Detection of a single base mutation in Mycobacterium tuberculosis DNA can provide fast and highly specific diagnosis of antibiotic-resistant tuberculosis. Mutation-specific ligation of padlock probes (PLPs) on the target followed by rolling circle amplification (RCA) is highly specific, but challenging to integrate in a simple microfluidic device due to the low temperature stability of the phi29 polymerase and the interference of phi29 with the PLP annealing and ligation. Here, we utilized the higher operation temperature and temperature stability of Equiphi29 polymerase to simplify the integration of the PLP ligation and RCA steps of an RCA assay in two different strategies performed at uniform temperature. In strategy I, PLP annealing took place off-chip and the PLP ligation and RCA were performed in one pot and the two reactions were clocked by a change of the temperature. For a total assay time of about 1.5 h, we obtained a limit of detection of 2 pM. In strategy II, the DNA ligation mixture and the RCA mixture were separated into two chambers on a microfluidic disc. After on-disc PLP annealing and ligation, the disc was spun to mix reagents and initiate RCA. For a total assay time of about 2 h, we obtained a limit of detection of 5 pM. Graphical abstract.

中文翻译：

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通过集成的DNA连接和扩增功能，对肺结核耐药性突变进行自动芯片分析。

检测结核分枝杆菌DNA中的单个碱基突变可以提供快速且高度特异性的抗生素耐药性结核诊断。挂锁探针（PLP）与靶标上的突变特异性连接，然后进行滚环扩增（RCA），具有很高的特异性，但是由于phi29聚合酶的低温稳定性和phi29的干扰，很难整合到简单的微流控设备中PLP退火和连接。在这里，我们利用较高的操作温度和Equiphi29聚合酶的温度稳定性，简化了在均匀温度下执行的两种不同策略中RCA分析的PLP连接和RCA步骤的整合。在策略一中 PLP退火在芯片外进行，PLP连接和RCA在一个反应​​釜中进行，两个反应通过温度变化进行计时。对于大约1.5小时的总测定时间，我们获得了2 pM的检测限。在策略II中，将DNA连接混合物和RCA混合物分成微流盘上的两个小室。盘上PLP退火和连接后，将盘旋转以混合试剂并启动RCA。对于大约2小时的总分析时间，我们获得了5 pM的检测限。图形概要。旋转光盘以混合试剂并启动RCA。对于大约2小时的总分析时间，我们获得了5 pM的检测限。图形概要。旋转光盘以混合试剂并启动RCA。对于大约2小时的总分析时间，我们获得了5 pM的检测限。图形概要。