Method for the detection and characterization of a toxinogenic clostridium difficile strain

Abstract

The invention relates to a method for the detection and characterization of a toxinogenic Clostridium difficile strain in a sample, wherein the following steps are performed, (i) a sample is provided for, (ii) in a multiplex PCR assay, (iii) the sample is analyzed with respect to the presence or absence of the cytotoxin tcdB gene, (iv) the sample is analyzed with respect to the presence or absence of one or more of the following deletions in the tcdC gene: (a) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347, (b) a 36 bp deletion in SEQ ID NO. 1 from nucleotide (301) to nucleotide (336), (c) a 39 bp deletion in SEQ ID NO. 1 from nucleotide (341) to nucleotide (370), (d) a 54 bp deletion in SEQ ID NO. 1 from nucleotide (313) to nucleotide (366) and (e) a single nucleotide deletion at position 117 of SEQ ID NO. 1. The invention also relates to respective kits and primers and probes.

1 . Method for the detection and characterization of a toxinogenic  Clostridium difficile  strain in a sample, wherein the following steps are performed,
 a. a sample is provided for;   b. in a multiplex PCR assay,
 i the sample is analyzed with respect to the presence or absence of the cytotoxin tcdB gene; 
 ii the sample is analyzed with respect to the presence or absence of one or more of the following deletions in the tcdC gene,
 a) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347; 
 b) a 36 bp deletion in SEQ ID NO. 1 from nucleotide 301 to nucleotide 336; 
 c) a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370; 
 d) a 54 bp deletion in SEQ ID NO. 1 from nucleotide 313 to nucleotide 366; 
 e) a single nucleotide deletion at position 117 of SEQ ID NO. 1 
 
   
     
     
         2 . Method according to  claim 1 , wherein the sample is additionally analyzed with respect to the presence or absence of the enterotoxin tcdA gene 1.8 kb deletion. 
     
     
         3 . Method according to  claim 1 , wherein the sample is additionally analyzed with respect to the presence or absence of the binary toxin cdtA and/or cdtB. 
     
     
         4 . Method according to  claim 1 , wherein the sample is analyzed with respect to,
 a. the presence or absence of all of the following deletions:   b. a 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347, a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370,   c. a single nucleotide deletion at position 117 of SEQ ID NO. 1,   d. the presence or absence of the cytotoxin tcdB gene,   e. the presence or absence of the 1.8 kb tcdA deletion and   f. the presence or absence of the cdtA/B binary toxin gene.   
     
     
         5 . Method according to  claim 1 , wherein
 a. if the tcdB gene sequence is present, the tcdA deletion is absent, neither the single nucleotide deletion at position 117 of SEQ ID NO. 1 is present, nor the 18 bp deletion is present, nor the 39 bp deletion is present, then the sample is scored as toxinogenic  Clostridium difficile,      b. if the tcdB gene sequence is present, the tcdA deletion is absent, the single nucleotide deletion at position 117 of SEQ ID NO. 1 is present, the 18 bp deletion is present, and the cdtA/B binary toxin gene is present, then the sample is scored as a ribotype 027  Clostridium difficile  strain,   c. if the tcdB gene sequence is present, the tcdA deletion is present, neither the single nucleotide deletion at position 117 of SEQ ID NO. 1 is present, nor the 18 bp deletion is present, nor the 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 is present, and the cdtA/B binary toxin gene is absent, then the sample is scored as a ribotype 017  Clostridium difficile  strain and   d. if the tcdB gene sequence is present, the tcdA deletion is absent, the 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 is present, and the cdtA/B binary toxin gene is present, then the sample is scored as a ribotype 078  Clostridium difficile  strain.   
     
     
         6 . Method according to  claim 1 , wherein the multiplex amplification reaction is done in a closed system in the presence of fluorescent indicators in the reaction mixture(s), the fluorescent indicators being capable of generating an optical signal related to a presence and/or quantity of each amplicon in the amplification reaction and monitoring the optical signal of the fluorescent indicators in the amplification reaction. 
     
     
         7 . Method according to  claim 6 , wherein the closed system gives an optical output for the user. 
     
     
         8 . Method according to  claim 1 , wherein the amplification products in the multiplex PCR assay are between 60 and 200 bp in size. 
     
     
         9 . Method according to  claim 1 , wherein the multiplex PCR amplification is quantitative real-time PCR. 
     
     
         10 . Method according to  claim 1 , wherein the sample is human or animal feces. 
     
     
         11 . Closed system amplification cartridge comprising one or more channels or chambers comprising primers and/or probes for amplifying and/or detecting (i) the cytotoxin tcdB gene, (ii) the 1.8 kb deletion in the tcdA gene, (iii) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347 of the tcdC gene, (iv) a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 of the tcdC gene, (v) a single nucleotide deletion at position 117 of SEQ ID NO. 1 and primers and/or probes for the detection of the binary toxin cdtA/B gene. 
     
     
         12 . Kit for performing the method according to  claim 11 , comprising primers and or probes for amplifying and/or detecting (i) the cytotoxin tcdB gene, (ii) the 1.8 kb deletion in the tcdA gene, (iii) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347 of the tcdC gene, (iv) a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 of the tcdC gene, (v) a single nucleotide deletion at position 117 of SEQ ID NO. 1 and primers and/or probes for (vi) the detection of the binary toxin cdtA/B gene.