB. The DRPA-LFS assay targets blaOXA−23 and blaOXA−51 genes, enabling isothermal amplification at 37 °C followed by a simple visual readout. The method demonstrated high sensitivity (102 CFU). Results can be obtained within 40 min without the need for complex instrumentation, making it suitable for resource-limited environments. the method offers a portable, user-friendly, and cost-effective solution for CRAB detection.

Pseudomonas aeruginosa (PA), which is a common Gram-negative bacterium, can become carbapenem-resistant Pseudomonas aeruginosa (CRPA) upon the development of antibiotic resistance, making clinical treatment difficult. CRPA with antibiotic resistance genes (ARGs), such as bla_NDM and bla_KPC, is common in China. The development of tests for ARGs would facilitate the more rapid identification of CRPA in China. Isothermal amplification research has improved, but limitations remain, including a lack of specialized equipment, the difficulty of developing sophisticated primers, and aerosol pollution. Thus, clinical examination methods must improve. We successfully integrated RPA with CRISPR/Cas12a, and we identified bla_NDM as our institution’s primary CRPA. RPA-CRISPR/Cas12a could accurately detect target DNA within 40 min without cross-reacting with other bacteria. The results showed high concordance with conventional culture-based methods, including 100

Candida parapsilosis is a common cause of candidiasis among hospitalized patients, often surpassing Candida albicans. Due to the recent increase in C. parapsilosis infections, there is an urgent need for rapid, sensitive, and real-time on-site detection of nucleic acids for timely diagnosis of candidiasis. We developed an assay for detection of C. parapsilosis by combining recombinase polymerase amplification (RPA) with a lateral flow strip (LFS). The RPA-LFS assay was used to amplify the beta-1,3-glucan synthase catalytic subunit 2 (FKS2) gene of C. parapsilosis with a primer–probe set optimized by introducing base mismatches (four bases modified by the probe and one by the reverse primer) to achieve specific and sensitive detection of clinical samples. The RPA assays can rapidly amplify and visualize a target gene within 30 min, while the entire process can be completed within 40 min by pre-processing the sample. The product of RPA has two chemical labels, FITC and Biotin, of the amplification product can be carefully on the strip. The sensitivity and specificity of the RPA-LFS assay were determined by analysis of 35 common clinical pathogens and 281 clinical samples against quantitative PCR. The results confirmed that the proposed RPA-LFS assay is a reliable molecular diagnostic method for the detection of C. parapsilosis to meet the urgent need for rapid, specific, sensitive, and portable field testing.

Abstract Background Candida parapsilosis is a common cause of candidiasis among hospitalized patients, often surpassing Candida albicans. Due to the recent increase in C. parapsilosis infections, there is an urgent need for rapid, sensitive, and real-time on-site detection of nucleic acids for timely diagnosis of candidiasis. Methods We developed an assay for detection of C. parapsilosis by combining recombinase polymerase amplification (RPA) with a lateral flow strip (LFS). The RPA-LFS assay was used to amplify the beta-1,3-glucan synthase catalytic subunit 2 (FSK2) gene of C. parapsilosis with a primer-probe set optimized by introducing base mismatches (4 bases modified by the probe and one by the reverse primer) to achieve specific and sensitive detection of clinical samples. Results The RPA assays can rapidly amplify and visualize a target gene within 30 min, while the entire process can be completed within 40 min by pre-processing the sample.The sensitivity and specificity of the RPA-LFS assay were determined by analysis of 35 common clinical pathogens and 281 clinical samples against quantitative PCR. Conclusions The results confirmed that the proposed RPA-LFS assay is a reliable molecular diagnostic method for the detection of C. parapsilosis to meet the urgent need for rapid, specific, sensitive, and portable field testing.

Streptococcus pneumoniae is a major pathogen that causes microbiological illness in humans. The introduction of polyvalent vaccines has resulted in a significant decrease in pneumococcal-related mortality. However, pneumococcal infections continue to be a leading cause of death in children under the age of 5 and adults over the age of 65 worldwide. A speedy and highly sensitive diagnostic tool is necessary for routine adoption to adequately manage patients and control the spread of infection. In this study, we investigated a new nucleic acid amplification technique, isothermal recombinase polymerase amplification (RPA), which amplifies DNA at 37°C under isothermal conditions with high specificity, efficiency, and rapidity. Using the autolysin gene lytA as the molecular diagnostic target, an RPA primer-probe combination was designed and optimized for the detection of S. pneumoniae. This RPA reaction produced amplification products labeled with specific chemical markers, to be detected with gold-nanoparticle-based lateral flow strips (LFS), reducing the reliance on equipment and trained personnel. The high specificity of the RPA-LFS technique was demonstrated with the specific detection of 22 strains of S. pneumoniae but not 25 closely related pathogenic bacteria. The assay showed good sensitivity, and detected S. pneumoniae down to 3.32 colony-forming units/μL. When used on clinical samples, the assay provided accurate and consistent results compared with PCR. The compliance with the culture-biochemistry method was 98.18% and the kappa index was 0.977. These results reveal that the RPA–LFS test significantly improved S. pneumoniae identification, particularly in resource-limited areas.

