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Methods for detecting mycobacteria with solvatochromic dye conjugates

A technology of chromogenic dyes and mycobacteria, applied in biochemical equipment and methods, biological material analysis, styrene-based dyes, etc., can solve problems such as poor specificity and low sensitivity

Pending Publication Date: 2019-05-21
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both approaches also require extensive processing to remove excess dye from debris and other bacteria while keeping the dye within the target mycobacteria
Therefore, these tests have poor specificity and low sensitivity

Method used

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  • Methods for detecting mycobacteria with solvatochromic dye conjugates
  • Methods for detecting mycobacteria with solvatochromic dye conjugates
  • Methods for detecting mycobacteria with solvatochromic dye conjugates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0277] Example 1. Detection of mycobacteria with DMN-Tre conjugates

[0278] First, a DMN-Tre conjugate was synthesized, along with two control compounds DMN-glucose (DMN-Glc) and a non-fluorescent analogue 6-fluorescein-trehalose (6-FITre), such as Figure 8 , where these structures are labeled (1), (2) and (3) accordingly. After confirming that the fluorescent properties of DMN-Tre were similar to those reported for the free dye, including an approximately 700-fold enhancement in fluorescence intensity when dissolved in organic solvents versus water, several strains from the phylum Actinomycetes were tested to evaluate The ability of DMN-Tre to label bacteria with mycobacterial membranes. Specifically, each of Mycobacterium smegmatis (Msmeg), Corynebacterium glutamicum (Cg) and Mycobacterium marinum (Mm) in exponential growth phase was treated with 100 μM DMN-Tre or its non-fluorescent analogous 6-FITre were incubated for 1, 2 and 6 hours, respectively, and then imaged w...

Embodiment 2

[0280] Example 2.Selectivity of DMN-Tre conjugates to mycobacteria

[0281] The potential of DMN-Tre as a diagnostic tool, including for the diagnosis of TB, depends on its selectivity for mycobacteria among other bacterial species. Therefore, to test its selectivity, classical Gram-negative and Gram-positive organisms (Bacillus subtilis (Bs), Escherichia coli (Ec), Listeria monocytogenes (Listeria) monocytogenes) (Lm) and Staphylococcus aureus (Sa)) were incubated with DMN-Tre. Figure 11A The results of this test are presented in which, without washing, none of the non-mycobacterial strains showed any detectable fluorescent labeling by the conjugate ("DMN" row), whereas by light microscopy in all tested samples The presence of bacteria was confirmed ("DIC" row) and the overall labeling result was further confirmed by the superposition of the two detection methods used ("Merge" row). In the same experiment, mycobacterial Cg was brightly labeled by DMN-Tre. This specifici...

Embodiment 3

[0282] Example 3. Metabolic properties of DMN-Tre conjugate uptake by mycobacteria

[0283] A series of experiments were performed to confirm that DMN-Tre labeling was due to metabolic transformation within the mycobacterial membrane or non-specific insertion of trehalose mycolic acid into the mycobacterial membrane. First, flow cytometry was used to confirm that Msmeg showed significant fluorescence above background within 20 minutes ( Figure 12 A), and >70% of cells were labeled within 30 minutes ( Figure 12 B). DMN-Glc with the same dye but mounted on glucose did not label Msmeg, suggesting that the trehalose scaffold is key to the observed fluorescent signal from DMN-Tre treatment ( Figure 12 C). Likewise, DMN-Tre labeling was reduced in the presence of excess trehalose, suggesting competition in the same biosynthetic pathway ( Figure 12 D). DMN-Tre labeling of a small panel of Msmeg trehalose transporter mutants was also assessed, and DMN-Tre incorporation was ...

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Abstract

A series of carbohydrate-dye conjugates, as well as a method for detection of pathogenic or other organisms (e.g., bacteria) using the same are provided. The carbohydrate-dye conjugate can be enzymatically incorporated into live and active (viable) bacteria of interest for facile detection of the bacteria. The conjugate incorporation is achieved by utilizing one or more of the enzymes that are endogenous to the bacteria of interest, which can incorporate the conjugate via the conjugate's carbohydrate. A detectable signal is produced by the conjugate's dye only upon incorporation into the bacteria of interest, due to the changes in the dye's local environment upon incorporation. The conjugate may be metabolically incorporated into the fatty outer membrane of a bacterial cell wall, which provides a distinctly hydrophobic environment for the conjugate's dye, causing it to produce a detectable signal.

Description

[0001] Statement Regarding Federally Funded Research [0002] This invention was made with Government support under contract AI051622 awarded by the National Institutes of Health. The government has certain rights in this invention. [0003] Cross References to Related Applications [0004] This application claims priority to US Provisional Patent Application No. 62 / 368928, filed July 29, 2016, the disclosure of which is incorporated herein by reference. field of invention [0005] The present disclosure relates to carbohydrate-dye conjugates and methods of using the same to detect pathogenic or other organisms. Background of the invention [0006] Mycobacteria, belonging to the Mycobacterium genus of the Actinobacteria phylum, are an important cause of morbidity and mortality, especially in immunocompromised or elderly individuals and in settings with limited medical resources country. 95% of human infections are caused by seven species: Mycobacterium tuberculosis, M. a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04
CPCG01N2333/34G01N2333/35C12Q1/04C09B23/14
Inventor C·R·贝尔托齐M·卡马里扎P·希
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV