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

• Published in: Frontiers in Bioengineering and Biotechnology
• Main Authors: Yi Wang, Wei-wei Jiao, Yacui Wang, Lin Sun, Jie-qiong Li, Ze-ming Wang, Jing Xiao, Chen Shen, Fang Xu, Hui Qi, Yong-hong Wang, Ya-jie Guo, A-dong Shen
• Format: Article in Journal/Newspaper
• Language: English
• Published: Frontiers Media S.A. 2019
• Subjects: isothermal nucleic acid amplification; polymerase spiral reaction; nanoparticle-based biosensor; Klebsiella pneumoniae; carryover contamination; LoD; Biotechnology; TP248.13-248.65; Antarctic; Antarc*
• Online Access: https://doi.org/10.3389/fbioe.2019.00401; https://doaj.org/article/5db895d9e3d448d98773e97e3a80b3f1

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.