Candida glabrata is the second or third most common Candida-associated species isolated from hospital-acquired infections, surpassing even C. albicans in some hospitals. With the rapid progression of the disease course of C. glabrata infections, there is an urgent need for a rapid and sensitive on-site assay for clinical diagnosis. Isothermal amplification is a recently developed method for rapid nucleic acid detection that is being increasingly used for on-site detection, especially recombinase polymerase amplification (RPA). RPA combined with lateral flow strips (LFS) can rapidly amplify and visually detect the target gene within 20 min. The whole detection process can be controlled within 30–60 min by rapid sample pre-treatment. In this study, RPA-LFS was used to amplify the internal transcribed spacer region 2 gene of C. glabrata. The primer–probe design was optimized by introducing base mismatches (probe modification of one base) to obtain a highly specific and sensitive primer–probe combination for clinical sample detection. RPA-LFS was performed on 23 common clinical pathogens to determine the specificity of the assay system. The RPA-LFS system specifically detected C. glabrata without cross-reaction with other fungi or bacteria. Gradient dilutions of the template were tested to explore the lower limit of detection of this detection system and to determine the sensitivity of the assay. The sensitivity was 10 CFU/µL, without interference from genomic DNA of other species. The RPA-LFS and qPCR assays were performed on 227 clinical samples to evaluate the detection performance of the RPA-LFS system. Eighty-five samples were identified as C. glabrata, representing a detection rate of 37.5%. The results were consistent with qPCR and conventional culture methods. The collective findings indicate a reliable molecular diagnostic method for the detection of C. glabrata, and to meet the urgent need for rapid, specific, sensitive, and portable clinical field-testing.

Acinetobacter baumannii is a worldwide, primary cause of respiratory tract infections, septicemia, urinary apparatus infections, and secondary meningitis. It can be fatal. Rapid and accurate detection methods are needed to control the spread of carbapenem-resistant A. baumannii (CRAB). Current molecular diagnostic methods are limited and not suitable for on-site detection. In this study, an isothermal detection method using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) was developed to target the bla OXA-51 and bla OXA-23 genes of A . baumannii . The reaction was completed in about 40 min at 37°C. This method can also effectively distinguish A. baumannii and CRAB. The limit of detection of 10 0 -10 1 CFU/reaction was equal to that of other detection methods. The detection accuracy was equal to that of the qPCR method with the use of clinical samples. The RPA-LFS assay is portable, rapid, and accurate and could replace existing detection methods for on-site detection of A . baumannii and CRAB.

Bacillus cereus is widely distributed in foods, especially dairy products, and can lead to diarrhea (non-emetic B. cereus) and emesis (emetic B. cereus). Although diarrhea due to B. cereus is usually mild, emesis can lead to acute encephalopathy and even death. To develop rapid and sensitive detection methods for B. cereus in foods, specific primers targeting the gyrase B (gyrB) and cereulide synthetase (ces) genes were designed and screened using recombinase polymerase amplification (RPA). Probes and base substitutions were introduced to improve specificity and eliminate primer-dependent artifacts. The 5′ ends of the reverse primers and probes were modified with biotin and fluorescein isothiocyanate for detection of RPA products on a lateral flow strip (LFS). The developed RPA-LFS assay allows detection within 20 min at 37°C with no cross-reactivity with other foodborne pathogens. The limit of detection was 104 copies/ml and 102 CFU/ml in pure cultures and milk, respectively. Comparisons with established methods using cream obtained similar results. A specific, rapid, and sensitive RPA-LFS assay was successfully developed for on-site detection of B. cereus in dairy products to distinguish emetic from non-emetic strains.

