KR20040032588A - Oligonucleotide primers and probes specific to Clostridium difficile and method for detecting Clostridium difficile - Google Patents

Abstract

PURPOSE: Oligonucleotide primers and probes specific to Clostridium difficile and a method for detecting Clostridium difficile using the same are provided, thereby rapidly, sensitively and specifically detecting Clostridium difficile using nucleic acid fragments specific to Clostridium difficile A or B toxin. CONSTITUTION: A method for detecting Clostridium difficile comprises the steps of: PCR amplifying a testing sample using nucleic acid fragments as a primer; and nucleotide sequencing the PCR amplified products, wherein the nucleic acid fragment comprises 6 or more consecutive nucleotides derived from the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 142, or combination thereof; the nucleic acid fragment is DNA, RNA or PNA; the combination comprises more than 6 consecutive nucleotides derived from one nucleotide sequence out of SEQ ID NO: 1 to SEQ ID NO: 30, and more than 6 consecutive nucleotides derived from one nucleotide sequence from SEQ ID NO: 31 to SEQ ID NO: 60, and the PCR product thereof is a more than 50bp of Clostridium difficile A toxin gene specific primer; the combination comprises more than 6 consecutive nucleotides derived from one nucleotide sequence out of SEQ ID NO: 61 to SEQ ID NO: 101, and more than 6 consecutive nucleotides derived from one nucleotide sequence from SEQ ID NO: 102 to SEQ ID NO: 142, and the PCR product thereof is a more than 50bp of Clostridium difficile B toxin gene specific primer; and the combination comprises more than 6 consecutive nucleotides derived from one nucleotide sequence out of SEQ ID NO: 31 to SEQ ID NO: 60, and more than 6 consecutive nucleotides derived from one nucleotide sequence from SEQ ID NO: 61 to SEQ ID NO: 101, and the PCR product thereof is a more than 50bp of Clostridium difficile A and B toxin genes specific primer. The alternative method for detecting Clostridium difficile comprises fusing the testing sample with a probe.

Description

Oligonucleotide primers and probes specific to Clostridium difficile and method for detecting Clostridium difficile}

The present invention relates to a nucleic acid fragment specific for Clostridium difficile A or B toxin, a method for diagnosing Clostridium difficile using the same, and a diagnostic kit comprising the same.

Clostridium difficile is an anaerobic apopoietic gram-positive bacillus that produces A-toxin (enterotoxin) and B-toxin (cytotoxin), causing colitis or antibiotic-induced diarrhea (Hyukmin Lee, MD et al., Korean J Clin Microbiol 2: 77-81, 1999). Clostridium difficile without toxins A and B does not cause disease. Clinical symptoms range from mild diarrhea to toxic megacolon. In addition, fibrin, mucus, leukocytes, etc. are increased in severe inflammation, epithelial cell necrosis and feces in the large intestine and accompanied by abdominal pain. In severe cases, it can lead to bowel perforation or death (Donna Zimmaro Bliss et al., Ann Intern Med 129 (12): 1012-1019, 1988). 10-20% of antibiotic-induced diarrhea is caused by Clostridium difficile and gastric colitis is mostly due to Clostridium difficile (Christoph Hogenauer et al., Clinical Infectious Diseases 27: 702-710, 1998). . Infection with Clostridium difficile is most common in patients who use antibiotics, including clindamycin, ampicillin, amoxicillin and cephalosporins. Commonly induced in The incidence is increasing gradually, which is believed to be due to the increase in immunosuppressive patients and elderly patients (Hyukmin Lee, MD et al ., Korean J Clin Microbiol 2: 77-81, 1999). In the United States, 300,000 to 3,000,000 people are infected with Clostridium difficile each year, with an additional cost of $ 6,000 to $ 10,000 per person for treatment (Eleftherios Mylonakis, MD et al., Arch Intern Med , 161: 525-). 533, 2001). Infections caused by Clostridium difficile can be treated with oral vancomycin or metronidazole. However, it is very difficult to discriminate between enteritis caused by Clostridium difficile and other causes only by clinical symptoms. Therefore, for the diagnosis of Clostridium difficile infection, development of a method for quickly and efficiently identifying and diagnosing Clostridium difficile that produces A and B toxins is urgently required.

