Detection and quantification of microorganisms using amplification and restriction enzyme analysis

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.

What is claimed is:  
     
         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.    
     
     
         2 . The method according to  claim 1 , wherein said microbiological flora is selected from the group consisting of bacterial flora, yeast flora, fungal flora, viral flora, and mixtures of any thereof  
     
     
         3 . The method according to  claim 1 , wherein said sample is taken from a source selected from the group consisting of digestive tract, organ, tissue, water, and food product.  
     
     
         4 . The method according to  claim 1  further comprising: 
 comparing the resulting pattern of the sample with at least one reference pattern.  
 
     
     
         5 . The method according to  claim 5 , wherein said reference pattern is derived from another source.  
     
     
         6 . The method according to  claim 1 , further comprising analyzing the resulting pattern of restriction fragments to determine the relative abundance of micro-organisms included in flora.  
     
     
         7 . The method according to  claim 1  wherein the sample is analyzed to determine hygienic status.  
     
     
         8 . The method according to  claim 4 , wherein said reference value is obtained from a database.  
     
     
         9 . A method of determining the relative ratios of microorganisms present in a microbiological flora, said method comprising: 
 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.    
     
     
         10 . A test kit for analyzing a composition of microbiological flora having more than one species of microorganism, the test kit comprising: 
 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.    
     
     
         11 . The method according to  claim 1 , wherein analyzing the resulting pattern of restriction fragments comprises determining presence or absence of equilibrium in intestinal bacterial flora.  
     
     
         12 . The method according to  claim 1 , wherein providing a sample comprises obtaining a sample from feces of a subject.  
     
     
         13 . The method according to  claim 1 , wherein said providing a sample comprises obtaining the sample from a broiler or human subject.  
     
     
         14 . The method according to  claim 1 , wherein said providing a sample comprises obtaining a sample from a tissue or organ of a subject, and further comprising analyzing the total bacterial content of said tissue or organ.  
     
     
         15 . The method of  claim 4 , wherein said reference pattern is a standardized baseline pattern derived from a plurality of subjects with healthy flora.  
     
     
         16 . The method of  claim 1 , wherein said subjecting the amplificate to restriction digestion comprises subjecting the amplificate to restriction enzymes recognizing nucleotide sequences of about 4 to about 8 nucleotides.  
     
     
         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.    
     
     
         18 . The method according to  claim 17 , wherein analyzing the resulting pattern of restriction fragments comprises determining presence or absence of equilibrium in intestinal bacterial flora.  
     
     
         19 . A method of analyzing a sample of microbiological flora containing more than one species of microorganism, said method comprising: 
 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.    
     
     
         20 . The method according to  claim 19  further comprising: 
 comparing the resulting pattern of the sample with at least one reference pattern.