As the use of pesticides increases year after year, so does the level of residual pesticides in the aquatic environment, posing a serious threat to non-target organisms. Difenoconazole (DFZ), a class of long-lasting fungicides and residues in the marine environment, has been shown to cause damaging effects on different organs of aquatic organisms. However, there is no research on the damage of DFZ to carp spleen tissue. This study aimed to investigate the acute toxic effects of DFZ on the spleen tissue of carp (Cyprinus carpio) by exposing juvenile carp to environmentally relevant concentrations of DFZ. We randomly selected 30 carp, divided them into the Control, Low, and High groups, and then exposed the three groups to 0, 0.488 mg/L DFZ, and 1.953 mg/L DFZ for 96 h respectively. We then investigated the toxic effects caused by DFZ on carp and spleen tissues by detecting changes in spleen histopathologic damage, apoptosis, oxidative stress, inflammation, and blood biochemical parameters. We found that DFZ causes severe histopathology in spleen tissue, including ballooning, structural relaxation, and giant mitochondria. In addition, we found that DFZ caused excessive apoptosis in spleen tissue by TUNEL staining and expression levels of apoptosis-related genes (caspase3, caspase8, caspase9, fas, bax, bcl-2, and p53). The activities and transcript levels of the antioxidant enzymes SOD, CAT, and GSH-Px were significantly down-regulated. In addition, DFZ led to a significant increase in activation of the NF-κB signaling pathway and mRNA levels of pro-inflammatory cytokines il-6, il-1β, and tnf-α, and a substantial decrease in mRNA levels of anti-inflammatory cytokines il-10 and tgf-β1 in spleen tissue. Blood biochemical parameters showed that DFZ exposure significantly reduced erythrocyte, leukocyte, hemoglobin, C3, and IgM levels. Collectively, DFZ exposure induced apoptosis, immunosuppression, oxidative stress, and inflammatory responses in the spleen tissue of carp, resulting in spleen tissue damage.

Candida tropicalis is one of the few Candida species besides Candida albicans that is able to produce true hyphae. At present, the commonly used clinical methods for the identification of this organism are traditional fungal culture, CTB staining, and color development. Polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) are also used to identify this fungus. Since the course of C . tropicalis infection progresses rapidly, there is an urgent need for rapid, sensitive, real-time field assays to meet the needs of clinical diagnosis. Recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) can rapidly amplify and visualize target genes within 20 min, and by pre-processing samples from different sources, the entire process can be controlled within 30 min. In this study, RPA-LFS was used to amplify the internal transcribed spacer-2 ( ITS2 ) gene of C . tropicalis , and primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical sample detection. LFS assay for 37 common clinical pathogens was performed, sensitivity and specificity of the detection system was determined, reaction temperature and time were optimized, and 191 actual clinical samples collected from different sources were tested to evaluate the detection performance of the established RPA-LFS system to provide a reliable molecular diagnostic method for the detection of C . tropicalis , the results show that the RPA-LFS system can specifically detect C. tropicalis without cross-reacting with other fungi or bacterial, with a sensitivity of 9.94 CFU/µL, without interference from genomic DNA of other species, at an optimal reaction temperature of 39°C, and the whole reaction process can be controlled within 20 min, and to meet the clinical need for rapid, sensitive, real-time, and portable field testing.

The incidence of Candida infections in intensive care units (ICU) has significantly increased in recent years, and these infections have become one of the most serious complications threatening the lives of ICU patients. The proportion of non-Candida albicans infections, such as Candida krusei and Candida glabrata infections, which are resistant to fluconazole, is increasing each year. Early identification of the strains causing Candida infections is important for the timely implementation of targeted treatments to save patients’ lives. However, the current methods of direct microscopy, culture, and histopathology, as well as other diagnostic methods, have many shortcomings, such as their low sensitivity and long assay times; therefore, they cannot meet the needs for early clinical diagnosis. Recombinant polymerase amplification (RPA) is a promising isothermal amplification technique that can be performed without sophisticated instruments and equipment, and is suitable for use in resource-poor areas. RPA combined with lateral flow strips (LFS) can be used to rapidly amplify and visualize target genes within 20 min. In this study, RPA-LFS was used to amplify the internal transcribed spacer 2 (ITS2) region of C. krusei. The primer-probe design was optimized by introduction of base mismatches (probe modification of five bases) to obtain a specific and sensitive primer-probe combination for the detection of clinical specimens. Thirty-five common clinical pathogens were tested with RPA-LFS to determine the specificity of the detection system. The RPA-LFS system specifically detected C. krusei without cross-reaction with other fungi or bacteria. A gradient dilution of the template was tested to explore the lower limit of detection and sensitivity of the assay. The sensitivity was 10 CFU/50 µL per reaction, without interference from genomic DNA of other species. The RPA-LFS and qPCR assays were performed on 189 clinical specimens to evaluate the detection performance of the RPA-LFS system. Seventy-six specimens were identified as C. krusei, indicating a detection rate of 40.2%. The results were consistent with those of qPCR and conventional culture methods. The RPA-LFS system established in our study provides a reliable molecular diagnostic method for the detection of C. krusei, thus meeting the urgent need for rapid, specific, sensitive, and portable clinical field testing.

