Method for classifying a microorganism in a biological sample

• US 7,479,375 B2
• Filed: 01/13/2006
• Issued: 01/20/2009
• Est. Priority Date: 01/13/2005
• Status: Expired due to Fees

• Current Assignee: Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Original Assignee: Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Inventors: Nutter, Dana M., Wainwright, Norman R.
• Primary Examiner(s): Weber; Jon P
• Assistant Examiner(s): Shen; Bin
• Application Number:
  US11/331,600
• Publication Number:
  US 20060216780A1
• Time in Patent Office: 1,103 Days
• Field of Search: None
• US Class Current: 435/7.2
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 33/579 involving limulus lysate

Abstract

The invention provides a method using a hemocyte preparation, for example, Limulus amebocyte lysate, for detecting in a single assay the presence of at least one of a Gram negative bacterium, a Gram positive bacterium, and a fungus in a sample of interest. The method exploits the differential reactivity of Gram negative bacteria, Gram positive bacteria, and fungi with the hemocyte preparation to produce measurable changes in a property, for example, an optical property, of the mixture. Because the Gram negative bacteria, Gram positive bacteria and fungi each produce different changes in a given property, for example, an optical property, it is possible to classify the type of microorganism present in the sample of interest.

Claims

1. A method of classifying a microorganism in a test sample between a Gram negative bacterium, a Gram positive bacterium, or a fungus, the method comprising the steps of:

(a) combining the test sample, which contains a standardized amount of the microorganism, with a hemocyte preparation to produce a mixture;

(b) measuring either (i) an optical property of the mixture at a preselected time or (ii) a time in which a preselected change occurs in an optical property of the mixture;

wherein the optical property is selected from the group consisting of absorbance, transmittance, optical density, and turbidity; and

(c) using the optical property of step (b)(i) or the time value of step (b)(ii) to determine whether the microorganism in the test sample is a Gram negative bacterium, a Gram positive bacterium, or a fungus.

2. The method of claim 1, wherein in step (c), a first value of the optical property is indicative of the presence of Gram negative bacteria in the sample, a second value of the optical property is indicative of the presence of fungi in the sample, and a third value of the optical property is indicative of the presence of Gram positive bacteria in the sample, wherein the first value of the optical property, the second value of the optical property, and the third value of the optical property are values of the same optical property.

3. The method of claim 1, wherein in step (c), a first time value is indicative of the presence of Gram negative bacteria in the sample, a second time value is indicative of the presence of fungi in the sample, and a third time value is indicative of the presence of Gram positive bacteria in the sample.

4. The method of claim 1, wherein in step (b)(i), the optical property is absorbance of light at a preselected wavelength or transmittance of light at a preselected wavelength.

5. The method of claim 1, wherein in step (b)(i), the optical property is turbidity.

6. The method of claim 1, wherein in step (b)(ii), the optical property is absorbance of light at a preselected wavelength or transmittance of light at a preselected wavelength.

7. The method of claim 1, wherein in step (b)(ii), the optical property is turbidity.

8. The method of claim 1, wherein in step (a), the hemocyte preparation is an amebocyte lysate.

9. The method of claim 8, wherein the amebocyte lysate is a Limulus amebocyte lysate.

10. The method of claim 1, wherein step (a) is performed in a cartridge.

11. The method of claim 1, wherein step (a) is performed in a well defined by a solid support.

12. The method of claim 1, wherein in step (a), the mixture further comprises a substrate for a clotting enzyme in the hemocyte preparation, wherein the substrate comprises a chromogenic moiety or a fluorogenic moiety.

13. The method of claim 12, wherein the chromogenic moiety comprises a para-nitroaniline chromophore.

14. The method of claim 12, wherein the chromogenic moiety comprises Ile-Glu-Ala-Arg-pNA (SEQ ID NO:

1), where pNA is a para-nitroaniline group.

15. The method of claim 1, wherein step (c) comprises comparing the measurement in step (b) with a standard value indicative of the presence of Gram negative bacteria in the sample.

16. The method of claim 1, wherein step (c) comprises comparing the measurement in step (b) with a standard value indicative of the presence of Gram positive bacteria in the sample.

17. The method of claim 1, wherein step (c) comprises comparing the measurement in step (b) with a standard value indicative of the presence of a fungus in the sample.

18. The method of any one of claims 15-17, wherein the standard value is a range of values of the optical property.

19. The method of any one of claims 15-17, wherein the standard value is a range of times in which a preselected change occurs in the optical property.

20. The method of claim 1, wherein the fungus is a yeast or a mold.