The current study reports on a cross-priming amplification (CPA) scheme that utilizes antarctic thermal sensitive uracilDNA-glycosylase (AUDG) for simultaneous detection of nucleic acids and prevention of carryover contamination. Amplification products were applied in a nanoparticle-based lateral flow biosensor (LFB). The method shows attractive features in that it only requires the use of a labeled primer, eliminating the use of labeled probes. Thus, it is able to remove false-positive results yielded by undesired hybridization between two labeled primers or between a probe and labeled primer. CPA amplification and AUDG cleavage are carried out in a single pot, and the use of a closed-vessel reaction eliminates unwanted results due to carryover contamination. Then, the assay devised in this report was applied to the detection of the hospital-acquired pathogen Klebsiella pneumoniae in pure cultures and artificial sputum samples. This biosensor can detect K. pneumoniae in pure cultures with a 100 fg · μL^-1 detection limit, and in artificial sputum samples with a 520 cfu · mL^-1 detection limit. The whole procedure, including specimen processing (20-min), CPA amplification (60-min), AUDG digestion (5-min) and result indicating (within 2-min), can be completed within 1.5 h. As a proof-of-concept technique, this method can be used for detecting a wide variety of other targets if the specific CPA primer set is available.

中文翻译：

---

核酸检测和交叉吸附扩增结合基于纳米颗粒的生物传感器和南极热敏尿嘧啶-DNA-糖基化酶，防止残留污染。

本研究报告了一种交叉引物扩增（CPA）方案，该方案利用南极热敏尿嘧啶DNA糖基化酶（AUDG）来同时检测核酸并防止残留污染。扩增产物被应用于基于纳米颗粒的侧向流生物传感器（LFB）。该方法具有吸引人的特征，因为它仅需要使用标记的引物，而无需使用标记的探针。因此，它能够消除由于两个标记的引物之间或探针与标记的引物之间不希望的杂交而产生的假阳性结果。CPA扩增和AUDG裂解在一个罐中进行，使用封闭容器反应可消除由于残留污染而产生的不良结果。然后，纯培养物和人工痰样本中的肺炎克雷伯菌。该生物传感器可检测肺炎克雷伯在纯培养物用100 FG· μ大号^-1检出限，并用520 CFU·毫升人工痰样品中的^-1检测限。整个过程，包括标本处理（20分钟），CPA扩增（60分钟），AUDG消化（5分钟）和结果指示（2分钟内），可以在1.5小时内完成。作为一种概念验证技术，如果可以使用特定的CPA引物组，则该方法可用于检测多种其他目标。