Method to amplify nucleic acids to generate fluorescence labeled fragments of conserved and arbitrary products
Disclosed herein are methods for the identification of the species, serotype, and strain of a microorganism. Also disclosed are primers for use in detecting such microorganisms and kits comprising such primers.
1. A method for the identification of the species, serotype, and strain of a microorganism comprising:
(a) amplifying DNA comprising variable sequences interspersed between highly conserved rDNA sequences by PCR and amplifying additional genomic sequences by random amplified polymorphic DNA (RAPD) PCR using a first primer of 13-14 bases in length and a second primer of 11-13 bases in length, said first primer comprising:
(i) 11-12 contiguous bases from a highly conserved 16S rDNA region;
(ii) 2 bases at the 5′ end of said first primer that are not complementary to said highly conserved 16S rDNA region; and
(iii) a fluorescent label; and
(b) separating the amplified DNA produced in step (a).
2. The method of claim 1 , wherein said first primer is a forward primer.
3. The method of claim 2 , wherein said first primer comprises SEQ ID NO:1.
4. The method of claim 3 , wherein said first primer is SEQ ID NO:2.
5. The method of claim 1 , wherein said second primer comprises 11-13 contiguous bases from 23S rDNA.
6. The method of claim 5 , wherein said second primer is SEQ ID NO:3 or SEQ ID NO:4.
7. The method of claim 1 , wherein said amplifying step utilizes a third primer of 11-13 bases in length, said third primer comprising 11-13 contiguous bases from 23S rDNA, wherein said third primer is a different length than said second primer, and wherein one of either the second primer or third primer comprises 13 contiguous bases from 23S rDNA.
8. The method of claim 1 , wherein step (b) is accomplished by capillary electrophoresis.