Method for classifying a microorganism in a biological sample
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.
1. A method of classifying a bacterium in a test sample, the method comprising the steps of:
(a) combining the test sample, which contains a standardized amount of the bacterium, 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) comparing the optical property of step (b)(i) or the time value of step (b)(ii) with a first standard value indicative of the presence of Gram negative bacteria and a second standard value indicative of the presence of Gram positive bacteria thereby to determine whether the bacterium is a Gram negative bacterium or a Gram positive bacterium.
2. The method of claim 1 , wherein in step (c), a first optical property is indicative of the presence of Gram negative bacteria in the sample and a second optical property is indicative of the presence of Gram positive bacteria in the sample.
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, and a second 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 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 substrate 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 one or more of the standard values is a range of values of the optical property.
16. The method of claim 1 , wherein one or more of the standard values is a range of times in which a preselected change occurs in the optical property.