Oligonucleotides for detecting bacteria and detection process

Abstract

A synthetic oligonucleotide which is complementary to a nucleotide sequence of a gene selected from the group consisting of the Shiga toxin gene of Shigella species, the ipaH gene of Shigella species and EIEC, the invE gene of Shigella species and EIEC, the araC gene of Salmonella species, the Verocytotoxin-1 gene of EHEC or VTEC, the Verocytotoxin-2 gene of EHEC or VTEC, the toxic shock syndrome toxin-1 gene of Staphylococcus aureus , the ctx gene of Vibrio cholerae , and the enterotoxin gene of Clostridium perfringens ; a method for detecting a bacterial strain by amplifying a region of the above gene by PCR using the above oligonucleotides as primers and detecting the amplified region; and a kit for the detection of the bacterial strain.

What is claimed is:  
     
         1 . A synthetic oligonucleotide of 10 to 30 bases which is complementary to a nucleotide sequence of a gene selected from the group consisting of the Shiga toxin gene of Shigella species, the ipaH gene of Shigella species and EIEC, the invE gene of Shigella species and EIEC, the arcC gene of Salmonella species, the toxic shock syndrome toxin-1 gene of  Staphylococcus aureaus , the ctx gene of  Vibrio cholerae , and the enterotoxin gene of  Clostridium perfringens.    
     
     
         2 . A synthetic oligonucleotide comprising a nucleotide sequence complementary to the synthetic oligonucleotide of  claim 1 .  
     
     
         3 . A method for detecting a bacterial strain selected from the group consisting of Shigella species, EIEC, Salmonella species, Staphylococcus aureus , vibrio cholerae, and  Clostridium perfringens,  wherein the method comprises 
 (1) hybridizing one primer to a single-stranded target DNA as a template DNA present in a specimen and carrying out a primer extension reaction to give a primer extension product;    (2) denaturing the resulting DNA duplex to separate the primer extension product from the template DNA, the primer extension product functioning as the other template DNA for the other primer;    (3) repeating a cycle of simultaneous primer extension reaction with the two primers, separation of the primer extension products from the templates, and hybridization of primers to amplify a region of the target DNA, in the steps from (1) to (3) said primers being selected from the group consisting of oligonucleotides of  claim 1  and a synthetic oligonucleotide comprising a nucleotide sequence complementary to the synthetic oligonucleotide; and    (4) detecting the amplified nucleotide sequence to determine whether a suspected bacterial strain is present in the specimen.    
     
     
         4 . A kit for detection of a bacterial strain comprising at least a pair of primers selected from the group consisting of oligonucleotides of claims  1 , and synthetic oligonucleotides comprising sequences complementary to the oligonucleotides of  claim 1 , a thermostable DNA polymerase, and dNTP solutions.  
     
     
         5 . The synthetic oligonucleotide according to  claim 1 , wherein said gene is the Shiga toxin gene of  Shigella dysenteriae  type 1, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 1 or oligonucleotide SEQ ID NO: 2.  
     
     
         6 . The synthetic oligonucleotide according to  claim 2 , wherein said nucleotide sequence is complementary to a synthetic oligonucleotide of 10 to 30 bases which in turn is complementary to a nucleotide sequence of a Shiga toxin gene of  Shigella dysenteriae  type 1, and wherein the synthetic oligonucleotide comprises bases complementary to at least 10 consecutive bases of oligonucleotide SEQ ID NO: 1 or oligonucleotide SEQ ID NO: 2.  
     
     
         7 . The synthetic oligonucleotide according to  claim 1 , wherein said gene is the ipaH gene of Shigella species and EIEC, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 3 or oligonucleotide SEQ ID NO: 4.  
     
     
         8 . The synthetic oligonucleotide according to  claim 2 , wherein said nucleotide sequence is complementary to a synthetic oligonucleotide of 10 to 30 bases which in turn is complementary to a nucleotide sequence of an ipaH gene of Shigella species and EIEC, and wherein the synthetic oligonucleotide comprises bases complementary to at least 10 consecutive bases of oligonucleotide SEQ ID NO: 3 or oligonucleotide SEQ ID NO: 4.  
     
