A dual RPA-LFD assay for the simultaneous detection of Salmonella typhimurium and Salmonella enteritidis.

Introduction: Salmonella was one of the most common bacteria that caused foodborne illness, with S. typhimurium (Salmonella typhimurium) and S. enteritidis (Salmonella enteritidis) infections accounting for more than 75% of human salmonella infections. Methods: In this study, we developed a method of dual recombinase polymerase amplification (RPA) combined with a lateral flow dipstick for the rapid detection of S. typhimurium and S. enteritidis in clinical specimens (stool). Results: The entire reaction process, including amplification and result reading, could be completed within 65 min. The detection limits of S. typhimurium and S. enteritidis in pure culture samples were 5.23 × 101 CFU/mL and 3.59 × 101 CFU/mL, respectively. The detection limits of S. typhimurium and S. enteritidis in artificially contaminated samples were 8.30 × 101 CFU/mL and 2.70 × 102 CFU/mL, respectively. In addition, the method had no cross-reaction with other pathogenic microorganisms. The results in clinical samples were fully consistent with those obtained using Bacterial Analysis Manual, with sensitivity and specificity were 100% (8/8) and 100% (17/17) for S. typhimurium and 100% (4/4) and 100% (21/21) for S. enteritidis, respectively. Discussion: The detection limits of S. typhimurium and S. enteritidis in artificially contaminated samples were higher than those in pure culture samples, which might be attributed to the inherent complex composition of artificially contaminated samples. In addition, the detection limits of S. typhimurium and S. enteritidis in the same sample were also different, which might be attributed to different amplification efficiency of two target genes in the same reaction system. Conclusion: This assay had potential application outdoors, as it could be performed within 1 h at 38°C without a complex instrument, and the results could be observed with the naked eye. In conclusion, the dual RPA-LFD assay established in this study had practical significance for the rapid detection of S. typhimurium and S. enteritidis in the future.

A multiplex RPA coupled with CRISPR-Cas12a system for rapid and cost-effective identification of carbapenem-resistant Acinetobacter baumannii.

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe nosocomial threat, prompting a need for efficient detection methods. Traditional approaches, such as bacterial culture and PCR, are time-consuming and cumbersome. The CRISPR-based gene editing system offered a potential approach for point-of-care testing of CRAB. Methods: We integrated recombinase polymerase amplification (RPA) and CRISPR-Cas12a system to swiftly diagnose CRAB-associated genes, OXA-51 and OXA-23. This multiplex RPA-CRISPR-Cas12a system eliminates bulky instruments, ensuring a simplified UV lamp-based outcome interpretation. Results: Operating at 37°C to 40°C, the entire process achieves CRAB diagnosis within 90 minutes. Detection limits for OXA-51 and OXA-23 genes are 1.3 × 10-6 ng/µL, exhibiting exclusive CRAB detection without cross-reactivity to common pathogens. Notably, the platform shows 100% concordance with PCR when testing 30 clinical Acinetobacter baumannii strains. Conclusion: In conclusion, our multiplex RPA coupled with the CRISPR-Cas12a system provides a fast and sensitive CRAB detection method, overcoming limitations of traditional approaches and holding promise for efficient point-of-care testing.

Decreased miR-127 promotes the occurrence of breast cancer via increasing the expression of SPP1.

BACKGROUND: The expression of miR-127 has been reported to be decreased in the breast tissue of patients with breast cancer (BRC). However, the mechanism of miR-127 involvement in the pathogenesis of BRC is still unclear and requires urgent clarification. OBJECTIVES: To explore the role of miR-127 in the pathogenesis of BRC. MATERIAL AND METHODS: In this study, we measured the expression of miR-127 in blood samples of 60 BRC patients and 60 controls, investigated the influence of miR-127 on the viability and apoptosis of MCF-7 and MDA-231 cells, identified a miR-127 target gene, and determined the expression level of the target gene in the blood samples of BRC patients and controls. RESULTS: We found that miR-127 expression was significantly decreased in the plasma of BRC patients compared to controls. Additionally, the upregulation of miR-127 in MCF-7 and MDA-231 cells inhibited their proliferation and promoted their apoptosis. Conversely, the downregulation of miR-127 promoted cell proliferation and inhibited their apoptosis. The SPP1 was successively predicted and validated as a target gene of miR-127. Finally, the expression level of SPP1 was significantly increased in the plasma of BRC patients compared to controls. CONCLUSIONS: Our study demonstrated that decreased miR-127 may promote BRC cell proliferation, inhibit apoptosis and promote the occurrence of BRC through increasing the SPP1 expression level.

