Method and components for the detection of yeasts and/or molds in a sample

• US 5,854,011 A
• Filed: 12/19/1996
• Issued: 12/29/1998
• Est. Priority Date: 12/19/1996
• Status: Expired due to Term

• Current Assignee: IDEXX Laboratories Incorporated
• Original Assignee: IDEXX Laboratories Incorporated
• Inventors: Chen, Chun-Ming, Gu, Haoyi
• Primary Examiner(s): Witz, Jean C.
• Assistant Examiner(s): HANLEY, SUSAN MARIE
• Application Number:
  US08/769,512
• Time in Patent Office: 740 Days
• Field of Search: 435/34, 435/24, 435/30, 435/31, 435/32, 435/231, 435/184, 435/195, 435/212, 435/219
• US Class Current: 435/24
• CPC Class Codes: C12Q 1/045 Culture media therefor
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 2334/22 4-Methylumbelliferyl, i.e. beta-methylumbelliferone, 4MU
• CPC Class Codes: C12Q 2337/22 7-Amino-4-methylcoumarin, i.e. AMC, MCA

Abstract

A composition and method for detecting the presence or amount of yeasts and molds in a test sample is presented. The composition contains a substrate and an inhibitor for an aminopeptidase. The substrate has a signal moiety capable of providing a detectable signal when cleaved by an aminopeptidase in yeasts or molds. The aminopeptidase inhibitor serves to reduce the endogenous aminopeptidase activity in the test sample. The method to detect yeasts or molds in a sample includes inoculating a test sample with the disclosed composition, incubating the sample and observing any detectable signal that indicates the presence of yeasts or molds.

Claims

1. A composition for use in a medium for detecting yeasts and molds in a test sample comprising:

a) a substrate for an aminopeptidase, wherein a signal moiety is linked to the substrate and said moiety is capable of providing a detectable signal when cleaved by said aminopeptidase in yeasts or molds;

b) an antibiotic capable of inhibiting the growth of bacteria in said sample; and

c) an inhibitor of aminopeptidase endogenous to the test sample in an amount effective to suppress endogenous aminopeptidase activity in said sample suspected of containing yeasts or molds.

2. The composition of claim 1, wherein said the enzyme inhibitor is H-leucine-chloromethylketone.

3. The composition of claim 1, wherein said aminopeptidase is L-leucine aminopeptidase or L-alanine aminopeptidase.

4. The composition of claim 3, wherein said substrate comprises a substrate of L-leucine aminopeptidase or L-alanine aminopeptidase, respectively.

5. The composition of claim 4, wherein said one of said substrates is hydrolyzed by L-leucine aminopeptidase, and the other said substrate is hydrolyzed by L-alanine aminopeptidase.

6. The composition of claim 3, wherein said enzyme substrate is L-leucine 7-amido-4-methylcoumarin, L-leucine β

-naphthylamide, L-alanine 7-amido-4-methylcoumarin, or L-alanine β

-naphthylamide.

7. The composition of claim 1, wherein said signal moiety is ortho-nitrophenyl, 4-methylumbelliferone, para-nitroanilide, 4-methoxy-β

-naphthylamide, 7-amido-4-methylcoumarin.

8. The composition of claim 1, wherein said enzyme substrate is:

N-o-Acetyl-lysine-7-amido-4-methylcoumarin acetate;

N-Acetyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin hydrochloride;

L-Alanine-7-amido-4-methylcoumarin;

β

-Alanine-7-amido-4-methylcoumarin TFA;

D-Alanine-7-amido-4-methylcoumarin TFA;

L-Alanine-7-amido-4-methylcoumarin TFA;

L-Alanine-7-amido-4-methylcoumarin TFA;

L-Alanine-7-amido-4-trifluoro-methylcoumarin TFA;

L-Alanyl-L-alanyl-L-phenylalanine-7-amido-4-methylcoumarin;

L-Alanyl-L-alanyl-L-phenylalanine-7-amido-4-methylcoumarin TFA;

D-Alanyl-L-leucyl-L-lysine-7-amido-4-methylcoumarin Salt;

L-Arginine-7-amido-4-methylcoumarin hydrochloride;

L-Arginyl-L-arginine-7-amido-4-methylcoumarin trihydrochloride;

L-Asparagine-7-amido-4-methylcoumarin TFA;

L-Aspartic acid-β

-(7-amido-4-methylcoumarin);

N-α

-Benzoyl-DL-arginine-7-amido-4-methylcoumarin;

N-α

-Benzoyl-L-arginine-7-amido-4-methylcoumarin;

N-Benzoyl-L-phenylalanyl-L-valyl-L-arginine-7-amido-4-methylcoumarin;

S-Benzyl-L-cysteine-7-amido-4-methylcoumarin;

