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

Yi Wang,Chen Shen,Lin Sun,Wei-Wei Jiao,Jie-Qiong Li,Ya-Jie Guo,Yong-Hong Wang,Ze-Ming Wang,A-Dong Shen,Hui Qi,Jing Xiao,Fang Xu

doi:10.3389/fchem.2019.00322

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.

Highlights

Nucleic acids were widely used for biological research and as powerful biomarkers for forensic science, agriculture, clinical diagnosis, environmental monitoring, food safety test, and so on
The biotin- and FITC-labeled cross-priming amplification (CPA) products were termed as double-labeled detectable amplicons, which could be visually detected by lateral flow biosensor (LFB)
We devised a new method on the basis of standard CPA assay, termed label-free CPA-endonuclease restriction (ER)-LFB

Summary

Introduction

Nucleic acids were widely used for biological research and as powerful biomarkers for forensic science, agriculture, clinical diagnosis, environmental monitoring, food safety test, and so on. Polymerase chain reaction (PCR)-based techniques, as the well-known molecular tools, have been applied for amplifying and analyzing low-abundance nucleic acids (Niemz et al, 2011; Feng et al, 2018a) These technologies require complex apparatus for amplification temperature adjustment and trained personnel, which significantly hinders their application in resource-limited settings (Reid et al, 2018). Device of inexpensive and simple techniques for nucleic acids amplification and detection is extremely important for on-site diagnostic applications in place of PCR-based assays To this end, extensive numbers of isothermal amplification technologies, including strand displacement amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), multiple cross displacement amplification (MCDA), loop-mediated isothermal amplification (LAMP) and cross-priming amplification (CPA), have been proposed (Wang et al, 2015, 2018a; Zhao et al, 2015; Wachiralurpan et al, 2018). The newer monitoring techniques, which are capable of simplifying and speeding up the total time of CPA-based methods, should be devised

Methods

Results

Discussion

Conclusion