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications.

After the outbreak of SARS-CoV-2, nucleic acid testing quickly entered people's lives. In addition to the polymerase chain reaction (PCR) which was commonly used in nucleic acid testing, isothermal amplification methods were also important nucleic acid testing methods. Among several common isothermal amplification methods like displaced amplification, rolling circle amplification, and so on, recombinase polymerase amplification (RPA) was recently paid more attention to. It had the advantages like a simple operation, fast amplification speed, and reaction at 37-42°C, et al. So it was very suitable for field detection. However, there were still some disadvantages to RPA. Herein, our review mainly summarized the principle, advantages, and disadvantages of RPA. The specific applications of RPA in bacterial detection, fungi detection, virus detection, parasite detection, drug resistance gene detection, genetically modified food detection, and SARS-CoV-2 detection were also described. It was hoped that the latest research progress on RPA could be better delivered to the readers who were interested in RPA.

Toxicity Evaluation of Three Imidazolium-based ionic liquids ([C6mim]R) on Vicia faba Seedlings Using an integrated biomarker response (IBR) index.

Ionic liquids (ILs) are regarded as green solvents and are frequently used in the chemical industry. However, ILs may impact plant growth if they are present in the soil environment. To compare toxicity of ILs with different anions in soil, three imidazolium-based ionic liquids (1-hexyl-3-methylimidazolium bromide, 1-hexyl-3-methylimidazolium nitrate, 1-hexyl-3-methylimidazolium tetrafluoroborate) were used to assess impact on Vicia faba. Following 10 d of exposure to these three ILs from 0 to 2500 mg kg-1, shoot length, root length and dry weight of Vicia faba were determined. Pot trials revealed that ILs inhibited Vicia faba growth and according to EC50 values, [C6mim]BF4 was the most toxic one. In addition, physiological indicators of Vicia faba were determined following 10 d of exposure at selected IL concentrations (0, 1, 10, 100 and 500 mg kg-1). ILs led to the generation of reactive oxygen species and then caused oxidative damage, including lipid peroxidation, protein damage and DNA damage, which triggered an increase in antioxidant content and enzyme activity. The experimental results indicated that oxidative stress may be the primary underlying toxic mechanism for Vicia faba. Furthermore, based on the data of physiological experiment, integrated biomarker response (IBR) was calculated to compare the toxicity of the three ILs and toxic order was: [C6mim]NO3＜[C6mim]Br＜[C6mim]BF4.

Phytotoxicity of imidazolium-based ILs with different anions in soil on Vicia faba seedlings and the influence of anions on toxicity.

To evaluate the toxic effects of ionic liquids (ILs) in soil on plants at the molecular and cellular levels and to assess the influence of anions on IL toxicity, the toxic effects of 1-decyl-3-methylimidazolium chloride ([Demim]Cl), 1-decyl-3-methylimidazolium bromide ([Demim]Br) and 1-decyl-3-methylimidazolium nitrate ([Demim]NO3) in soil on Vicia faba (V. faba) seedlings were studied for the first time. Our results show that these ILs had little impact on the growth of V. faba seedlings at 1, 5 and 25 mg kg(-1); however, the shoot length, root length, dry weight and pigment contents of the seedlings were significantly affected at 50 mg kg(-1). Furthermore, the EC50 values for effects of [Demim]Cl, [Demim]Br and [Demim]NO3 on the shoot length, root length and dry weight induced were consistent, indicating that the anion may have little influence on IL toxicity. ROS levels were also significantly enhanced at 50 mg kg(-1), resulting in cellular lipid peroxidation, DNA damage and oxidative damage.