Materials and methods for detection of Oxalobacter formigenes
First Claim
1. A method for detecting Oxalobacter formigenes in a sample, comprising the steps of:
- (a) contacting said sample with a polynucleotide probe under conditions sufficient for selective hybridization of said polynucleotide probe with DNA of Oxalobacter formigenes that comprises a nucleotide sequence of an oxalyl-CoA decarboxylase gene, wherein said polynucleotide probe is substantially complementary with a nucleotide sequence of an oxalyl-CoA decarboxylase gene of Oxalobacter formigenes; and
(b) detecting said probe hybridized to said DNA.
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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.
8 Citations
49 Claims
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1. A method for detecting Oxalobacter formigenes in a sample, comprising the steps of:
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(a) contacting said sample with a polynucleotide probe under conditions sufficient for selective hybridization of said polynucleotide probe with DNA of Oxalobacter formigenes that comprises a nucleotide sequence of an oxalyl-CoA decarboxylase gene, wherein said polynucleotide probe is substantially complementary with a nucleotide sequence of an oxalyl-CoA decarboxylase gene of Oxalobacter formigenes; and
(b) detecting said probe hybridized to said DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 38, 39, 40, 41, 42, 43)
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13. A method for detecting Oxalobacter formigenes in a sample, comprising the steps of:
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(a) amplifying DNA of Oxalobacter formigenes by polymerase chain reaction (PCR) using a polynucleotide PCR primer, wherein said primer is substantially complementary with a polynucleotide sequence comprising an oxalyl-CoA decarboxylase gene of Oxalobacter formigenes;
(b) contacting said amplified DNA with a polynucleotide probe under conditions sufficient for selective hybridization of said polynucleotide probe with DNA of Oxalobacter formigenes that comprises a nucleotide sequence of an oxalyl-CoA decarboxylase gene, wherein said polynucleotide probe is substantially complementary with a nucleotide sequence of an oxalyl-CoA decarboxylase gene of Oxalobacter formigenes; and
(c) detecting said probe hybridized to said DNA. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 44, 45, 46, 47, 48, 49)
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- 33. A polynucleotide probe that selectively hybridizes with DNA of Oxalobacter formigenes, comprising a nucleotide sequence that is substantially complementary with a polynucleotide sequence present in an Oxalobacter formigenes genome, wherein the polynucleotide sequence present in the Oxalobacter formigenes genome comprises a gene selected from the group consisting of the formyl-CoA transferase gene and the oxalyl-CoA decarboxylase gene.
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34. The polynucleotide probe, according to clam 33, wherein said polynucleotide probe hybridizes under high-stringency conditions with a nucleotide sequence of said formyl-CoA transferase gene or said oxalyl-CoA decarboxylase gene.
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35. An oligonucleotide comprising a nucleotide sequence that is substantially complementary with a polynucleotide sequence present in an Oxalobacter formigenes genome, wherein said polynucleotide sequence present in said Oxalobacter formigenes genome comprises a gene selected from the group consisting of the formyl-CoA transferase gene and the oxalyl-CoA decarboxylase gene, and wherein said oligonucleotide is capable of priming polymerase chain reaction amplification of said polynucleotide sequence present in said Oxalobacter formigenes genome.
Specification