Triplex real-time quantitative fluorescence PCR method for detecting drug resistance genes

With the global overuse of antibiotics, the emergence of multidrug-resistant (MDR) bacteria and "superbugs" has become a major threat to human health and food safety. Among these, drug-resistant bacteria carrying mcr-1 (polymyxin resistance gene), vanA (vancomycin resistance gene), and bla_NDM-1 (New Delhi metallo-β-lactamase gene) can spread via plasmids, exacerbating the dissemination of resistance. However, existing detection methods are often limited to single-gene analysis and suffer from inefficiency and long processing times.

In a recent study published in the KeAi journal Biomedical Analysis, a team from the Beijing Academy of Science and Technology Institute of Analysis and Testing designed and screened specific primers and probes for the three resistance genes.

“By optimizing reaction systems and amplification conditions, we  established a triplex real-time quantitative fluorescence PCR detection method,” shares Qiushui Wang, corresponding author of the study. “Validation tests confirmed a detection limit as low as 10³ copies/μL, linear correlation coefficients (R²) exceeding 0.99 for standard curves, and intra- and inter-group reproducibility with relative standard deviations (RSD) below 3%.”

Furthermore, the method was successfully applied to 42 real-world samples (including aquatic products, meats, and environmental samples), detecting five positive samples. Notably, three resistance genes were simultaneously identified in river water samples.

“Traditional antibiotic susceptibility testing only detects phenotypes and is time-consuming, while existing molecular methods cannot simultaneously detect multiple genes,” emphasized Wang. “Our method not only shortens the detection cycle, but also accurately quantifies gene concentrations in diverse samples, providing critical technical support for monitoring drug resistance.”

The research team highlighted the method’s applicability across food, environmental and clinical samples. For instance, the bla_NDM-1 gene detected in river water samples reached concentrations as high as 7.94×10² copies/μL, underscoring environmental transmission risks.

“We plan to further optimize multi-gene detection systems and expand their applications in drug-resistant bacteria prevention,” added co-first author Jie Deng.

In view of this, circDNAs can be used as a potential ideal biomarker for cancer diagnosis and prognosis, offering advantages over existing RNA biomarkers, such as superior stability, preservation and tissue-specific expression.

Contact author:  Qiushui Wang, Institute of Analysis and Testing, Beijing Academy of Science and Technology (Beijing Center for Physical and Chemical Analysis), Beijing 100089, China. wangqius9182901@hotmail.com;  Lijuan Gao, Institute of Analysis and Testing, Beijing Academy of Science and Technology (Beijing Center for Physical and Chemical Analysis), Beijing 100089, China. aglj889@163.com

Funder: This study was supported by NHC Key Laboratory of Food Safety Risk Assessment (China National Center for Food Safety Risk Assessment, 2024-15) and the Beijing Academy of Science and Technology Municipal Finance Program (24CA001-04).

Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

See the article: Jie Deng, Rong Guo, Qiushui Wang, Yue Liu, Lijuan Gao, Establishment of a triplex real-time quantitative fluorescence PCR method for detecting drug resistance genes mcr-1, bla_NDM-1, and vanA, Biomedical Analysis, Volume 2, Issue 5, 2025, Pages 1-7, https://doi.org/10.1016/j.bioana.2024.11.003.