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.

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Contact the 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

The publisher KeAi was established by Elsevier and China Science Publishing & Media Ltd to unfold quality research globally. In 2013, our focus shifted to open access publishing. We now proudly publish more than 200 world-class, open access, English language journals, spanning all scientific disciplines. Many of these are titles we publish in partnership with prestigious societies and academic institutions, such as the National Natural Science Foundation of China (NSFC).