Combining CRISPR-Cas12a-Based Technology and Metagenomics Next Generation Sequencing: A New Paradigm for Rapid and Full-Scale Detection of Microbes in Infectious Diabetic Foot Samples.

Yixin Chen,Lin Li,Chao Liu,Yaping Xie,Ya Shi,Shunwu Fan,Hanxin Zhao,Weifen Zhu,Jie Yang,Jiaxing You,Hongye Li

doi:10.3389/fmicb.2021.742040

Yixin Chen, Lin Li + Show 9 more

Open Access

https://doi.org/10.3389/fmicb.2021.742040

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Abstract

Introduction: Diabetic foot infections (DFIs) pose a huge challenge for clinicians. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is one of the most significant pathogens of DFI. Early pathogen identification will greatly benefit the diagnosis and treatment of the disease. However, existing diagnostic methods are not effective in early detection.Methods: We developed an assay that coupled loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) techniques to enable quick and specific detection of Staphylococcus aureus and differentiate MRSA in samples from patients with DFI. Furthermore, the results were compared using a reference culture, quantitative real-time polymerase chain reaction (qRT-PCR), and metagenomics next generation sequencing (mNGS).Results: The CRISPR-LAMP assay targeting nuc and mecA successfully detected S. aureus strains and differentiated MRSA. The limit of detection (LoD) of the real-time LAMP for nuc and mecA was 20 copies per microliter reaction in comparison to two copies per μL reaction for the qRT-PCR assay. The specificity of the LAMP-CRISPR assay for nuc was 100%, without cross-reactions with non-S. aureus strains. Evaluating assay performance with 18 samples from DFI patients showed that the assay had 94.4% agreement (17/18 samples) with clinical culture results. The results of mNGS for 8/18 samples were consistent with those of the reference culture and LAMP-CRISPR assay.Conclusion: The findings suggest that the LAMP-CRISPR assay could be promising for the point-of-care detection of S. aureus and the differentiation of MRSA in clinical samples. Furthermore, combining the LAMP-CRISPR assay and mNGS provides an advanced platform for molecular pathogen diagnosis of DFI.

Highlights

Diabetic foot infections (DFIs) pose a huge challenge for clinicians
We developed an assay coupling loop-mediated isothermal amplification (LAMP) and trans-cleavage of Cas12a for the quick and specific detection of S. aureus and the differentiation of methicillin-resistant S. aureus (MRSA) in samples of patients with diabetic foot infections (DFI)
We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system to realize the visual readout of the targeted amplicons, based on the collateral DNA

Summary

Introduction

Diabetic foot infections (DFIs) pose a huge challenge for clinicians. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is one of the most significant pathogens of DFI. Pathogen identification will greatly benefit the diagnosis and treatment of the disease. One of the most critical complications of diabetes mellitus, has become a significant public health problem. When patients with diabetic foot ulcers first consult physicians, over half of them have complications with diabetic foot infections (DFIs) (Prompers et al, 2006; Jia et al, 2017). Treatment of DFI requires precise evaluation of infection conditions, pathogen confirmation of infectious ulcers, appropriate selection of antibiotics, and surgical intervention, if necessary (Lipsky et al, 2012). Broad-spectrum empirical antimicrobials would be the primary option for clinicians to hinder the further deterioration of infectious conditions and lower medical risks, which increases antibiotic exposure and selective pressure that contribute to the development of multi-drug resistant microorganisms. A rapid, accessible, and accurate diagnostic test for pathogenic identification is valuable for clinical procedures

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