Materials and methods for detection of oxalobacter

• US 5,912,125 A
• Filed: 09/27/1996
• Issued: 06/15/1999
• Est. Priority Date: 06/20/1994
• Status: Expired due to Term

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: University of Florida (State University System of Florida)
• Inventors: Sidhu, Harmeet, Peck, Ammon B.
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/717,587
• Time in Patent Office: 991 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/24.1, 536/24.33, 536/23.7, 935/76, 935/77, 935/78
• US Class Current: 435/6.12
• 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 1/689 for bacteria

Abstract

The subject invention concerns the novel use of formyl-CoA transferase enzyme together with oxalyl-CoA decarboxylase enzyme for the detection and measurement of oxalate in biological samples. The use of the enzyme system according to the subject invention results in the conversion of oxalate into carbon dioxide and formate. Because the production of formate is directly correlated to the concentration of oxalate present in a sample, the determination of the resulting formate concentration provides an accurate, sensitive and rapid means for detecting even low levels of oxalate. The subject invention further concerns the cloning, sequencing and expression of the genes that encode the formyl-CoA transferase enzyme and the oxalyl-CoA decarboxylase enzyme of Oxalobacter formigenes. The subject invention also concerns methods for detecting the presence of Oxalobacter formigenes organisms in a sample, and the polynucleotide probes and primers used in the detection method.

Claims

1. An isolated polynucleotide molecule, comprising a nucleotide sequence that encodes a formyl-CoA transferase polypeptide derived from Oxalobacter formigenes.

2. The polynucleotide molecule, according to claim 1, wherein said nucleotide sequence encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 2.

3. The polynucleotide molecule, according to claim 1, comprising the nucleotide sequence shown in SEQ ID NO. 1.

4. The polynucleotide molecule, according to claim 1, wherein said polynucleotide molecule hybridizes under standard high-stringency conditions with a polynucleotide molecule comprising the nucleotide sequence shown in SEQ ID NO. 1.

10. The polynucleotide molecule, according to claim 1, comprising nucleotides 162 through 1445 of SEQ ID NO. 1.

5. A polynucleotide probe complementary with a polynucleotide sequence encoding a formyl-CoA transferase polypeptide of Oxalobacter formigenes, said probe comprising a nucleotide sequence shown in SEQ ID NO. 10.

6. A method for detecting Oxalobacter formigenes in a sample, comprising the steps of:

(a) contacting said sample with a polynucleotide probe of claim 5 under high stringency conditions sufficient for selective hybridization of said polynucleotide probe with a DNA fragment of Oxalobacter formigenes; and

(b) detecting said polynucleotide probe hybridized to said DNA fragment.

7. The method, according to claim 6, wherein said DNA fragment is immobilized on a membrane.

8. The method according to claim 6, wherein said polynucleotide probe contains a detectable label.

9. A kit for detecting the presence of Oxalobacter formigenes in a sample, comprising in one or more separate containers:

(a) the polynucleotide probe of claim 5.