Additional file 1 of A colorimetric, photothermal, and fluorescent triple-mode CRISPR/cas biosensor for drug-resistance bacteria detection

Supplementary Material

DataSheet1_Rapid Detection and Antimicrobial Susceptibility Testing of Pathogens Using AgNPs-Invertase Complexes and the Personal Glucose Meter.DOCX

Rapid detection of pathogens and assessment of antimicrobial susceptibility is of great importance for public health, especially in resource-limiting regions. Herein, we developed a rapid, portable, and universal detection method for bacteria using AgNPs-invertase complexes and the personal glucose meter (PGM). In the presence of bacteria, the invertase could be released from AgNPs-invertase complexes where its enzyme activity of invertase was inhibited. Then, the enzyme activity of invertase was restored and could convert sucrose into glucose measured by a commercially PGM. There was a good linear relationship between PGM signal and concentration of E. coli or S. aureus as the bacteria model with high sensitivity. And our proposed biosensor was proved to be a rapid and reliable method for antimicrobial susceptibility testing within 4 h with consistent results of Minimum Inhibitory Concentrations (MICs) testing, providing a portable and convenient method to treat infected patients with correct antibiotics and reduce the production of antibiotic-resistant bacteria, especially for resource-limiting settings.</p

An Ultrasensitive Colorimetric Foodborne Pathogenic Detection Method Using a CRISPR/Cas12a Mediated Strand Displacement/Hybridization Chain Reaction

Accurate, rapid, and sensitive pathogenic detections play an important role in food safety. Herein, we developed a novel CRISPR/Cas12a mediated strand displacement/hybridization chain reaction (CSDHCR) nucleic acid assay for foodborne pathogenic colorimetric detection. A biotinylated DNA toehold is coupled on avidin magnetic beads and acts as an initiator strand to trigger the SDHCR. The SDHCR amplification allowed the formation of long hemin/G-quadruplex-based DNAzyme products to catalyze the TMB-H2O2 reaction. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a was activated to cleave the initiator DNA, resulting in the failure of SDHCR and no color change. Under optimal conditions, the CSDHCR has a satisfactory linear detection of DNA targets with a regression equation Y = 0.0531*X – 0.0091 (R2 = 0.9903) in the range of 10 fM to 1 nM, and the limit of detection was determined as 4.54 fM. In addition, Vibrio vulnificus, one foodborne pathogen, was used to verify the practical application of the method, and it showed satisfactory specificity and sensitivity with a limit of detection at 1.0 × 100 CFU/mL coupling with recombinase polymerase amplification. Our proposed CSDHCR biosensor could be a promising alternative method for ultrasensitive and visual detection of nucleic acids and the practical application of foodborne pathogens

Data_Sheet_1_A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a.PDF

Aeromonas hydrophila is an emerging waterborne and foodborne pathogen with pathogenicity to humans and warm water fishes, which severely threatens human health, food safety and aquaculture. A novel method for the rapid, accurate, and sensitive detection of pathogenic A. hydrophila is still needed to reduce the impact on human health and aquaculture. In this work, we developed a rapid, accurate, sensitive, and visual detection method (dRAA-CRISPR/Cas12a), without elaborate instruments, integrating the dualplex recombinase-assisted amplification (dRAA) assay and CRISPR/Cas12a system to detect pathogenic A. hydrophila expressing aerA and/or hlyA virulence genes. The dRAA-CRISPR/Cas12a method has high sensitivity, which can rapidly detect (about 45 min) A. hydrophila with the limit of detection in 2 copies of genomic DNA per reaction, and has high specificity for three pathogenic A. hydrophila strains (aerA+hlyA−, aerA−hlyA+, and aerA+hlyA+). Moreover, dRAA-CRISPR/Cas12a method shows satisfactory practicability in the analysis of the spiked human blood and stool and fish samples. These results demonstrate that our developed pathogenic A. hydrophila detection method, dRAA-CRISPR/Cas12a, is a promising potential method for the early diagnosis of human A. hydrophila infection and on-site detection of A. hydrophila in food and aquaculture.</p

Table_1_Rapid and Sensitive Detection of Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay.DOC

Vibrio vulnificus is an important zoonotic and aquatic pathogen and can cause vibriosis in humans and aquatic animals (especially farmed fish and shrimp species). Rapid and sensitive detection methods for V. vulnificus are still required to diagnose human vibriosis early and reduce aquaculture losses. Herein, we developed a rapid and sensitive diagnostic method comprising a recombinase-aided amplification (RAA) assay and the CRISPR/Cas12a system (named RAA-CRISPR/Cas12a) to detect V. vulnificus. The RAA-CRISPR/Cas12a method allows rapid and sensitive detection of V. vulnificus in 40 min without a sophisticated instrument, and the limit of detection is two copies of V. vulnificus genomic DNA per reaction. Meanwhile, the method shows satisfactory specificity toward non-target bacteria and high accuracy in the spiked blood, stool, and shrimp samples. Therefore, our proposed rapid and sensitive V. vulnificus detection method, RAA-CRISPR/Cas12a, has great potential for early diagnosis of human vibriosis and on-site V. vulnificus detection in aquaculture and food safety control.</p

Data_Sheet_1_One-step synthesized antimicrobial peptide-functionalized gold nanoclusters for selective imaging and killing of pathogenic bacteria.DOCX

The development of multifunctional nanomaterials with bacterial imaging and killing activities is of great importance for the rapid diagnosis and timely treatment of bacterial infections. Herein, peptide-functionalized gold nanoclusters (CWR11-AuNCs) with high-intensity red fluorescence were successfully synthesized via a one-step method using CWR11 as a template and by optimizing the ratio of CWR11 to HAuCl4, reaction time, pH, and temperature. The CWR11-AuNCs bound to bacteria and exhibited selective fluorescence microscopy imaging properties, which is expected to provide a feasible method for locating and imaging bacteria in complex in vivo environments. In addition, CWR11-AuNCs not only retained the antibacterial and bactericidal activities of CWR11 but also exhibited certain inhibitory or killing effects on gram-negative and gram-positive bacteria and biofilms. The MICs of CWR11-AuNCs against Escherichia coli and Staphylococcus aureus were 178 and 89 μg/ml, respectively. Surprisingly, cell viability in the CWR11-AuNC-treated group was greater than that in the CWR11-treated group, and the low cytotoxicity exhibited by the CWR11-AuNCs make them more promising for clinical applications.</p