Method for detecting bacteria
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 or β-glucan in a sample,
wherein the sample is selected from the group consisting of urine, a urine product, and cerebrospinal fluid, the method comprising:
(a) incubating the sample with (i) hemolymph from a lepidopteran insect; and (ii) a phenoloxidase substrate that generates a reaction product, and 3-methyl-2-benzothiazolinone hydrazone or other hydrazone derivative thereof; and,
(b) detecting the formation of a colored-reaction product by absorbance, wherein formation of the colored-reaction product indicates the presence of peptidoglycan or β-glucan in the sample, and the absence of the colored-reaction product indicates the absence of peptidoglycan or β-glucan in the sample.
2. The method of claim 1 , wherein the sample is a suspension or a liquid.
3. The method of claim 2 , wherein prior to step (b), the sample is processed by centrifugation.
4. 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.
5. The method of claim 1 , further comprising the step of exposing the sample to an extraction solution at an elevated temperature, prior to incubating the sample with the hemolymph.
6. The method of claim 5 , wherein the extraction solution is an alkaline extraction solution.
7. The method of claim 5 , further comprising the step of exposing the sample to a neutralization buffer prior to incubating the sample with the hemolymph from a lepidopteran insect, the phenoloxidase substrate that generates a quinone reaction product, and 3-methyl-2-benzothiazolinone hydrazone or derivative thereof.
8. The method of claim 7 , wherein the hemolymph from a lepidopteran insect is lyophilized.
9. The method of claim 7 , wherein the neutralization buffer comprises 3-methyl-2-benzothizolinone hydrazone or other hydrazone derivative thereof.
10. The method of claim 1 , further comprising the step of exposing the sample to a stop reagent prior to step (b).
11. The method of claim 10 , wherein the stop reagent is an acid reagent, a phenoloxidase inhibitor, or a strong anionic detergent alone or in combination with a phenoloxidase inhibitor.
12. A method for detecting bacteria or bacterial fragments in a sample, wherein the sample is selected from the group consisting of urine, a urine product, and cerebrospinal fluid, the method comprising the steps of:
a. extracting the sample in an extraction solution,
b. incubating the sample with hemolymph from a lepidopteran, dopamine, and 3-methyl-2-benzothiazolinone hydrazone or other hydrazone derivative dissolved in neutralization buffer, and
(c) detecting the formation of a colored-reaction product by absorbance, wherein the formation of the colored-reaction product indicates the presence of bacteria or bacterial fragments in the sample, and the absence of the colored-reaction product indicates the absence of bacteria or bacterial fragments in the sample.
13. The method of claim 12 , further comprising the step of exposing the sample to a neutralization buffer prior to incubating the sample with the hemolymph, and 3-methyl-2-benzothiazolinone or other hydrazone derivative thereof.
14. The method of claim 13 , wherein the hemolymph, dopamine, and 3-methyl-2-benzothiazolinone hydrazone or other hydrazone derivative are lyophilized.
15. The method of claim 1 , wherein the lepidopteran insect is a hornworm.
16. The method of claim 1 , wherein formation of the colored-reaction product indicates the presence of peptidoglycan or β-glucan in the sample and provides a positive diagnosis of urinary tract infections, bacterial meningitis, or other bacterial infections of the CNS.
17. The method of claim 1 , wherein the absorbance of the colored reaction product indicating the presence of peptidoglycan or β-glucan is monitored continuously over time.
18. The method of claim 17 , wherein the absorbance of the colored reaction product is monitored continuously over time until a threshold absorbance is reached, indicating the presence of peptidoglycan or β-glucan in the sample.