Data_Sheet_1_Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction.pdf

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.

本报告设计了一种新颖的等温诊断检测方法，命名为基于纳米颗粒的生物传感器（NB）和南极热敏感尿苷-DNA-糖基化酶（ATSU）补充的聚合酶螺旋反应（PSR；NB-ATSU-PSR）。该技术融合了残留污染物酶解消化与等温核酸扩增技术（PSR），实现了核酸序列的同时检测和残留污染物的消除。具体而言，核酸扩增与残留污染物的消除在同一反应体系中完成，因此，封闭管路反应的使用能够有效去除因残留污染物引起的非期望结果。为展示目的，以肺炎克雷伯菌作为模型，展示了NB-ATSU-PSR检测方法的实用性。通过纯培养物和痰液样本的成功评估，该检测方法在敏感性、特异性和实用性方面均表现出色。扩增产物在基因组DNA低至100 fg和1 ml掺杂物痰液中约550个形成菌落单位（CFU）的浓度下即可被检测到。所有经NB-ATSU-PSR检测的肺炎克雷伯菌菌株均为阳性，而所有非肺炎克雷伯菌菌株经NB-ATSU-PSR技术检测均为阴性。整个流程，包括快速模板制备（20分钟）、PSR扩增（60分钟）、ATSU处理（5分钟）和结果报告（2分钟以内），可在90分钟内完成。作为一种概念验证方法，NB-ATSU-PSR技术可通过重新设计PSR引物集，重新配置以检测各种目标核酸序列。