Single-tube detection of nine bacterial antibiotic-resistance genes by a 2-dimensional multiplex qPCR assay based on flu
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ORIGINAL ARTICLE
Single-tube detection of nine bacterial antibiotic-resistance genes by a 2-dimensional multiplex qPCR assay based on fluorescence and melting temperature Yingxue Li1,2,3 · Ping Xu4 · Zhenzhou Wan5 · Hong Du6 · Xia Jin1 · Chiyu Zhang1,2 Received: 16 December 2019 / Accepted: 28 August 2020 © Springer Nature B.V. 2020
Abstract Simple, multiplex qPCR methods are advantages for rapid molecular diagnosis of multiple antibiotics-resistant genes simultaneously. However, the number of genes can be detected in a single reaction tube is often limited by the fluorescence channels of a real-time PCR instrument. In this study, we developed a simple 2-D multiplex qPCR method by combining the probe colors and amplicon Tm values to overcome the mechanical limit of the machine. The principle of the novel assay was validated by detection of nine bacterial antibiotic-resistance genes (KPC, NDM, VIM, OXA-48, GES, CIT, EBC, ACC and DHA) in a single reaction tube. This assay is highly sensitive within a range of 30–3000 copies per reaction. The simplicity, rapidity, high sensitivity and specificity, and low cost of the novel method make it a promising tool for developing clinical diagnostic kits for monitoring resistance and other genetic determinants of infectious diseases. Keywords 2-D multiplex qPCR · Melting temperature (Tm) · Fluorescence probe · Antibiotic-resistance gene · Amplicon · Melting curve
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11033-020-05789-6) contains supplementary material, which is available to authorized users. * Hong Du [email protected] * Chiyu Zhang [email protected] 1
Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
2
Pathogen Discovery and Evolution Unit, Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai 200031, China
3
School of Life Sciences, Shanghai University, Shanghai 200444, China
4
Department of Clinical Laboratory, The Fifth People’s Hospital of Suzhou, Suzhou 215000, China
5
Medical Laboratory of Taizhou Fourth People’s Hospital, Taizhou 225300, Jiangsu, China
6
Department of Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou 215000, China
The multiplex PCR that can detect simultaneously more than one target in a single-tube reaction has wide application in the detection of multiple genes, such as drug resistance genes and genetic material of pathogens [1–4]. Early multiplex assays were mainly based on different amplicon sizes that can be distinguished by gel electrophoresis [5, 6]. The development of real-time PCR revolutionized the multiplex nucleic acid detection by increasing the sensitivity and specificity and avoiding contamination during postPCR gel electrophoresis [7]. Previously developed multiplex qPCR strategies mainly include fluorescent color-based and melting curve-based methods for product separation [8, 9]. Because of the limit of the fluorescence channel number of an instrument, the
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