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.

本研究构建了一种新型等温诊断检测方法，命名为基于纳米颗粒的生物传感器（nanoparticle-based biosensor, NB）与南极热敏感性尿嘧啶DNA糖基化酶（antarctic thermal sensitive uracil-DNA-glycosylase, ATSU）联合聚合酶螺旋反应（polymerase spiral reaction, PSR；简称NB-ATSU-PSR）。该技术将残留污染物酶解处理与等温核酸扩增技术（PSR）相结合，可同时实现核酸序列检测与残留污染清除。其独特优势在于，核酸扩增与残留污染清除可在单管内同步进行，因此采用闭管反应即可规避因残留污染引发的非预期检测结果。

为验证该检测方法的实用性，本研究以肺炎克雷伯菌（Klebsiella pneumoniae）作为模型菌种。通过纯培养物与痰液样本对该检测方法的灵敏度、特异性及实际应用可行性进行评估，结果均表现优异。该检测方法可检出的最低基因组DNA量为100 fg，且可在1 ml加标痰液样本中检出约550个菌落形成单位（colony-forming unit, CFU）。所有受试肺炎克雷伯菌菌株均呈NB-ATSU-PSR检测阳性，而所有受试非肺炎克雷伯菌菌株均呈检测阴性。

整个检测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）与结果判读（2分钟以内），总耗时不超过90分钟。作为一种概念验证型方法，NB-ATSU-PSR技术可通过重新设计PSR引物组，适配对多种目标核酸序列的检测。