Related Experiment Video
Updated: Oct 28, 2025
Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip
Published on: March 29, 2024
Rapid and Sensitive Detection of the Colistin Resistance Gene mcr-3 by Loop-Mediated Isothermal Amplification and
Zhihai Liu1, Changmei Guo2, Yaru Zhang3
1College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, China.
Abstract:
The distribution of colistin resistance in mcr-carrying bacteria poses a threat to global public health. In particular, the newly identified mcr-3 allele has spread globally, especially in China, second only to mcr-1. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid, sensitive, and visual detection of the presence of the mcr-3 gene. A total of 13 clinical bacterial strains and 11 negative strains were used in this study. We designed LAMP Primers, optimized reaction conditions, used three different methods to detect LAMP amplification products: (1) agarose gel electrophoresis, (2) LAMP-hydroxy naphthol blue (HNB) detection, (3) LAMP-SYBR Green I (LAMP-SGI) visual inspection, and evaluated its specificity and sensitivity. The amplification reaction was completed in 1 hr at 62°C under isothermal conditions. The final optimized mixtures contained 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100, 1.2 μL HNB, and 0.5 μL SYBR Green I as additives to the initial reaction mixture. LAMP detection, including two visual methods, LAMP-HNB and LAMP-SGI, of mcr-3 possessed the same specificity and a 10-fold higher sensitivity compared with a conventional polymerase chain reaction assay using the same samples. We successfully established an mcr-3 LAMP detection with portability and rapidity of the reaction by the easily distinguishable color changes in the reaction tubes. This visual LAMP assay for mcr-3 detection was simple, time saving, and economical, especially suited to field laboratories.
Insights
A new loop-mediated isothermal amplification (LAMP) assay rapidly detects the mcr-3 gene, a colistin resistance threat. This visual method offers high sensitivity and specificity, suitable for field use.
Area of Science:
- Microbiology
- Molecular Biology
- Public Health
Background:
- Colistin resistance in bacteria, particularly due to the mcr-3 gene, is a growing global health concern.
- The mcr-3 allele has shown significant spread, especially in China, posing a substantial threat to public health.
Purpose of the Study:
- To develop a rapid, sensitive, and visual loop-mediated isothermal amplification (LAMP) assay for detecting the mcr-3 gene.
- To evaluate the specificity and sensitivity of the developed LAMP assay.
Main Methods:
- Designed LAMP primers and optimized reaction conditions for mcr-3 gene detection.
- Evaluated three detection methods: agarose gel electrophoresis, LAMP-hydroxy naphthol blue (HNB), and LAMP-SYBR Green I (LAMP-SGI).
- Tested the assay using 13 clinical bacterial strains and 11 negative strains.
Main Results:
- The optimized LAMP assay detected the mcr-3 gene within 1 hour at 62°C under isothermal conditions.
- Visual detection methods (LAMP-HNB and LAMP-SGI) demonstrated 10-fold higher sensitivity than conventional PCR.
- The assay exhibited high specificity and sensitivity, with easily distinguishable color changes for visual inspection.
Conclusions:
- A rapid, portable, and economical visual LAMP assay for mcr-3 gene detection was successfully established.
- This assay is well-suited for resource-limited settings and field laboratories.
- The developed method contributes to combating the spread of colistin resistance.
