Detection of fungal pathogens using the polymerase chain reaction

Abstract

The present invention relates to the use of primers in polymerase chain reaction assays for the detection of fungal pathogens Colletotrichum acutatum, Ateniaria spp., and Cladosporium carpophilum. Specific primers are identified as being useful for the identification of fungal isolates using PCR based techniques. Also described are novel extraction buffer solutions for use in isolating DNA from an organism, methods of extracting DNA from tissue, and methods of performing PCR analysis on DNA extracted from tissue.

1 . An oligonucleotide primer consisting of SEQ ID NO: 16 or 12.  
     
     
         2 . A pair of oligonucleotide primers, wherein said pair consists of said primer of  claim 1 .  
     
     
         3 . A pair of oligonucleotide primers, consisting of: 
 a) SEQ ID NO:16 and SEQ ID NO:12;.    
     
     
         4 - 7 . (canceled)  
     
     
         8 . A method for the detection of  Alternaria  spp., comprising the steps of: 
 (a) isolating DNA from a plant tissue infected with a pathogen;    (b) subjecting said DNA to polymerase chain reaction amplification using at least one primer according to  claim 1;  and    (c) detecting  Alternaria  spp. by visualizing the product or products of said polymerase chain reaction amplification.    
     
     
         9 . (canceled)  
     
     
         10 . A method for the detection of  Alternaria  spp., comprising the steps of: 
 (a) isolating DNA from a plant tissue infected with  Alternaria  spp.;    (b) amplifying a part of the Internal Transcribed Spacer sequence of  Alternaria  spp. using said DNA as a template in a polymerase chain reaction with a pair of primers according to  claim 2;  and    (c) detecting  Alternaria  spp. by visualizing the amplified part of the Internal Transcribed Spacer sequence.    
     
     
         11 . (canceled)  
     
     
         12 . The method of  claim 10 , wherein said pair of primers is according to  claim 3 .  
     
     
         13 - 16 . (canceled)  
     
     
         17 . A diagnostic kit used in detecting a fungal pathogen, comprising the primer of  claim 1 .  
     
     
         18 . A diagnostic kit used in detecting a fungal pathogen, comprising the pair of primers of  claim 2 .  
     
     
         19 . A diagnostic kit used in detecting a fungal pathogen, comprising the pair of primers of  claim 3 .  
     
     
         46 . (canceled)