Visualized Genotyping from “Sample to Results” Within 25 Minutes by Coupling Recombinase Polymerase Amplification (RPA) With Allele-Specific Invasive Reaction Assisted Gold Nanoparticle Probes Assembling

Abstract

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from “samples to results” within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.

Ultrafast and Highly Specific Detection of One-Base Mutated Cell-Free DNA at a Very Low Abundance

“…Although FARPA was very fast, gDNA extraction and purification from blood samples was time-consuming and labor-intensive. To achieve ultrafast genotyping, we used oral swabs to noninvasively get samples from the patient’s mouth and used 100 mM NaOH or QuickExtract solution (QE for short) to rapidly lyse the oral swabs. After 3 min of lysis by NaOH or 6 min of lysis by QE, 1 μL of NaOH lysates or 2 μL of QE lysates were directly used for FARPA.…”

“…We have proved that invasive reactions could be used to detect single nucleotide polymorphisms (SNPs) when coupling RPA to amplify the targets; however, it is not a one-pot format, and open-tube operation is necessary after RPA due to the incompatibility between the two reaction systems. Recently, we have developed a novel method called FEN1-aided RPA (FARPA) to solve this issue, and the two reaction systems could be compatible in a homogeneous way.…”

FARPA-based tube array coupled with quick DNA extraction enables ultra-fast bedside detection of antibiotic-resistant pathogens

“…Unlike PCR, isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification, 18,19 are quite time-saving since there is no need for thermal cycling. Since RPA can yield enough amplicons for downstream analysis within 20 min at 37-42 °C, 20 it is an ideal tool for quickly amplifying antibioticresistant genes if the amplicons can be efficiently sensed.…”

Visualised genotyping assay with oral swabs in a closed tube by nested invasive reaction assisted with gold nanoparticle probes

“…Commonly, methods for SNP typing are mainly based on the amplification of nucleic acid coupled with the detection of amplicons. Polymerase chain reaction (PCR) is a classical method of nucleic acid amplification, and recently emerging isothermal amplification technology such as recombinase polymerase amplification (RPA) [ 11 , 12 ] and loop‐mediated isothermal amplification (LAMP) [ 13 , 14 ] is also increasingly used for nucleic acid amplification. Sequencing [ 15 , 16 ] and fluorescence‐based methods [ 17 , 18 , 19 , 20 , 21 , 22 ], such as molecular beacons, TaqMan probes, and CRISPR/Cas systems, are usually used for the detection of amplificons.…”