N-BOC-L-phenylalanyl-L-seryl-L-arginine-7-amido-4-methylcoumarin acetate;

N-BOC-L-vanyl-glycyl-L-arginine-7-amido-4-methylcoumarin hydrochloride;

N-BOC-L-vanyl-L-leucyl-L-lysine-7-amido-4-methylcoumarin Salt;

N-α

-CBZ-L-arginine-7-amido-4-methylcoumarin hydrochloride;

N-CBZ-glycylglycyl-L-leucine-7-amido-4-methylcoumarin;

N-CBZ-glycyl-L-proline-7-amido-4-methylcoumarin;

N-CBZ-glycyl-L-prolyl-L-arginine-7-amido-4-methylcoumarin;

N-β

-CBZ-L-lysine-7-amido-4-methylcoumarin;

N-CBZ-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin hydrochloride;

N-CBZ-L-prolyl-L-arginine-7-amido-4-methylcoumarin hydrochloride;

L-Citrulline-7-amido-4-methylcoumarin hydrochloride;

L-Citrulline-7-amido-4-methylcoumarin hydrochloride TFA;

D-Glutamic acid-γ

-(7-amido-4-methylcoumarin);

L-Glutamic acid-α

-(7-amido-4-methylcoumarin);

L-Glutamine-7-amido-4-methylcoumarin hydrochloride;

Glutaryl-glycyl-L-arginine-7-amido-4-methylcoumarin hydrochloride;

Glutaryl-glycylglycyl-L-phenylalanine-7-amido-4-methylcoumarin;

Glutaryl-L-phenylalanine-7-amido-4-methylcoumarin;

Glycine-7-amido-4-methylcoumarin hydrochloride;

Glycyl-L-alanine-7-amido-4-methylcoumarin hydrochloride;

Glycyl-L-arginine-7-amido-4-methylcoumarin Salt;

Glycylglycine-7-amido-4-methylcoumarin hydrochloride;

Glycyl-L-phenylalanine-7-amido-4-methylcoumarin;

Glycyl-L-proline-7-amido-4-methylcoumarin hydrochloride;

L-Histidine-7-amido-4-methylcoumarin;

L-Isoleucine-7-amido-4-methylcoumarin;

L-Isoleucine-7-amido-4-methylcoumarin TFA;

L-Leucine-7-amido-4-methylcoumarin;

L-Leucine-7-amido-4-methylcoumarin hydrochloride;

L-Leucyl-1-valvyl-1-tyrosine-7-amido-4-methylcoumarin;

L-Lysine-7-amido-4-methylcoumarin acetate;

L-Methionine-7-amido-4-methylcoumarin acetate;

L-Ornithine-7-amido-4-methylcoumarin carbonate;

L-Phenylalanine-7-amido-4-methylcoumarin TFA;

L-Proline-7-amido-4-methylcoumarin hydrochloride;

L-Prolyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin Salt;

L-Pyroglutamic acid-7-amido-4-methylcoumarin;

L-Serine-7-amido-4-methylcoumarin hydrochloride;

L-Seryl-L-tyrosine-7-amido-4-methylcoumarin hydrate;

or, L-Tyrosine-7-amido-4-methylcoumarin.

9. The composition of claim 1 wherein said substrate comprises a nutrient moiety, and the nutrient moiety is linked to the signal moiety by a covalent bond.

10. The composition of claim 9 wherein the covalent bond is a peptide bond.

11. The composition of claim 1, wherein the composition comprises two or more substrates for the aminopeptidase, each substrate comprising a signal moiety that causes or produces a substantially identical detectable signal.

12. The composition of claim 1, wherein said detectable moiety is a fluorescent moiety, and said fluorescent moiety is capable of providing a fluorescent signal.

13. The composition of claim 1, wherein said detectable moiety is a chromogen moiety, and said chromogen moiety is capable of providing a signal in the visible, ultraviolet or infrared spectrum.

14. The composition of claim 1 is gel.

15. The composition of claim 1 is liquid.

16. The composition of claim 1 comprised in a powder form.

17. The yeast and/or mold growth medium comprising the composition of claim 1.

18. A method of detecting the presence or amount of yeasts or molds in a test sample, comprising the step of:

inoculating a medium comprising the composition of claim 1 with said test sample;

incubating the inoculated medium under conditions which support the growth of yeasts or molds; and

,

observing any detectable signal, whereby the presence of a detectable signal indicates the presence of yeasts or molds in the test sample.

19. The method of claim 18 wherein the medium is added to aliquots of serial dilutions of the test sample.

20. The method of claim 19 wherein the medium is added to aliquots of 10⁰, 10^-1, and 10^-2 dilutions of the test sample.

21. The method of claim 16 conducted at a temperature whereby yeasts and molds will grow.

22. The method of claim 21 conducted at a temperature in a temperature range of 20°

C. to 35°

C.