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Single-tube detection of nine bacterial antibiotic-resistance genes by a 2-dimensional multiplex qPCR assay based on fluorescence and melting temperature

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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.

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Funding

The work was supported by the grants from the National Science and Technology Major Project of China (2017ZX10103009-002), the “One Belt One Road” project (153831KYSB20170043) of the Chinese Academy of Sciences, and the 133 projects of Institut Pasteur of Shanghai, CAS.

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Authors and Affiliations

  1. Shanghai Public Health Clinical Center, Fudan University, Shanghai, 201508, China

    Yingxue Li, Xia Jin & Chiyu Zhang

  2. Pathogen Discovery and Evolution Unit, Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai, 200031, China

    Yingxue Li & Chiyu Zhang

  3. School of Life Sciences, Shanghai University, Shanghai, 200444, China

    Yingxue Li

  4. Department of Clinical Laboratory, The Fifth People’s Hospital of Suzhou, Suzhou, 215000, China

    Ping Xu

  5. Medical Laboratory of Taizhou Fourth People’s Hospital, Taizhou, 225300, Jiangsu, China

    Zhenzhou Wan

  6. Department of Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou, 215000, China

    Hong Du

Authors
  1. Yingxue Li
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  2. Ping Xu
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  3. Zhenzhou Wan
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  4. Hong Du
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  5. Xia Jin
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Contributions

CZ and HD conceived and designed the study. YL carried out the experiments. ZW, PX and HD collected the clinical samples. CZ, YL, ZW, PX and HD analyzed data. CZ, YL and XJ interpreted the results. CZ and YL wrote the manuscript. XJ contributed to critical revision of the manuscript. CZ supervised the study.

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Correspondence to Hong Du or Chiyu Zhang.

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Li, Y., Xu, P., Wan, Z. et al. Single-tube detection of nine bacterial antibiotic-resistance genes by a 2-dimensional multiplex qPCR assay based on fluorescence and melting temperature. Mol Biol Rep 47, 7341–7348 (2020). https://doi.org/10.1007/s11033-020-05789-6

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  • DOI: https://doi.org/10.1007/s11033-020-05789-6

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