Rapid peptidoglycan-based assay for detection of bacterial contamination

1. A method for detecting peptidoglycan, β-glucan, bacteria, bacterial fragments, or fungi in a sample, comprising steps of:

(a) providing the sample;

(b) providing reagents, comprising:

(i) a hemolymph from a lepidopteran insect;

(ii) a phenoloxidase substrate; and

(iii) 3-methyl-2-benzothiazolinone hydrazone or a 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;

(c) combining the sample and reagents;

(d) spectrophotometrically detecting a measurable absorbance in the combination,

wherein when peptidoglycan, bacteria, bacterial fragments, ß-glucan, or fungi are present in the sample, a colored reaction product forms in the combination, and

(e) quantifying the concentration of peptidoglycan, bacteria, bacterial fragments, ß-glucan, or fungi present in the sample, comprising steps of:

determining the detected absorbance of the combination at a time elapsed from the step of combining to the step of detecting; and

correlating with a standard curve having absorbance and concentration.

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 sample is a clinical sample, which is an anesthetic, blood, a blood product, a dialysis fluid, a hydration fluid, an imaging agent, a nutrient fluid, a pharmacologically active agent, platelets, or a vaccine.

4. The method of claim 2 , wherein the 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 combining 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 the step of exposing the sample to an extraction solution at an elevated temperature, prior to the step (c).

9. The method of claim 8 , wherein the extraction solution is an alkaline extraction solution.

10. The method of claim 8 , further comprising the step of exposing the sample to a neutralization buffer prior to the step of combining 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.

11. The method of claim 1 , wherein at least one of the provided reagents is in a lyophilized form.

12. The method of claim 1 , further comprising the step of exposing the sample to a stop reagent.

13. The method of claim 12 , wherein the stop reagent is an acid reagent, a phenoloxidase inhibitor, or a strong anionic detergent alone or in combination with a phenoloxidase inhibitor.

14. 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.

15. The method of claim 1 , wherein the absorbance of the colored reaction product indicating the presence of bacteria is monitored continuously over time.

16. The method of claim 14 , wherein the lepidopteran insect is hornworm or silkworm.

17. The method of claim 7 , wherein the phenoloxidase substrate is dopamine.