Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction.

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

doi:10.3389/fbioe.2019.00401

Abstract

The current report devised a novel isothermal diagnostic assay, termed as nanoparticle-based biosensor (NB)- and antarctic thermal sensitive uracil-DNA-glycosylase (ATSU)-supplemented polymerase spiral reaction (PSR; NB-ATSU-PSR). The technique merges enzymatic digestion of carryover contaminants and isothermal nucleic acid amplification technique (PSR) for simultaneous detection of nucleic acid sequences and elimination of carryover contamination. In particular, nucleic acid amplification and elimination of carryover contamination are conducted in a single pot and, thus, the use of a closed-tube reaction can remove undesired results due to carryover contamination. For demonstration purpose, Klebsiella pneumoniae is employed as the model to demonstrate the usability of NB-ATSU-PSR assay. The assay's sensitivity, specificity, and practical feasibility were successfully evaluated using the pure cultures and sputum samples. The amplification products were detectable from as little as 100 fg of genomic DNAs and from ~550 colony-forming unit (CFU) in 1 ml of spiked sputum samples. All K. pneumoniae strains examined were positive for NB-ATSU-PSR detection, and all non-K. pneumoniae strains tested were negative for the NB-ATSU-PSR technique. The whole process, including rapid template preparation (20 min), PSR amplification (60 min), ATSU treatment (5 min), and result reporting (within 2 min), can be finished within 90 min. As a proof-of-concept methodology, NB-ATSU-PSR technique can be reconfigured to detect various target nucleic acid sequences by redesigning the PSR primer set.

Highlights

Nucleic acid amplification is essential to most molecule testing strategies (Breaker, 2004)
The Ft∗ primer is employed as the model primer to validate the availability of nanoparticle-based biosensor (NB)-polymerase spiral reaction (PSR) methodology
The Ft∗ primer is employed as the model primer to validate the availability of NB-assisted PSR technique (NB-PSR) methodology, which is labeled with hapten at the 5′ end

Summary

Introduction

Nucleic acid amplification is essential to most molecule testing strategies (Breaker, 2004). Polymerase chain reaction (PCR) is the most common methodology for nucleic acid amplification, but PCR-based techniques (such as conventional PCR, multiplex PCR, and real-time PCR) require complex experimental processes, sophisticated instruments, or trained personnel (Navarro et al, 2015). To solve these problems posed by classical PCR-based technologies, many isothermal amplification techniques [e.g., loop-mediated isothermal amplification (LAMP), multiple cross-displacement amplification (MCDA), cross-priming amplification (CPA), isothermal genome exponential amplification, and rolling circle amplification] have been reported, which rely on simpler apparatus, using only a single, moderate temperature (Wang et al, 2015; Zhao et al, 2015). These isothermal amplification techniques involve rigorous optimization to obtain excellent performance

Results

Discussion

Conclusion