Kit for detecting 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 kit for detecting peptidoglycan, β-glucan, bacteria, or bacterial fragments in a sample, comprising:
A. a prophenoloxidase cascade system,
B. a phenoloxidase substrate that generates a quinone reaction product, and
C. 3-methyl-2-benzothiazolinone hydrazone or derivative thereof.
2. The kit of claim 1 , wherein the prophenoloxidase cascade system is obtained from insect plasma or hemolymph.
3. The kit of claim 2 , wherein the prophenoloxidase cascade system is obtained from tobacco hornworm hemolymph.
4. The kit of claim 3 , wherein the prophenoloxidase cascade system comprises prophenoloxidase activating enzyme, prophenoloxidase, and a serine proteinase cascade.
5. The kit of claim 4 , further comprising a peptidoglycan binding protein or a β-glucan binding protein.
6. The kit of claim 1 , wherein the phenoloxidase substrate that generates a quinone reaction product is L-3,4-dihydroxyphenylalanine, dopamine, 3,4-dihydroxyphenyl propionic acid, 3,4-dihydroxyphenyl acetic acid, or catechol.
7. The kit of claim 1 , further comprising a peptidoglycan, β-glucan, bacterial, or bacterial fragment standard.
8. The kit of claim 7 , comprising a peptidoglycan standard, which is isolated bacterial peptidoglycan, whole bacterial extract, or inactivated whole bacteria.
9. The kit of claim 1 , further comprising instructions for spectrophotometric detection or a color coded scale for visual evaluation.
10. The kit of claim 1 , further comprising a sterile sample receptacle.
11. The kit of claim 1 , further comprising an extraction solution.
12. The kit of claim 11 , wherein the extraction solution is an alkaline extraction solution.
13. The kit of claim 1 , further comprising a neutralization buffer.
14. The kit of claim 13 , wherein component (c) is dissolved in the neutralization buffer.
15. The kit of claim 13 , wherein component (c) is co-lyophilized with a prophenoloxidase cascade system and a phenoloxidase substrate that generates a quinone reaction product.
16. The kit of claim 1 , further comprising a stop reagent.
17. The kit of claim 16 , wherein the stop reagent is an acid reagent.
18. The kit of claim 16 , wherein the stop reagent is phenoloxidase inhibitor.
19. The kit of claim 16 , wherein the stop reagent is a strong anionic detergent alone or in combination with a phenoloxidase inhibitor.
20. The kit of claim 1 , wherein the sample is a clinical sample, an environmental sample, an agricultural sample, a manufacturing sample, or a medical product.
21. The kit of claim 20 , wherein the sample is a clinical sample, which is a bodily fluid, tissue specimen, hydration fluid, nutrient fluid, blood, blood product, vaccine, anesthetic, pharmacologically active agent, or an imaging agent.
22. The kit of claim 21 , wherein the sample is selected from the group consisting of urine, a urine product, and cerebrospinal fluid.
23. The kit of claim 20 , wherein the sample is a suspension or a liquid.
24. The kit of claim 23 , wherein the sample is processed by centrifugation and bacteria or fungi present in the sample are pelleted during centrifugation.
25. The kit 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.
26. The kit of claim 11 , wherein the sample is exposed to an extraction solution at an elevated temperature and then incubated with the hemolymph.
27. The kit of claim 13 , wherein the sample is exposed to a neutralization buffer and then incubated 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.
28. The kit of claim 13 , wherein the neutralization buffer comprises 3-methyl-2-benzothizolinone hydrazone or other hydrazone derivative thereof.
29. The kit of claim 27 , wherein the hemolymph, and 3-methyl-2-benzothiazolinone hydrazone or other hydrazone derivative are lyophilized.
30. The kit of claim 27 , wherein the lepidopteran insect is a hornworm.
31. The kit of claim 23 , wherein the sample is prepared by filtration and then incubated with hemolymph.
32. The kit of claim 1 , for use in diagnosing urinary tract infections, bacterial meningitis, bacterial infections of the CNS, or bacterial infections.
33. The kit of claim 32 , further comprising a sterile filter for filtering the sample during sample preparation.