     
         9 . The synthetic oligonucleotide according to  claim 1 , wherein said gene is the invE gene of Shigella species and EIEC, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 5 or oligonucleotide SEQ ID NO: 6.  
     
     
         10 . The synthetic oligonucleotide according to  claim 2 , wherein said nucleotide sequence is complementary to a synthetic oligonucleotide of 10 to 30 bases which in turn is complementary to a nucleotide sequence of an invE gene of Shigella species and EIEC, and wherein the synthetic oligonucleotide comprises bases complementary to at least 10 consecutive bases of oligonucleotide SEQ ID NO: 5 or oligonucleotide SEQ ID NO: 6.  
     
     
         11 . The method according to  claim 3 , wherein said target DNA is a gene of Shigella species and EIEC and said primers are selected from the group consisting of oligonucleotides of  claims 5  to  10 .  
     
     
         12 . The kit according to  claim 4 , wherein said pair of primers are selected from the group consisting of oligonucleotides of  claims 5  to  10  for detection of Shigella species and EIEC.  
     
     
         13 . The synthetic oligonucleotide according to  claim 1 , wherein said gene is the araC gene of Salmonella species, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of the oligonucleotide selected from the group consisting of oligonucleotide SEQ ID NO: 7, oligonucleotide SEQ ID NO: 8, oligonucleotide SEQ ID NO: 9, oligonucleotide SEQ ID NO: 10, and oligonucleotide SEQ ID NO: 11.  
     
     
         14 . The synthetic oligonucleotide according to  claim 2 , wherein said nucleotide sequence is complementary to the synthetic oligonucleotide of  claim 13 .  
     
     
         15 . The method according to  claim 3 , wherein said target DNA is a gene of a Salmonella species and said primers are selected from the group consisting of oligonucleotides comprising at least 10 consecutive bases of the oligonucleotide of SEQ ID NO: 7, the oligonucleotide of SEQ ID NO: 8, the oligonucleotide of SEQ ID NO: 9, the oligonucleotide SEQ ID NO: 10, the oligonucleotide of SEQ ID NO: 11, the oligonucleotide complementary to SEQ ID NO: 7, the oligonucleotide complementary to SEQ ID NO: 8, the oligonucleotide complementary to SEQ ID NO: 9, the oligonucleotide complementary to SEQ ID NO: 10 and the oligonucleotide complementary to SEQ ID NO: 11.  
     
     
         16 . The method according to  claim 15 , wherein said two primers are selected from the following oligonucleotide combinations: a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 7 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 8; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 9 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 10; and a combination in which one comprises at least 10 consecutive bases of Oligonucleotide SEQ ID NO: 11 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 8.  
     
     
         17 . The kit according to  claim 4 , wherein said pair of primers is selected from the group consisting of oligonucleotides comprising at least 10 consecutive bases of the oligonucleotide of SEQ ID NO: 7, the oligonucleotide of SEQ ID NO: 8, the oligonucleotide of SEQ ID NO: 9, the oligonucleotide SEQ ID NO: 10, the oligonucleotide of SEQ ID NO: 11, the oligonucleotide complementary to SEQ ID NO: 7, the oligonucleotide complementary to SEQ ID NO: 8, the oligonucleotide complementary to SEQ ID NO: 9, the oligonucleotide complementary to SEQ ID NO: 10 and the oligonucleotide complementary to SEQ ID NO: 11.  
     
     
         18 . The synthetic oligonucleotide according to  claim 1 , wherein said gene is the toxic shock syndrome toxin-1 gene of  Staphylococcus aureus , and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of the oligonucleotide selected from the group consisting of oligonucleotide SEQ ID NO: 19, oligonucleotide SEQ ID NO: 20, oligonucleotide SEQ ID NO: 21, and Oligonucleotide SEQ ID NO: 22.  
     
     
         19 . The synthetic oligonucleotide according to  claim 2 , wherein said nucleotide sequence is complementary to the synthetic oligonucleotide of  claim 18 .  
     
     
         20 . The method according to  claim 3 , wherein said target DNA is a gene of  Staphylococcus aureus  and said primers are selected from the group consisting of oligonucleotides of claims  18  and  19 .  
     
