Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the simple, quick, and low-cost detection of various viral genes. 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. 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). 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. The detection of complementary DNAs transcribed from the hepatitis C virus (HCV) RNA was performed by the PEG-nGO-based LAMP. 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 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). 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. 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. 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.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5′-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.