Thus, PEG-nGO can significantly improve the performance of LAMP assays by facilitating the specific amplification of target DNA with a decrease in background signal.įoodborne pathogens pose serious risks to human health and food safety. In addition, the PEG-nGO-based LAMP method significantly improves the detection precision for the false-positive decision by 1.75-fold as compared to the LAMP without PEG-nGO. Among the 20 clinical samples tested, all 10 HCV-positive samples are detected as positive in the PEG-nGO-based LAMP, while only 7 samples are detected as HCV-positive in the RT-qPCR. ![]() The PEG-nGO-based LAMP assay greatly facilitates the detection of HCV-positive clinical samples, with superior precision to the conventional quantitative real-time PCR (RT-qPCR). We observed that the inclusion of PEG-nGO significantly enhances the specificity and sensitivity of the LAMP assay through the augmented difference in fluorescence signals between the target and non-target samples. The detection of complementary DNAs transcribed from the hepatitis C virus (HCV) RNA was performed by the PEG-nGO-based LAMP. By adsorbing surplus ssDNA primers, PEG-nGO minimizes the non-specific annealing of ssDNAs, including erroneous priming and primer dimerization, leading to the enhanced specificity of LAMP. Herein, we reported that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly enhance the specificity of LAMP, owing to its ability to adsorb single-stranded DNA (ssDNA). LAMP assays are susceptible to generating non-specific amplicons, as high concentrations of DNA primers can give rise to primer dimerization and mismatched hybridizations, resulting in false-positive signals. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the simple, quick, and low-cost detection of various viral genes. Subsequent initiations at FIP give rise to additional release of Fd, resulting in exponential signal detection. (4) The resulting structure undergoes exponential amplification in the LAMP reaction. The newly synthesized strand is displaced by extension from the B3 primer. This releases the quenching resulting in a gain of signal. ![]() (3) Synthesis from the primer annealed to the B2c sequence displaces the Fd probe. (2) The BIP primer anneals to the B2c site in the newly synthesized strand. ![]() This new strand is displaced by upstream synthesis from the F3 primer. (1) LAMP initiates at the F2c sequence of the target, with the Fd probe quenched through annealing to Q-FIP. For clarity, LoopF and LoopB primers are not shown. (B) Outline of DARQ LAMP reactions, with core LAMP primers FIP (F1c + F2), BIP (B1c + B2), F3 and B3, and the DARQ oligonucleotide, Fd (Q, black F, red). The quencher and fluorophore moieties are represented by Q and F, respectively. (A) Schematic depiction of a DARQ probe, with a 5′-quencher FIP (F1c + F2 sequence) annealed to a 3′-fluorophore Fd.
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