     
         21 . The method according to  claim 20 , wherein said two primers are selected from the following oligonucleotide combinations: a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 20 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 21; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 19 and the other comprises at least TO consecutive bases of oligonucleotide SEQ ID NO: 22; and a combination in which one comprises at least 10 consecutive bases of Oligonucleotide SEQ ID NO: 20 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 22.  
     
     
         22 . The kit according to  claim 4 , wherein said pair of primers are selected from the group consisting of oligonucleotides of claims  18  and  19  for detection of  Staphylococcus aureus.    
     
     
         23 . The synthetic oligonucleotide according to  claim 1 , wherein said gene is the cholera toxin gene of  Vibrio cholerae , and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of the oligonucleotide selected from the group consisting of oligonucleotide SEQ ID NO: 23, oligonucleotide SEQ ID NO: 24, oligonucleotide SEQ ID NO: 25, and oligonucleotide SEQ ID NO: 26.  
     
     
         24 . The synthetic oligonucleotide according to  claim 2 , wherein said nucleotide sequence is complementary to the synthetic oligonucleotide of  claim 23 .  
     
     
         25 . The method according to  claim 3 , wherein said target DNA is a gene of  Vibrio cholerae  and said primers are selected from the group consisting of oligonucleotides of claims  23  and  24 .  
     
     
         26 . The method according to  claim 25 , wherein said two primers are selected from the following oligonucleotide combinations: a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 23 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 25; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 24 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 26.  
     
     
         27 . The kit according to  claim 4 , wherein said pair of primers are selected from the group consisting of oligonucleotides of claims  23  and  24  for detection of  Vibrio cholerae.    
     
     
         28 . The synthetic oligonucleotide according to  claim 1 , wherein said gene is the enterotoxin gene of  Clostridium perfringens,  and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of the oligonucleotide selected from the group consisting of oligonucleotide SEQ ID NO: 27, oligonucleotide SEQ ID NO: 28, oligonucleotide SEQ ID NO: 29, oligonucleotide SEQ ID NO: 30, oligonucleotide SEQ ID NO: 31, oligonucleotide SEQ ID NO: 32, oligonucleotide SEQ ID NO: 33, oligonucleotide SEQ ID NO: 34, and oligonucleotide SEQ ID NO: 35.  
     
     
         29 . The synthetic oligonucleotide according to  claim 2 , wherein said nucleotide sequence is complementary to the synthetic oligonucleotide of  claim 28 .  
     
     
         30 . The method according to  claim 3 , wherein said target DNA is a gene of  Clostridium perfringens  and said primers are selected from the group consisting of oligonucleotides comprising at least 10 consecutive bases of the oligonucleotide of SEQ ID NO: 27, the oligonucleotide of SEQ ID NO: 28, the oligonucleotide of SEQ ID NO: 29, the oligonculeotide SEQ ID NO: 30, the oligonucleotide of SEQ ID NO: 31, the oligonucleotide of SEQ ID NO: 32, the oligonucleotide of SEQ ID NO: 33, the oligonucleotide of SEQ ID NO: 34, the oliogonucleotide of SEQ ID NO: 35, the oligonucleotide complementary to SEQ ID NO: 27, the oligonucleotide complementary to SEQ ID NO: 28, the oligonucleotide complementary to SEQ ID NO: 29, the oligonucleotide complementary to SEQ ID NO: 30 and the oligonucleotide complementary to SEQ ID NO: 31, the oligonucleotide complementary to SEQ ID NO: 32, the oligonucleotide complementary to SEQ ID NO: 33, the oligonucleotide complementary to SEQ ID NO: 34 and the oligonucleotide complementary to SEQ ID NO: 35.  
     
     
         31 . The method according to  claim 30 , wherein said two primers are selected from the following oligonucleotide combinations: a combination in which one. oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 27 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 32; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 28 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 33; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 29 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 33; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 30 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 34; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 31 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 35.  
     
     
         32 . The kit according to  claim 4 , wherein said pair of primers are selected from the group consisting of oligonucleotides of claims  28  and  29  for detection of  Clostridium perfringens .