We are excited to see this new preprint focused on antimicrobial resistance (AMR).
The preprint features Bento Lab as part of a molecular detection toolbox for bacterial antibiotic resistance gene markers, with different workflows for a) well-equipped labs, b) equipment-poor labs, and c) portable uses with Bento Lab and lateral flow strips.
The molecular toolbox is reported in Vargas-Reyes et al. (2024) and was developed by a collaboration of scientists from Universidad Peruna de Ciencias Aplicadas, Lima, Peru, and the University of Camerino, Camiro, Italy.
The group wanted to address the global health problem of antimicrobial resistance (AMR), where the use of antibiotics plus environmental factors create a selective increase in bacterial strains resistant to antibiotic drugs — a big problem if any of these bacteria become involved in human or animal infections!
“The development of a simplified detection system for ARGs [antimicrobial resistance genes] is crucial for investigating AMR across a spectrum of contexts, from clinical settings to environmental and veterinary surveillance.”
This problem can be particularly notable in low- and middle-income countries where sanitation and hygiene standards may be limited and molecular laboratories can be less well-equipped to run surveillance and tracking programs.
To help address this problem, the team set out to develop a testing “toolbox” that could combine the sensitivity, accuracy, and speed of molecular techniques such as qPCR and DNA sequencing, with the low costs of more conventional antimicrobial susceptibility testing. They also wanted to allow their toolbox to be used in a wide range of test scenarios, from well-equipped laboratories to portable on-site applications.
“This toolbox is composed of a collection of primers, crRNAs, and well-defined protocols to be applied in laboratories with different equipment availability.”
The solution the team came up with was a set of workflows that first amplified specific antimicrobial target genes using conventional PCR, and then detected specific nucleic acid sequences of antimicrobial genes or plasmids using CRISPR technology. This combination of techniques is rapid, extremely sensitive and specific, and low-cost.
The CRISPR detection system involves a targeting sequence called crRNA that is synthesised to match the gene region of interest. When crRNA binds to the complementary target DNA:
- The Cas12a enzyme is activated and exhibits collateral nuclease activity
- Collateral nuclease activity non-specifically cuts a reporter probe composed of a fluorescence donor/quencher FRET (fluorescence resonance energy transfer) pair placed into single-stranded DNA ✂️
- The released fluorophore present in the FRET pair can now fluoresce in the presence of blue light 🌟
- The light released can be detected and measured, and the brightness is related to the target DNA’s concentration within the sample
The authors adapted this technique so it could be used in a range of lab conditions:
- In a high-equipped laboratory scenario, fluorescence can be detected and quantified precisely for large numbers of samples using a multimode microplate reader.
- In a less-well-equipped laboratory, fluorescence can be qualitatively detected using a blue light transilluminator
- In an on-site scenario, the Bento Lab portable PCR workstation can be used for PCR and CAS12a/crRNA incubations, and a Lateral Flow Assay platform (from Milenia Biotec GmbH) used to detect the cleaved reporter probe. In this scenario, the reporter probe contains biotin which (when the probe is cleaved by Cas12a) binds tightly to the streptavidin protein present in the lateral flow test (T) band. This allows the results to be visualised on the lateral flow strip by a colour change rather than by fluorescence.
Using these methods, the authors were able to detect less than 3x 10-7 ng of DNA or 100 colony-forming units per mL, detecting antibiotic-resistance genes conferring resistance to the antibiotics cefotaxime, other β lactams, and also amphenicols. Impressively, the assays’ sensitivity was 10x to 100x the lowest target concentration that could be detected using PCR and agarose gel electrophoresis analysis.
The workflows designed for different lab scenarios worked equally well in detecting the presence of two antimicrobial resistance genes in the authors’ test panels, although transilluminator-based detection and Lateral Flow Strip assays were only capable of qualitative, rather than quantitative, detection.
The authors say that their research “presents an optimized molecular detection toolbox for two of the most widespread genes conferring resistance to beta-lactams (blaCTX-M-15) and amphenicols (floR) and Int1 integrase, using the CRISPR-Cas technology.”
“The technical features of our toolbox are valuable because they enable accurate, consistent, and cost effective monitoring/surveillance efforts. These aspects are particularly beneficial for resource-limited settings lacking an adequate, cutting-edge, laboratory infrastructure.“
It’s a very cool toolbox of techniques, and it’s great to see Bento Lab was used as part of the proof-of-concept! It’s also a toolbox that should be useful for the detection of a wide range of other antibiotic resistance markers, as well as other markers, with minor modifications!
Congratulations to the team!
You can read the preprint here:
Vargas-Reyes et al. (2024). Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers. bioRxiv, 2024-11.
https://www.biorxiv.org/content/10.1101/2024.11.14.623642v1.abstract
