GALGT2

Escherichia coli can cause gastrointestinal infection, urinary tract infection and other infectious diseases. Accurate detection of Escherichia coli 16S rDNA (Ec-16S rDNA) in clinical practice is of great significance for the identification and treatment of related diseases. At present, there are various types of sensors that can achieve accurate detection of Ec-16S rDNA. Electrochemiluminescence (ECL) has attracted considerable attention from researchers, which causes excellent performance in bioanalysis. Based on the previous research, it is significance to develop a novel, sensitive and efficient ECL biosensor.

In this work, an ECL biosensor for the detection of Ec-16S rDNA was constructed by integrating CRISPR/Cas12a technology with the cascade signal amplification strategy consisting of strand displacement amplification (SDA) and dual-particle three-dimensional (3D) DNA rollers. The amplification products of SDA triggered the operation of the DNA rollers, and the products generated by the DNA rollers activated CRISPR/Cas12a to cleave the signal probe, thereby realizing the change of the ECL signal. The cascade amplification strategy realized the exponential amplification of the target signal and greatly improved the sensitivity. Manganese dioxide nanoflowers (MnO₂ NFs) as a co-reaction promoter effectively enhanced the ECL intensity of tin disulfide quantum dots (SnS₂ QDs). A new ternary ECL system (SnS₂ QDs/S₂O₈^2-/MnO₂ NFs) was prepared, which made the change of ECL intensity of biosensor more significant. The proposed biosensor had a response range of 100 aM-10 nM and a detection limit of 27.29 aM (S/N = 3).

Herein, the cascade signal amplification strategy formed by SDA and dual-particle 3D DNA rollers enabled the ECL biosensor to have high sensitivity and low detection limit. At the same time, the cascade signal amplification strategy was integrated with CRISPR/Cas12a to enable the biosensor to efficiently detect the target. It can provide a new idea for the detection of Ec-16S rDNA in disease diagnosis and clinical analysis.

Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field.

Neisseria gonorrhoeae is the only pathogen that causes gonorrhea, and can have serious consequences if left untreated. A simple and accurate detection method for N. gonorrhoeae is essential for the diagnosis of gonorrhea and the appropriate prescription of antibiotics. The application of isothermal recombinase polymerase amplification (RPA) to detect this pathogen is advantageous because of its rapid performance, high sensitivity, and minimal dependency on equipment. However, this simplicity is offset by the risk of false-positive signals from primer-dimers and primer-probe dimers. In this study, RPA-initiated strand displacement amplification (SDA) was established for the detection of N. gonorrhoeae, and eliminated false-positive signals from primer-dimers and primer-probe dimers. The developed biosensor allows for the reduced generation of nonspecific RPA amplification through the design of enzyme cleavage sites on primers, introduction of SDA, and detection of the final product using a molecular beacon (MB). Using this system, the DNA double strand is transformed into single-stranded DNA following SDA, thereby providing a more suitable binding substrate and improving the efficiency of MB detection. Amplification can be conducted below 37 °C, and the process can be completed within 90 min. The limit of detection was determined to be 0.81 copies/μL. This system is highly specific for N. gonorrhoeae and exhibits no cross-reactivity with other common urogenital pathogens. The results of this study are consistent with those of real-time PCR performed on clinical specimens of urogenital secretions. In summary, the biosensor is a simple and specific detection method for N. gonorrhoeae.