The conventional cytotoxicity assay by tissue culture was regarded as the standard method of detecting toxin producing Clostridium difficile. However, this method was difficult to perform routinely in the laboratory. In addition, culturing Clostridium difficile using Cycloserine Cefoxitin Fructose Agar (CCFA) as a selective medium, and then detecting Clostridium difficile by cytotoxin neutralization Although the method was commonly used, this method had the disadvantage of taking 3-4 days for diagnosis (David et al., Ann Intern Med 125 (6): 515-516, 1996). In addition, the conventional intestinal endoscopy method was a method that can be used only when the infectious disease has developed into gastric colitis. Recently, a method for detecting A or B toxin directly from fecal specimens has been developed by immunoassay, but its sensitivity varies from 34 to 100% depending on the report (Dieter HM Groschel, Crit Rev Clin Lab). Sci 33 (3): 203-45, 1996). The toxins of Clostridium difficile are very unstable at room temperature, so the fecal sample must be tested immediately. To solve these diagnostic problems, high sensitivity, rapid results can be obtained, and PCR, strand displacement amplification, and nucleic acid sequence amplification, which are less affected by the room temperature exposure time of the sample. Nucleic acid amplification and molecular amplification methods such as sequence-based amplification, rolling circle amplification, invaders and DNA chips (Barry Schweitzer et al., Current opinion in biotechnology 12: 21-27, 2001 Molecular biological diagnostics method is developed.

It is an object of the present invention to provide nucleic acid fragments specific for Clostridium difficile.

Still another object of the present invention is to provide a method for amplifying a nucleic acid specific for Clostridium difficile using the nucleic acid fragment.

Still another object of the present invention is to provide a method for detecting Clostridium difficile in a sample by PCR using the nucleic acid fragment.

Still another object of the present invention is to provide a method for detecting Clostridium difficile in a specimen through a hybridization method using the nucleic acid fragment.

It is also an object of the present invention to provide a kit for diagnosing Clostridium difficile comprising the nucleic acid fragment.

The present invention also provides a composition comprising the nucleic acid fragment.

1 is a diagram showing the positions of Clostridium difficile A toxin and B toxin genes and the position of the nucleic acid fragment of the present invention,

FIG. 2A shows PCR after PCR for Clostridium difficile standard strains and general pathogenic microbial standard strains using primary primers CDA7F and CDA13R and secondary primers CDA10F and CDA13R specific for Clostridium difficile A toxin gene. It's a permanent picture,

FIG. 2B shows PCR of the primary primers CDB38F and CDB40R specific to the Clostridium difficile B toxin gene and the Clostridium difficile standard strain and the general pathogenic microorganism standard strain using CDB38F and CDB39R. It is a picture of electrophoresis of the obtained PCR product,

Figure 3a is a picture of the electrophoresis of the PCR product obtained by PCR using the first primers CDA7F and CDA13R and the second primers CDA10F and CDA13R specific for Clostridium difficile A toxin gene in clinical specimens,

Figure 3b is a picture of the electrophoresis of the PCR product obtained by PCR using the primary primers CDB38F and CDB40R and the secondary primers CDB38F and CDB39R specific to the Clostridium difficile B toxin gene in clinical specimens,

Figure 4 shows the electrophoresis of the PCR product obtained by performing nested PCR using a pair of translocation primers specific for the Clostridium difficile B toxin gene and inverted primers specific for the Clostridium difficile A toxin gene in clinical samples. One picture,

5 is obtained by performing nested double PCR using clinical specimens and enterobacteria as a template using primer pairs specific for Clostridium difficile A toxin gene and primer pairs specific for Clostridium difficile B toxin gene. Picture of electrophoresis of PCR product,

Fig. 6 is a photograph showing electrophoresis of a PCR product obtained by performing PCR using a continuous dilution of the cultured toxin producing Clostridium difficile as a template.

The present invention provides a nucleic acid fragment comprising at least six consecutive nucleotides or a combination thereof derived from any one of the nucleotide sequences of SEQ ID NOs: 1 to 142.

Such nucleic acids include, for example, DNA, RNA or PNA. In addition, the nucleic acid includes RNA having a nucleotide sequence in which T is substituted with U in the nucleotide sequences of SEQ ID NOs: 1 to 142. The size of the nucleic acid fragment is in the range of 6 to SEQ ID NO: 1 to 142 of the nucleotide sequence of the base sequence. In addition, the combination of the nucleic acid fragment of this invention means the collection of the nucleic acid fragment of this invention containing 2 or more nucleic acid fragments of the said nucleic acid fragment.

