RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min

Abstract

Loop-mediated isothermal amplification (LAMP) is a commonly used alternative to PCR for point-of-care detection of nucleic acids due to its rapidity, sensitivity, specificity, and simpler instrumentation. While dual-labeled TaqMan probes are widely used in PCR for single-nucleotide polymorphism (SNP) genotyping, real-time LAMP primarily relies on turbidimetry or intercalator fluorescence measurements, which can be non-specific and generate false-positive results. In this study, we propose a closed-tube, dual-labeled RNA-modified probes and RNase H II-assisted real-time LAMP (RART-LAMP) method for SNP genotyping. Our findings indicate that (1) fluorescence signals were predominantly derived from probe hydrolysis rather than hybridization, (2) temperature-controlled hybridization between the probe and template ensured the specificity of SNP analysis, and (3) RNase H II hydrolysis between the target containing SNP sites and probes did not exhibit sequence specificity. Our RART-LAMP approach demonstrated excellent performance in genotyping C677T clinical samples, including gDNA extracted from blood, saliva, and swabs. More importantly, saliva and swab samples could be directly analyzed without any pretreatment, indicating promising prospects for nucleic acid analysis at the point of care in resource-limited settings.

Introducing Triplex Forming Oligonucleotide into Loop-Mediated Isothermal Amplification for Developing a Lateral Flow Biosensor for Streptococci Detection

“…In contrast, PCR methods employ sequence-specific probes, such as TaqMan, which utilize the 5 ′ to 3 ′ exonuclease activity of the polymerase for cleavage and fluorescent signal production [10]. To address this, researchers have introduced sequence-specific signal strategies into LAMP, incorporating enzymes like endonucleases [11][12][13][14], high-fidelity polymerases [15], and CRISPR-Cas proteins [16,17]. These additions enable the selective cleavage of probes bound to correctly amplified target sequences within the LAMP process.…”