Rapid peptidoglycan-based assay for detection of bacterial contamination
View Patent ↗The invention relates to a colorimetric method for detecting bacterial or fungal pathogens by detecting peptidoglycan or (1-3)-β-D-glucan in a sample.
1. A method for detecting peptidoglycan, bacteria, bacterial fragments, β-glucan, or fungi in a sample, comprising the steps of:
(a) incubating the sample with (i) hemolymph from a lepidopteran insect, (ii) a phenoloxidase substrate, and (iii) 3-methyl-2-benzothiazolinone hydrazone or derivative thereof having the general structure
wherein R 1 represents H, alkyl, halide, —NO 2 , —CO 2 , or —SO 3 ; wherein R 2 represents H, or —SO 2 R 3 ; and wherein R 3 represents alkyl, aryl and heteroaryl; and
(b) detecting the formation of a colored-reaction product by absorbance, wherein formation of the colored-reaction product indicates the presence of peptidoglycan, bacteria, bacterial fragments, β-glucan, or fungi in the sample, and the absence of colored-reaction product indicates the absence of peptidoglycan, bacteria, bacterial fragments, β-glucan, or fungi in the sample.
2. The method of claim 1 , wherein the sample is a clinical sample, an environmental sample, an agricultural sample, a manufacturing sample, or a medical product.
3. The method of claim 2 , wherein the clinical sample is selected from the group consisting of a tissue specimen, a hydration fluid, a nutrient fluid, blood, a blood product, a vaccine, an anesthetic, a pharmacologically active agent, a dialysis fluid, platelets, and an imaging agent.
4. The method of claim 2 , wherein the clinical sample is selected from the group consisting of urine, a urine product, and cerebrospinal fluid.
5. The method of claim 1 , wherein the sample is a suspension or a liquid.
6. The method of claim 5 , further comprising a step of centrifugating the sample prior to the step of incubating, wherein bacteria or fungi present in the sample are pelleted during the centrifugation step.
7. The method of claim 1 , wherein the phenoloxidase substrate is L-3,4-dihydroxyphenylalanine, dopamine, 3,4-dihydroxyphenyl propionic acid, 3,4-dihydroxyphenyl acetic acid, a dihydroxyphenol, a monophenol, or catechol.
8. The method of claim 1 , further comprising a step of exposing the sample to an extraction solution prior to the step of incubating.
9. The method of claim 8 , wherein the extraction solution is an alkaline extraction solution.
10. The method of claim 8 , further comprising a step of exposing the sample to a neutralization buffer prior to the step of incubating.
11. The method of claim 10 , wherein the hemolymph is lyophilized.
12. The method of claim 10 , wherein the neutralization buffer comprises the 3-methyl-2-benzothizolinone hydrazone or derivative thereof.
13. The method of claim 1 , further comprising a step of exposing the sample to a stop reagent.
14. The method of claim 13 , wherein the stop reagent is selected from the group consisting of an acid reagent, a phenoloxidase inhibitor, an anionic detergent, and a combination of a phenoloxidase inhibitor and an anionic detergent.
15. The method of claim 1 , wherein the 3-methyl-2-benzothiazolinone hydrazone or derivative thereof is lyophilized.
16. The method of claim 1 , wherein the lepidopteran insect is selected from the group consisting of a hornworm, a silkworm, a flesh fly, a house fly, an Emmafield cricket and a beetle.
17. The method of claim 1 , further comprising a step of filtering the sample prior to the step of incubating.
18. The method of claim 1 , wherein formation of the colored-reaction product provides a positive diagnosis of a urinary tract infection, bacterial meningitis, or other bacterial infection of the CNS.
19. The method of claim 1 , wherein the absorbance of the colored reaction colored-reaction product is monitored continuously over time.
20. The method of claim 19 , wherein the absorbance of the colored-reaction product is monitored continuously over time until a threshold absorbance is reached, wherein the threshold absorbance indicates the presence of peptidoglycan, bacteria, bacterial fragments, β-glucan, or fungi in the sample.
21. The method of claim 7 , wherein the phenoloxidase substrate is dopamine.
22. The method of claim 21 , wherein the dopamine is lyophilized.
23. The method of claim 16 , wherein the lepidopteran insect is a hornworm.