Visual and rapid detection of Acinetobacter baumannii by a multiple cross displacement amplification combined with nanoparticles-based biosensor assay
Visual and rapid detection of Acinetobacter baumannii by a multiple cross displacement amplification combined with nanoparticles-based biosensor assay
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DOI：
10.1186/s13568-019-0754-0
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发表时间：
2019-02-26
期刊：
影响因子：
3.7
通讯作者：
Tang, Yijun
中科院分区：
文献类型：
--
作者：
Cheng, Xueqin;Yang, Jing;Tang, Yijun
传统的鲍曼不动杆菌微生物学检测方法费时费力。因此，我们试图建立一种新的目标病原体的快速检测方法。设计了一套多重交叉置换扩增(MCDA)引物，用于识别该细菌pGAD基因的10个不同区域，这些区域是该细菌所特有的。在MCDA体系中，扩增引物D_1和R_1分别用荧光素和生物素标记。在扩增阶段形成了大量的FITC和生物素连接的双链扩增产物，通过免疫反应(双链FITC和LFB测试线上的抗FITC)和生物素/链霉亲和素的相互作用(双链上的生物素和纳米颗粒上的链霉亲和素)，由基于纳米颗粒的横向流动生物传感器(LFB)检测到这些扩增产物。结果表明，MCDA-LFB法的最佳反应条件为反应温度62℃，反应时间25min。与Non-A无交叉反应。对鲍曼不动杆菌属和非不动杆菌属的检测下限为100fg/反应。对135份痰标本的检测结果表明，MCDA-LFB法的检测能力优于培养法和常规PCR法。因此，MCDA-LFB法有望成为临床标本和低来源地区鲍曼不动杆菌快速检测的一种新方法。
The traditional microbiological methods used for detecting Acinetobacter baumannii were usually time-consuming and labor-intensive. Thus, we sought to establish a novel rapid detecting method for target pathogen. A set of multiple cross displacement amplification (MCDA) primers was designed to recognize 10 different regions of the pgaD gene, which was conservative and specific for the bacterium. In the MCDA system, amplification primers D1 and R1 were 5-labeled with FITC (fluorescein) and biotin, respectively. Numerous FITC- and biotin-attached duplex amplicons were formed during the amplification stage, which were detected by nanoparticles-based lateral flow biosensors (LFB) through immunoreactions (FITC on the duplex and anti-FITC on the LFB test line) and biotin/streptavidin interaction (biotin on the duplex and streptavidin on the nanoparticles). The results showed that the optimized reaction condition of MCDA-LFB method was 62 degrees C within 25min. There was no cross reaction with non-A. baumannii species and the non-Acinetobacter genera, and the detection limit for DNA samples was 100fg/reaction. For 135 sputum samples, the detection results showed that the detection ability of MCDA-LFB assay was superior to the culture methods and conventional PCR. Therefore, MCDA-LFB assay could be a potential tool for the rapid detection of A. baumannii in clinical samples and low resource areas.