Cryptococcus neoformans (C. neoformans)/C. gattii can easily invade the human central nervous system and cause cryptococcal meningitis (CM). The clinical fatality rate of these fungi is extremely high and causes more than 180,000 deaths worldwide every year. At present, the common clinical identification methods of these fungi are traditional culture methods and Indian ink staining. In addition, enzyme-linked immunosorbent assay (ELISAs), polymerase chain reaction (PCR), real-time quantitative PCR detecting system (qPCR), mass spectrometry, and metagenomic next-generation sequencing (mNGS) have also been applied to detect these fungus. Due to the rapid progress of meningitis caused by C. neoformans/C. gattii infection, there is a desperate need for fast, sensitive, and on-site detection methods to meet the clinical diagnosis. Recombinase polymerase amplification (RPA) is a promising isothermal amplification technique that can compensate for the shortcomings of the above techniques, featuring short reaction time, high specificity, and high sensitivity, thus meeting the demand for in-field detection of C.neoformans/C. gattii. In our study, RPA- lateral flow strip (LFS) was used to amplify the capsule-associated gene, CAP64, of C. neoformans/C. gattii, and the primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical testing, and specificity of the detection system was determined for 26 common clinical pathogens. This system was developed to obtain results in 20 min at an isothermal temperature of 37°C with a lower limit of detection as low as 10 CFU/μL or 1 fg/μL. A total of 487 clinical samples collected from multicenter multiplexes were tested to evaluate the detection performance of the RPA-LFS system, which revealed that the system could specifically detect C. neoformans/C. gattii, meeting the need for rapid, specific, and sensitive detection.

Enterococcus faecalis is a serious problem for hospitals and can spread from patient to patient. Most of the current detection methods are associated with limitations associated with the need for trained personnel; they are also time-consuming. Thus, it is necessary to develop rapid and accurate detection methods to control the spread of E. faecalis. In this study, we developed a rapid and accurate detection method for E. faecalis using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS). This method could be completed in approximately 35 min at 37°C. The limit of detection was 10 CFU/µL, irrespective of whether the templates were pure or complex. This method also showed good specificity and compatibility. In total, 278 clinical samples were tested using the RPA-LFS method; the detection accuracy was equal to that of the conventional qPCR method. This visualized isothermal amplification method could be useful for the future on-site detection of E. faecalis.

Drinking culture has high significance in both China and the world, whether in the entertainment sector or in social occasions; according to the World Health Organization's 2018 Global Alcohol and Health Report, about 3 million people died from excessive drinking in 2016, accounting for 5.3% of the total global deaths that year. Oxidative stress and inflammation are the most common pathological phenomena caused by alcohol abuse (Snyder et al., 2017). Scutellarin, a kind of flavonoid, is one of the main active ingredients extracted from breviscapine. It exerts anti-inflammatory, antioxidant, and vasodilation effects, and has been used to treat cardiovascular diseases and alcoholic liver injury. Although scutellarin can effectively alleviate multi-target organ injury induced by different forms of stimulation, its protective effect on alcoholic brain injury has not been well-defined. Therefore, the present study established an acute alcohol mice brain injury model to explore the effect of scutellarin on acute alcoholic brain injury. The study was carried out based on the targets of oxidative stress and inflammation, which is of great significance for the targeted therapy of clinical alcohol diseases.

Based on the characteristics of low investment and quick effect, co-branding has been widely applied in the marketing circle, but it still faces enormous challenges. The traditional joint model with high asset and low popularity cannot keep pace with the increasingly diversified social environment. The two parties of the co-branding need to transform and upgrade the traditional brand association, stabilize the combination of high-asset brands and lowpopularity brands, improve consumer's evaluation, and expand the influence of the association. Keywords—co-branding; consumer's evaluation; influence

Objective To study the expression of Notch signaling pathway-related microRNAs in T cells of collagen-induced arthritis (CIA) mice. Methods First, the mice model was established with bovine collagen type II and immune adjuvant DBA/1 J mice. Next, peripheral blood mononuclear cells (PBMCs) were isolated and CD4+ T and CD8+ T cells were sorted from the peripheral blood, spleen, lymph node in CIA mice by using flow cytometry. Finally, real-time quantitative PCR was used to detect the expression levels of Notch signaling pathway-related microRNAs in PBMCs and T cells. Results The levels of miR-200b-3p, miR-30c-5p, miR-30b-5p and miR-23b-3p in the PBMCs of CIA mice were significantly decreased; miR-200b-3p, miR-30c-5p and miR-30b-5p in peripheral blood, spleen, lymph node CD4+ T cells and CD8+ T cells of CIA mice was not significantly different from that of normal controls; but the expression level of miR-23b-3p in peripheral blood and spleen CD4+ T cells of CIA mice was significantly lower than that of normal control mice. Conclusion miR-23b-3p may be involved in the immunoregulation of Notch signaling to T cells of CIA mice.