Amplification of trp1 for specific detection of phytophthora ramorum

Abstract

Phytophthora ramorum is currently a devastating disease for many plant species and infection presents significant economic problems, and in particular has lead to devastating effects on many specie of oak trees. The present invention provides methods and kits for selective detection of Phytophthora ramorum by amplification of indole-3-glycerol-phosphate synthase N-5′-phosphoribosyl anthranilate isomerase (trp1) in order to provide a method for detection of the presence of P. ramorum infection in a biological sample.

What is claimed is: 
     
         1 . A kit for the detection of  Phytophthora ramorum  in a biological sample, the kit comprising:
 (i) an upstream and downstream amplification primer pair for amplifying a region of the  P. ramorum  genome within the indole-3-glycerol-phosphate synthase N-5′-phosphoribosyl anthranilate isomerase (trp1) gene where the primer pair is 5′-1 (SEQ ID NO:3) and 3′-1 (SEQ ID NO:8), or 5′-5 (SEQ ID NO:7), and 3′-1 (SEQ ID NO:8); and,   (ii) non-specific amplification reagents for isothermal amplification of the trp1 gene.   
     
     
         2 . A kit of  claim 1  where the primers are:
 5′-1 (SEQ ID NO:3) and 3′-1 (SEQ ID NO:8). 
 
     
     
         3 . A kit of  claim 1  where the primers are:
 5′-5 (SEQ ID NO:7) and 3′-1 (SEQ ID NO:8). 
 
     
     
         4 . A kit of  claim 1  further comprising a nucleic acid probe that hybridizes to the amplified region of the trp1 gene. 
     
     
         5 . A kit of  claim 4  wherein the nucleic acid probe that hybridizes to the amplified region of the trp1 gene binds to nucleotides 163 to 213 of SEQ ID NO:1. 
     
     
         6 . A kit of  claim 4  wherein the nucleic acid probe that hybridizes to the amplified region comprises SEQ ID NO:2. 
     
     
         7 . A kit of  claim 4  where the probe is fluorescently labeled. 
     
     
         8 . A kit of  claim 1  wherein the non-specific amplification reagents include a bacterial recombinase. 
     
     
         9 . A kit of  claim 1  further comprising a DNA intercalating agent to detect amplified DNA.