On-site detection demands are quickly increasing to control foodborne pathogenic bacteria along with the long food supply chains. Combining the isothermal recombinase polymerase amplification (RPA) with lateral flow strips (LFSs) is a promising molecular detection approach for the short reaction time, low isothermal condition, and simple and “instrument-free” procedure. However, the method comes with a non-negligible intrinsic risk of the primer-dependent artifacts. In this study, with an important foodborne pathogenic bacterium Salmonella enterica serotype Typhimurium (S. Typhimurium) as the model, system measures including the careful selection of primers targeting unique virulence genes, use of a probe in the RPA reaction, introducing base substitutions with specific guidelines in the primer and probe sequences, and analyzing and screening the primer–probe complex formation were taken to eliminate the primer-dependent artifacts. The measures were strictly tested for the efficacy, and the standardized method was able to specifically detect S. typhimurium within 30 min at 42°C without any interference of probe–primer signals. The established RPA-LFS method shared high sensitivity with the detection limit of 1 CFU/μl of unpurified culture. Our study provided practical measures for the prevention of false positive signals from primer–dimers or primer–probe complexes when using the RPA–LFS method in pathogen detections, and also established a readily applicable method for S. Typhimurium detection.

The interaction between the single-stranded DNA and the homologous duplex DNA is essential for DNA homologous repair. Here, we report that parallel triplex structure can form spontaneously between a mechanically extended ssDNA and a homologous dsDNA in protein-free condition. The triplex has a contour length close to that of a B-form DNA duplex and remains stable after force is released. The binding energy between the ssDNA and the homologous dsDNA in the triplex is estimated to be comparable to the basepairing energy in a B-form dsDNA. As ssDNA is in a similar extended conformation within recombinase-coated nucleoprotein filaments, we propose that the parallel triplex may form and serve as an intermediate during recombinase-catalyzed homologous joint formation.

摘要

Polyethylene glycol modification (PEGylation) is a widely used strategy to improve the physicochemical properties of various macromolecules, especially protein drugs. However, its application in enhancing the performance of enzymes for molecular biology remains underexplored. This study explored the PEGylation of Bst DNA polymerase, determining optimal modification reaction conditions. In comparison to the unmodified wild-type counterpart, the modified Bst DNA polymerase exhibited significantly improved of activity, thermal stability and inhibitor tolerance during loop-mediated isothermal amplification (LAMP). When applied for the detection of Salmonella in crude samples, the modified enzyme demonstrated a notably accelerated reaction rate. Therefore, PEGylation emerges as a viable strategy for refining DNA polymerases, helping in the development of novel molecular diagnostic reagents.

Background: The DNA polymerase of Thermus thermophilus (Tth pol) presents reverse transcriptase activity with Mn2+, and can be used for one-step RT-qPCR. However, Mn2+ would reduce amplification fidelity and cause nonspecific products. Objective: Eliminating the Mn2+ dependence of the reverse transcriptase activity of Tth pol by point mutations. Methods: We constructed three variants I640F, I709K, and I640F/I709K, and measured their DNA polymerase and reverse transcriptase activities without Mn2+. Their enzymatic characteristics and PCR inhibitor resistance were also tested. Finally, these variants were applied in one-step RT-qPCR. Results: All three variants presented reverse transcriptase activity with Mg2+ only and increased DNA polymerase activity. The variants, except I709K, showed no significant difference in thermostability, optimal pH, optimal NaCl concentration, storage stability and PCR inhibitor resistance compared to the wild type. Variant I640F/I709K had good performance in one-step RT-qPCR with Mg2+ only, whereas both variants with single substitution exhibited nonspecific amplification. Conclusion: We successfully constructed three Tth pol variants possessing Mn2+ independent reverse transcriptase activity. The variant I640F/I709K was suitable for one-step RT-qPCR because of its good performance.