Genetic Affinity of Microorganisms and Viruses

Abstract

Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism.

What is claimed is: 
     
         1 . A method of determining the genetic affinity of organisms or viruses in a test sample containing a nucleic acid comprising the steps of:
 A) Detecting signals generated as a result of the presence in the nucleic acid of a plurality of signature sequences or their complements;   B) Comparing the signature sequences found in the nucleic acid to a database of signature sequences found in a plurality of organisms or viruses   C) Determining which Domain, Kingdom, Phylum, Subphylum, Class, Subclass, Order, Suborder, Family, Subfamily, Genus, Species, or Subspecies shares specific genetic affinity with the unknown organism or virus by considering the quality and number of signal-generating signature sequences that are shared with members of each grouping in the database.   
     
     
         2 . In a method of determining the genetic affinity of organisms or viruses in a test sample comprising detecting the presence of a plurality of signature sequences; the improvement comprising: detecting the presence by means comprising mass spectrometry. 
     
     
         3 . A method of  claim 1  in which the database of signature sequences comprises a library of signature masses. 
     
     
         4 . A method of  claim 1  in which signature sequences are generated from a target nucleic acid by enzymatic digestion with a nuclease. 
     
     
         5 . A method of  claim 1  in which signature genes are used as signature sequences. 
     
     
         6 . Method of  claim 14  in which fragmentation is accomplished using ribonuclease T1. 
     
     
         7 . Method of  claim 1  in which masses are determined by any suitable mass spectrometric method including, matrix assisted laser desorption ionization, electrospray, time of flight, or resonance methods. 
     
     
         8 . A method of  claim 1  in which signature sequences are generated from a target nucleic acid by enzymatic digestion with a nuclease and identified by mass spectrometry. 
     
     
         9 . A method according to  claim 2  for identifying or detecting organisms such as bacteria, eukaryotes, archaebacteria, or viruses comprising: isolating a characteristic nucleic acid or protein component of an organism, determining at least a portion of the monomer or molecular composition of a sequence derived from said characteristic nucleic acid or protein; and identifying or detecting the micro-organism from which said characteristic nucleic acid or protein was derived by reference to a database of compositions of nucleic acids and proteins produced by organisms. 
     
     
         10 . The method of  claim 1  in which the characteristic molecule is DNA encoding ribosomal RNA or a fragment thereof. 
     
     
         11 . The method of  claim 1  in which the characteristic molecule is a protein or DNA encoding a protein, or a fragment thereof. 
     
     
         12 . The method of  claim 1  in which the composition is determined by mass spectrometry. 
     
     
         13 . The method of  claim 2  in which the method of mass spectrometry comprises matrix assisted laser desorption ionization (MALDI). 
     
     
         14 . A system for identifying or detecting organisms such as bacteria, viruses, archaebacteria or eukaryotes comprising: a chemical isolator or amplifier for identifying the characteristic nucleic acid or protein of an organism present in a specimen; a controlled fragmentation reactor that generates sub-fragments of said characteristic acid or protein; a mass spectrometer that measures the molecular weight of said sub-fragments and generates a set of representative data; a computer that processes said data and compares said measured weights with known predicted sub-fragment masses to make an identification. 
     
     
         15 . The system of  claim 14  in which the characteristic molecule has been amplified by PCR, RT-PCR, NASBA or equivalent methods. 
     
     
         16 . The system of  claim 14  where the predicted sub-fragment masses are obtained from an available database. 
     
     
         17 . The system of  claim 14  in which the mass of the signature is determined within 2%. 
     
     
         18 . A method according to  claim 9  for identifying or detecting organisms such as bacteria, eukaryotes, archaebacteria, or viruses comprising: determining known fragment sequences for a pre-determined set of nucleic acid or proteins; isolating a characteristic nucleic acid or protein component of an organism present in a specimen, determining at least a portion of the monomer composition of a sequence derived from said characteristic nucleic acid or protein; and identifying or detecting the micro-organism. 
     
     
         19 . A method according to  claim 1  for determining the genetic affinity of organisms or viruses in a test sample containing a target RNA, comprising in combination the steps of:
 A. Obtaining or creating a database of homologous RNA sequences from all organisms or viruses that will be incorporated into the determination; 
 B. Obtaining or developing a bifurcating phylogenetic tree having multiple nodes that establishes the genetic affinity between the organisms or viruses included in the database of homologous RNA sequences; 
 C. Computationally fragmenting each sequence in the database of homologous sequences; such fragmentation being performed in a programmed computer so as to create a catalog of all subsequences of length 7 or larger for each sequence in the database of homologous sequences. 
 D. Calculating in a programmed computer the molecular mass associated with each subsequence in each catalog 
 E. Tabulating in a programmed computer the extent to which the presence of a particular molecular mass is characteristic of each specific nodes in the bifurcating phylogenetic tree of genetic relationship by examining the occurrence frequency of a molecular mass in the target nucleic acid of the organisms and viruses and calculating a signature quality index which measures the extent to which that mass is characteristic of each node in the bifurcating node phylogenetic tree of genetic relationships. 
 F. Collecting the target nucleic acid from a test sample and fragmenting it and determining by mass spectrometry the molecular masses that are present. 
 G. Identifying the nodes in the bifurcating phylogenetic tree of genetic relationship that are represented by the molecular masses that are produced by the organism or virus in the test sample. 
 
     
     
         20 . A Method of  claim 19  wherein the subsequences are selected to end in G, A, C or U.