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

TITLE=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

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.742040

DOI=10.3389/fmicb.2021.742040

ISSN=1664-302X

ABSTRACT=<p><bold>Introduction:</bold> Diabetic foot infections (DFIs) pose a huge challenge for clinicians. <italic>Staphylococcus aureus</italic>, including methicillin-resistant <italic>S. aureus</italic> (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.</p><p><bold>Methods:</bold> 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 <italic>Staphylococcus aureus</italic> 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).</p><p><bold>Results:</bold> The CRISPR-LAMP assay targeting <italic>nuc</italic> and <italic>mecA</italic> successfully detected <italic>S. aureus</italic> strains and differentiated MRSA. The limit of detection (LoD) of the real-time LAMP for <italic>nuc</italic> and <italic>mecA</italic> 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 <italic>nuc</italic> was 100%, without cross-reactions with non-<italic>S. aureus</italic> 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.</p><p><bold>Conclusion:</bold> The findings suggest that the LAMP-CRISPR assay could be promising for the point-of-care detection of <italic>S. aureus</italic> 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.</p>