Multiplex amplification of target genes of periodontal pathogens in continuous flow PCR microfluidic chip
Multiplex amplification of target genes of periodontal pathogens in continuous flow PCR microfluidic chip
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DOI：
10.1039/d1lc00457c
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发表时间：
2021-06-10
期刊：
影响因子：
6.1
通讯作者：
Yamaguchi, Yoshinori
中科院分区：
文献类型：
--
作者：
Li, Zhenqing;Liu, Jiahui;Yamaguchi, Yoshinori
牙龈卟啉单胞菌 (P.g)、齿垢密螺旋体 (T.d) 和连翘坦纳菌 (T.f) 被认为是引起牙龈炎的主要牙周病原体，牙龈炎影响全球 50-90% 的成年人。基于连续流PCR（CF-PCR）的微流控芯片是传统热循环仪的理想替代品，因为它可以有效减少温度转换所需的时间。在此，我们首次使用 CF-PCR 微流控芯片探索了 P.g、T.d 和 T.f 的多重 PCR。通过一系列实验，我们获得了适合对这三种牙周病原菌进行多重PCR的两种最佳引物组合，扩增子大小分别为（197 bp、316 bp、226 bp）和（197 bp、316 bp、641 bp）。结果还表明，通过使用多重PCR，对于短尺寸扩增子，扩增时间可缩短至短至3'48''，而对于T.f（641bp），所需的最短时间为8'25''。这项工作提供了一种在短时间内同时扩增P.g、T.d和T.f靶基因的有效方法，并可能促进CF-PCR作为牙龈炎即时检测的实用工具。
Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f) are believed to be the major periodontal pathogens that cause gingivitis, which affects 50-90% of adults worldwide. Microfluidic chips based on continuous flow PCR (CF-PCR) are an ideal alternative to a traditional thermal cycler, because it can effectively reduce the time needed for temperature transformation. Herein, we explored multi-PCR of P.g, T.d and T.f using a CF-PCR microfluidic chip for the first time. Through a series of experiments, we obtained two optimal combinations of primers that are suitable for performing multi-PCR on these three periodontal pathogens, with amplicon sizes of (197 bp, 316 bp, 226 bp) and (197 bp, 316 bp, 641 bp), respectively. The results also demonstrated that by using multi-PCR, the amplification time can be reduced to as short as 3 ' 48 '' for the short-sized amplicons, while for T.f (641 bp), the minimum time required was 8 ' 25 ''. This work provides an effective way to simultaneously amplify the target genes of P.g, T.d and T.f within a short time, and may promote CF-PCR as a practical tool for point-of-care testing of gingivitis.