Method for the identification of microorganisms by the utilization of directed and arbitrary DNA amplification
First Claim
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.
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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.
171 Citations
11 Claims
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1. A method for the identification of the species, serotype and strain of a microorganism, comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification