Cellular detection by providing high energy phosphate donor other than ADP to produce ATP

• US 6,703,211 B1
• Filed: 09/15/1999
• Issued: 03/09/2004
• Est. Priority Date: 03/13/1998
• Status: Expired due to Fees

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Wood, Keith V., Hall, Mary, Shultz, John William, Pratt, Tamara Sue
• Primary Examiner(s): Naff, David M.
• Assistant Examiner(s): Ware, Deborah K.
• Application Number:
  US09/396,334
• Time in Patent Office: 1,637 Days
• Field of Search: 435/4, 435/8, 435/243, 435/29, 435/34
• US Class Current: 435/8
• 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: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: C12Q 2535/131 Allele specific probes

Abstract

Processes and materials are disclosed for the detection of cells using endogenous enzymes to catalyze the conversion of AMP and/or ADP to ATP in the presence of a high energy phosphate donor other than ADP, then the ATP is detected using luciferase.

Claims

1. A method for determining the presence of cells in an aqueous sample composition that comprises the steps of:

(a) admixing said aqueous sample composition with a high energy phosphate donor other than ADP to form a reaction mixture, said cells, when present, being treated to form ATP in said reaction mixture;

(b) maintaining said reaction mixture for a time period sufficient for enzymes endogenous to the cells to convert adenosine present to ATP; and

(c) assaying for the presence of ATP, the presence of ATP indicating the presence of cells in said aqueous sample.

2. The method according to claim 1 wherein AMP is further admixed in the reaction mixture of step (a).

3. The method according to claim 1 wherein ATP is assayed using a luciferase enzyme.

4. The method according to claim 3 wherein said luciferase enzyme is added to the admixture of step (a).

5. The method according to claim 3 wherein said luciferase enzyme is a thermostable luciferase.

6. The method according to claim 5 wherein said thermostable luciferase enzyme is heat-treated.

7. The method according to claim 6 wherein said thermostable luciferase enzyme is Luc146-1H2.

8. The method according to claim 1 wherein the high energy phosphate donor other than ADP is a nucleotide or deoxynucleotide triphosphate other than ADP.

9. The method according to claim 8 wherein the high energy phosphate donor is dCTP.

10. The method according to claim 1 wherein the cells in a test sample are treated to form an aqueous sample composition.

11. The method according to claim 10 wherein a sample source is filtered to form a test sample on a filter.

12. The method according to claim 10 wherein the cells are treated by admixing a test sample with an extractant.

13. The method according to claim 12 wherein the extractant and high energy phosphate donor are admixed together with said test sample.

14. The method according to claim 12 wherein the test sample is a surface to be analyzed.

15. The method according to claim 12 wherein said extractant comprises polymixin B sulfate.

16. The method according to claim 15 wherein said extractant further comprises chlorhexidine.

17. The method according to claim 16 wherein said polymixin B sulfate is present at a concentration of about 0.5 percent to about 5 percent and said chlorhexidine is present at a concentration of about 0.1 percent to about 2 percent.

18. The method according to claim 1 wherein magnesium ion is also admixed in step (a).

19. The method according to claim 1 wherein the aqueous sample composition is derived from a liquid sample source.

20. The method according to claim 1 wherein the aqueous sample composition is derived from a sample source that is a food or beverage.