Method for the identification of microorganisms by the utilization of directed and arbitrary DNA amplification

• US 5,753,467 A
• Filed: 08/22/1996
• Issued: 05/19/1998
• Est. Priority Date: 12/04/1991
• Status: Expired due to Term

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Straus, Neil Alexander, Jensen, Mark Anton
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/701,290
• Time in Patent Office: 635 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method for the identification of microorganisms is disclosed, comprising first isolating genomic DNA from the suspect microorganism. The isolated DNA has variable sequences interspersed between highly conserved rDNA sequences. The variable sequences are amplified. The amplification reaction is carried out in such manner as to either amplify the variable sequences interspersed between highly conserved rDNA sequences or to amplify arbitrary genomic regions in conjunction with the variable sequences. The resulting amplified DNA fragments are polymorphic with respect to both size and number in a manner which is specific to species, serotype and strain. The distribution of the polymorphic fragments is analyzed and compared to an established database to determine the species, serotype and strain of the suspect microorganism.

Claims

1. A method for the identification of the species, serotype and strain of a microorganism, comprising:

(a) isolating genomic DNA from the microorganism, said genomic DNA comprising variable sequences interspersed between highly conserved rDNA sequences in addition to arbitrary regions along the genome thereof;

(b) amplifying said variable sequences by annealing only a single pair of oligonucleotide primer of 10-12 bases in length to said highly conserved rDNA sequences at a temperature of about 43°

to about 46°

C. and during a period of about 5 to about 10 minutes, and introducing a nucleic acid ploymerase and nucleoside triphosphates to said primers, suitable to amplify said variable sequences, thereby producing ribosomal sequence fragments having particular distributions in size and number; and

(c) amplifying said arbitrary regions by annealing as set forth in step (b) suitable to amplify said arbitrary regions, thereby producing arbitrary genomic fragments having particular distributions in size and number simultaneously with (b); and

(d) separating said ribosomal sequence fragments and said arbitrary genomic fragments by size and analyzing the distribution of said ribosomal sequence fragments and said arbitrary genomic fragments both for elements common to all members of the species and for elements which differentiate the unique strains within that species only, and comparing to a database of previously visualized product thereby identifying the species, serotype and strain of the microorganism.

2. The method of claim 1 wherein said variable sequences are spacer regions.

3. The method of claim 1 wherein step (a) isolation comprises cellular lysis and deproteinization.

4. The method of claim 1 wherein said amplification is performed at least twice.

5. The method of claim 1 wherein the nucleic acid polymerase is a DNA polymerase and the nucleoside triphosphates are deoxyribonucleoside triphosphates.

6. The method of claim 5 wherein the DNA polymerase is thermostable.

7. The method of claim 1 wherein step (d) comprises resolving said ribosomal sequence fragments and said arbitrary genomic fragments by electrophoresis and thereafter visualizing said ribosomal sequence fragments and said arbitrary genomic fragments.

8. The method of claim 1 wherein the database contains fragment distributions which are characteristic for species, serotype and strain of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria welshimeri, Salmonella typhimurium, Salmonella enteritidis, Salmonella newport, Salmonella infantis, Staphylococcus aureus, Staphylococcus sciuri, Staphylococcus saprophyticus, Staphylococcus warneri, Staphylococcus epidermidus, Escherichia coli, Escherichia blattae, Escherichia fergusonii, Escherichia hermanii, Escherichia vulneris, Citrobacter freundii, Citrobacter diversus, Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Proteus vulgari, and Yersinia enterocolitica.

9. The method of claim 1 wherein in step (d) visualization is conducted by staining said ribosomal sequence fragments and said aribitrary genomic fragments with ethidium bromide or detecting the position of said ribosomal sequence fragments and said arbitrary genomic fragments by spectrophotometric procedures.

10. The method of claim 1 wherein in step (d) visualization is conducted by fLuorescence-labelling deoxynucleotide during amplification (b) and (c).

11. The method of claim 1 wherein said microorganism is bacteria.