Label-Free Cross-Priming Amplification Coupled With Endonuclease Restriction and Nanoparticles-Based Biosensor for Simultaneous Detection of Nucleic Acids and Prevention of Carryover Contamination

Abstract

Here, we reported on a label-free cross-priming amplification (CPA) scheme that utilized endonuclease restriction for simultaneous detection of nucleic acids and elimination of carryover contamination. Reaction mixtures were detected in a nanoparticle-based lateral flow biosensor (LFB). The assay exhibited attractive traits in that it did not require the use of labeled primers or labeled probes, and thus, the technique could prevent undesired results arising from unwanted hybridization between labeled primers or between a probe and labeled primer. Isothermal amplification and endonuclease restriction digestion were conducted in a single pot, and the use of a closed-tube amplification removed false-positive results due to contaminants. To validate the assay's applicability, we employed the novel technique to detect the pathogen Staphylococcus aureus in pure cultures and artificial blood samples. The assay could detect target bacterium in pure culture with a 100 fg.μL −1 detection limit, and in spiked blood samples with a 700 cfu.mL −1 detection limit. The whole process, including sample procedure (20-min), isothermal amplification (60-min), endonuclease digestion (10-min) and result reporting (within 2-min), could be finished within 95-min. As a poof-of-concept assay, the technique devised in the current report could be employed for detecting various other sequences if the specific CPA primers were available.

Research Progress of Nucleic Acid Detection Technology for Genetically Modified Maize

“…The system relies on only one ring structure for replication [128]. The CPA assay enables the amplification of nucleic acid sequences at a constant temperature and requires only an enzyme with strand displacement activity and a set of five primers to perform the CPA reaction, without the need for an initial denaturation step or the addition of a nickase [129]. At an assay temperature of 63 • C, the formation of primer template hybrids under transient spontaneous denaturing bubbles in the DNA template are more favorable than the re-annealing of the template strands by high concentrations of primers relative to the template DNA.…”

“…Yi et al [129] studied label-free cross-priming amplification coupled with a nanoparticlebased lateral flow biosensor. This technology combines CPA determination with the restriction endonuclease cleavage of pollutants and side-flow biosensor analysis of the reaction products.…”

Lateral Flow Recombinase Polymerase Amplification Assays for the Detection of Human Plasmodium Species

“…7 Principally, within a lateral flow strip/cassette, the amplicon is detected via a primer or probe containing a tag, with the most common ones being fluorescence isothiocyanate, digoxigenin (DIG), and biotin, which are complementary to an antibody bound to the surface of the "test" point (on nitrocellulose membrane), and thus allowing for visualization. [8][9][10] The results can be read in 5 to 10 minutes. The two types of lateral flow-based detection devices commonly available in the market are dipsticks and cassettes.…”

CRISPR/Cas12a-Enhanced Loop-Mediated Isothermal Amplification for the Visual Detection of Shigella flexneri

“…Although CRISPR/Cas has the potential for accurate diagnosis, a sufficient amount of target is still needed for the IVD method to ensure detection sensitivity. Various nucleic acid amplification techniques have been used to produce enough and specific targets, including polymerase chain reaction (PCR) ( S Wang et al, 2021 ), loop-mediated isothermal amplification (LAMP) ( Crone et al, 2020 ), recombinase polymerase amplification (RPA) ( Xianfeng Wang et al, 2021 ), rolling circle amplification (RCA) ( Ruixuan Wang et al, 2020 ), crossing priming amplification (CPA) ( Wang et al, 2019 ), strand displacement amplification (SDA) ( Dong-Xia Wang et al, 2020 ), and nucleic acid sequence-based amplification (NASBA) ( Ju et al, 2021 ).…”