Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay

Abstract

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10−11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

Capillary Tube Based Molecular Diagnostic Test for Naked Eye Detection of Antibiotic Resistant Bacteria

“…In addition to real‐time analysis, the generation of amplified DNA products was confirmed by gel electrophoresis compared to NC. Detection limit of our system with pM range was similar to previously reported systems such as netlike rolling circle amplification (NRCA) for miRNA analysis and recombinase polymerase amplification (RPA) for DNA detection …”

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

“…Most published RPA papers have applied amplicon lengths between 100 and 250 nucleotides, which usually incur fast and efficient amplification. However, shorter amplicons (79 nucleotides; 37 94 nucleotides [38][39][40] and longer amplicon up to 1500 nucleotides 6 and 1941 nucleotides 41 have also been reported. Unlike PCR, the length of RPA primers is relatively long (a recommended minimum of 30 nucleotides, but typically between 32 and 35 nucleotides).…”

“…Such nucleic acid labelling can be achieved terminally using 5′-labelled primers or internally via labelled nucleotides. 39,40,[98][99][100][101][102][103][104] The labels used for nucleic acid labelling can be fluorescent entity (e.g. fluorescein), ligand (e.g.…”

Recombinase polymerase amplification: Basics, applications and recent advances

“…An alternative RPA-lateral flow assay used tailed primers (primer containing a carbon stopper to generate double stranded DNA flanked by single stranded tails), to generate double tailed amplicons. Oligo-functionalised AuNPs were used as reporter probes and oligonucleotides as capture probes in the test and control line, instead of the conventional antigen label and antibody capture approach [67], decreasing the cost of the strip. Fig.…”