Detection and quantification of microorganisms using amplification and restriction enzyme analysis
First Claim
1. A method of analyzing a microbiological flora containing more than one species of microorganism, said method comprising:
- providing a sample of said microbiological flora;
amplifying nucleic acid from the more than one species of microorganism presenting in said sample to produce an amplificate;
subjecting the amplificate to at least one restriction digestion to produce restriction fragments from the more than one species of microorganism; and
preparing a resulting pattern based on the restriction fragments of the more than one species of microorganism in the sample.
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Abstract
In livestock, a number of pathological conditions and/or syndromes have been noticed that do not seem to be attributable to, for example, an infection with a single microorganism. These syndromes however do seem to have an effect on the equilibrium which normally exists within the different bacteria constituting the bacterial flora. A number of these syndromes are referred to as dysbacteriosis and bacterial overgrowth. To enable at least in part the evaluation of the “general” health of the livestock the invention provides means and methods for analyzing the composition of a microbiological flora. In one aspect a method of the invention comprises providing a sample of the flora, selectively amplifying nucleic acid present in the sample, subjecting the amplificate to restriction digestion and analyzing the resulting pattern of restriction fragments.
9 Citations
20 Claims
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1. A method of analyzing a microbiological flora containing more than one species of microorganism, said method comprising:
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providing a sample of said microbiological flora;
amplifying nucleic acid from the more than one species of microorganism presenting in said sample to produce an amplificate;
subjecting the amplificate to at least one restriction digestion to produce restriction fragments from the more than one species of microorganism; and
preparing a resulting pattern based on the restriction fragments of the more than one species of microorganism in the sample. - View Dependent Claims (2, 3, 4, 6, 7, 8, 11, 12, 13, 14, 15, 16)
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5. The method according to claim 5, wherein said reference pattern is derived from another source.
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9. A method of determining the relative ratios of microorganisms present in a microbiological flora, said method comprising:
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providing a sample of said microbiological flora;
amplifying nucleic acid from the more than one species of microorganism present in said sample to produce an amplificate;
subjecting the amplificate to at least one restriction digestion to produce restriction fragments from the more than one species of microorganism;
preparing a resulting pattern based on the restriction fragments of the more than one species of microorganism in the sample; and
analyzing the resulting pattern of restriction fragments to determine the relative ratios of microorganisms present in said flora.
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10. A test kit for analyzing a composition of microbiological flora having more than one species of microorganism, the test kit comprising:
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two primers for amplification of nucleic acids in a the composition of microbiological flora to produce an amplificate;
at least one restriction enzyme for digesting an amplificate of a microbiological flora sample to produce restriction fragments from more than one species of microorganism; and
at least one reference pattern for comparative analysis of a resulting pattern of restriction fragments from digestion of a microbiological flora sample.
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17. A method of analyzing the composition of a bacterial flora, comprising:
- obtaining a sample of bacterial flora taken from the digestive tract of a subject;
amplifying nucleic acid from more than one species of bacteria present in said sample with at least one primer of about 20 to 30 nucleotides directed to a nucleic acid believed to be present in said sample to produce an amplificate;
subjecting the amplificate to restriction digestion;
analyzing a resulting pattern of restriction fragments by comparing the restriction pattern of the sample with at least one reference restriction pattern; and
determining relative abundance and relative ratios of said bacterial flora. - View Dependent Claims (18)
- obtaining a sample of bacterial flora taken from the digestive tract of a subject;
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19. A method of analyzing a sample of microbiological flora containing more than one species of microorganism, said method comprising:
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providing the sample of said microbiological flora;
subjecting the sample to at least one restriction digestion to produce restriction fragments from the more than one species of microorganism to produce a restriction digestion product;
amplifying the restriction digestion product to produce an amplificate; and
preparing a resulting pattern based on the restriction fragments of the more than one species of microorganism in the sample. - View Dependent Claims (20)
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Specification