Pcr diagnostics of dermatophytes and other pathogenic fungi

Abstract

Dermatophytes which belong to one of the three genera Epidermophyton, Trichophyton and Microsporum are the main cause of fungal infections of skin, hair and nails. Traditional diagnostic procedures consist of microscopy and culture, but due to the slow growth rate of dermatophytes typically two to four weeks are needed before a final diagnosis is obtained. The present invention is a rapid DNA extraction method extracting nucleic acids from fungi (e.g. dermatophytes and other pathogenic fungi) which can be performed from directly on hair, nail or skin specimens from humans, from naturally or experimentally infected animals or from cultured fungal colonies for the use in PCR amplification and detection assays. The present invention also includes specific primer sets for detection of any dermatophyte and for species specific detection of Trichophyton rubrum and Epidermophyton floccosum by PCR and a kit for diagnosing fungal infections.

1 . A method of extracting nucleic acid from fungi comprising the following steps
 a) Heating of the sample in a lysis buffer and   b) Mixing the solution with a neutralizing buffer   
     
     
         2 . A method of extracting nucleic acid from fungi according to  claim 1  where the fungus is a species belonging to the genera  Trichophyton, Epidermophyton  and  Microsporum  or a yeast and the nucleic acid from fungi can be extracted from cultured fungi and from patient samples (e.g. hair, skin, nails). 
     
     
         3 . A method of extracting nucleic acid from fungi according to  claim 1  where the lysis buffer con-sists of a reducer, a salt and a buffering compound in an aqueous solution. 
     
     
         4 . A method of extracting nucleic acid from fungi according to  claim 3 , where the reducer is chosen from sodium carbonate, sodium sulfite, beta-mercaptoethanol, dithiotreitol, sodium sulfide, sodium chlorate, sodium chlorite, sodium iodate and sodium bicarbonate. 
     
     
         5 . A method of extracting nucleic acid from fungi according to  claim 3 , where the salt is chosen from potassium chloride and ammonium persulfate. 
     
     
         6 . A method of extracting nucleic acid from fungi according to  claim 3 , where the buffering com-pound is chosen from CAPSO, TRIS, HEPES and phosphate buffer. 
     
     
         7 . A method of extracting nucleic acid from fungi according to  claim 3  where the lysis buffer consists of potassium chloride (KCl), sodium bicarbonate (NaHCO 3 ) and tris(hydroxymethyl) ami-nomethane (TRIS). 
     
     
         8 . A method of extracting nucleic acid from fungi according to  claim 1  where the sample is heated to between 80 and 100° C., preferably 95° C. for 10 minutes. 
     
     
         9 . A method of extracting nucleic acid from fungi according to  claim 1  where the neutralizing buffer is an aqueous 0.5-3% W/V protein solution, where the protein is preferably BSA or ovalbumin. 
     
     
         10 . A method of extracting nucleic acid from fungi according to any  claim 1 , with the use of Extract-N-Amp Plant Kit (SIGMA) or Red Extract-N-Amp Plant Kit (SIGMA). 
     
     
         11 . A method of detecting fungi infections in patient samples comprising the following steps:
 A) Extracting nucleic acid from the sample by
 a) Heating of the sample in an lysis buffer and 
 b) Mixing the solution with an neutralizing buffer 
   B) Amplifying the nucleic acid with PCR using fungus specific primers,   C) Detecting fungus specific genes.   
     
     
         12 . A method of detecting fungal infections in patient samples according to  claim 11  where the primers are chosen from panDerm1, panDerm2, uni, Trubrum-rev, Ef-rev, TmTt-for, TmTt-rev, Mc-for, Mc-rev, Micr773-for, Micr885-rev, Trichopyros-for and Trichopyros-rev. 
     
     
         13 . Primers for PCR comprising the nucleotide sequences SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. 
     
     
         14 . A multiplex PCR kit for detecting dermatophytes comprising
 a) a lysis buffer   b) a neutralizing buffer   c) fungus specific primers   d) agents for performing a PCR reaction   
       where the lysis buffer consists of a reducer (e.g. sodium carbonate, sodium sulfite, beta-mercaptoethanol, dithiotreitol, sodium sulfide, sodium chlorate, sodium chlorite, sodium iodate and sodium bicarbonate), a salt (e.g. potassium chloride and ammonium persulfate) and a buffering compound (e.g. CAPSO, TRIS, HEPES or phosphate buffer) in an aqueous solution, the neutraliz-ing buffer comprising a 0.5-3% w/v, preferably a 2% w/v protein solution (e.g. BSA or ovalbu-min) and the fungus specific primers are chosen from panDerm1 (SEQ ID NO. 1), panDerm2 (SEQ ID NO. 2), uni (SEQ ID NO: 3), Trubrum-rev (SEQ ID NO. 4), Ef-rev (SEQ ID NO. 5), Micr773-for SEQ ID NO. 10), Micr885-rev (SEQ ID NO. 11), Trichopyros-for (SEQ ID NO. 12) and Trichopy-ros-rev (SEQ ID NO.13).