The nucleic acid fragments of the present invention can be used as primers or probes. In the present invention, "primer" refers to the starting point of nucleic acid synthesis from a template under appropriate buffer and temperature under appropriate conditions (ie, the presence of four kinds of nucleoside triphosphate, DNA or RNA polymerase or polymerase such as reverse transcriptase). It represents a single stranded oligonucleotide that can act as. In addition, in the present invention, "probe" means a hybridization probe, and means an oligonucleotide capable of sequence-specific binding to the complementary strand of a nucleic acid. The primer or probe may be attached with a labeling material, for example, a labeling material selected from the group consisting of radioisotopes, fluorescent materials, enzymes, chemiluminescent materials, coenzymes, and hapten.

Nucleic acid fragments of the present invention, the combination is derived from any one or more of the nucleotide sequence of SEQ ID NO: 31 to 60 and six or more consecutive nucleotides derived from any one of the nucleotide sequence of SEQ ID NO: 1 to 30 It may be Clostridium difficile A toxin gene specific primer consisting of one or more each of six or more consecutive nucleotides, the PCR product obtained therefrom is 50bp or more in size.

In addition, the nucleic acid fragment of the present invention is a combination of six or more consecutive nucleotides derived from any one of the nucleotide sequence of SEQ ID NO: 61 to 101 and the base sequence of any one of the nucleotide sequence of SEQ ID NO: 102 to 142 It may be a Clostridium difficile B toxin gene specific primer comprising at least one of six or more contiguous nucleotides derived from each other and having a PCR product of 50 bp or more in size.

In addition, the nucleic acid fragment of the present invention is a combination of six or more consecutive nucleotides derived from any one of the nucleotide sequence of SEQ ID NO: 31 to 60 and the base sequence of any one of the nucleotide sequence of SEQ ID NO: 61 to 101 It may be Clostridium difficile A toxin and B gene specific primer consisting of one or more each of six or more consecutive nucleotides derived.

The present invention relates to Clostridium difficile toxin A, B or A and B gene specific region comprising the step of PCR using any one of the Clostridium difficyl toxin A, B or A and B gene specific primers Provides a way to amplify. The PCR can be performed using conventional PCR instruments and reagents. Such PCRs include commonly used PCR techniques such as single PCR, multiplex PCR, nested PCR, semi-nested PCR, and sequencing PCR.

The present invention comprises the steps of PCR using a sample obtained from the sample Clostridium difficyl toxin A, B or any one of A and B gene specific primers as a primer; And it provides a method for diagnosing the presence of Clostridium difficile in the sample comprising the step of analyzing the base sequence of the product amplified by the PCR. The sample may include a sample derived from a clinical patient. In addition, the sequencing may be used a conventional sequencing method or kit. As a result of sequencing, it is determined that Clostridium difficile is present when Clostridium difficile toxin A, B or A and B gene specific regions are present.

The present invention also provides a method for diagnosing the presence of Clostridium difficile in a sample comprising hybridizing a sample obtained from the sample with a probe of the present invention. The hybridization process may be performed according to conventional hybridization methods under appropriate conditions, taking into account the type of probe or the sequence of the target region.

The present invention also provides a diagnostic kit for diagnosing the presence of Clostridium difficile in a sample containing a nucleic acid fragment of the present invention. In addition to the nucleic acid fragment, the diagnostic kit of the present invention may include reagents for PCR reaction, for example, four types of dNTP solutions, DNA polymerases, and buffers. In addition, in the case of a diagnostic kit including a nucleic acid fragment of the present invention as a probe, various solutions and buffers required for hybridization may be included.

The present invention also provides a composition comprising the nucleic acid fragment of the present invention. Compositions of the present invention include pharmaceutically acceptable salts of the nucleic acid fragments. In addition, ingredients that can be used in conventional pharmaceutical compositions such as buffers, excipients and carriers can be included.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

Example 1 Culture of Clostridium difficile and Genomic DNA Isolation

Clostridium difficile standard strains were purchased from the Gene Bank (Korea Collection for type Culture, KCTC), and clinical specimens were obtained from Yonsei Medical Center and Kosin Medical Center. DNA separation from the standard strain and clinical specimens was done using an InstaGene matrix (Bio-Rad Co.).

The strain to be used in this example was incubated in a selective medium (Cycloserine Cefoxitin Fructose Agar, CCFA), and then suspended in a 1.5 ml tube containing 100 μl of instagin matrix using platinum. This reaction was carried out at 56 ° C. for 30 minutes, followed by well mixing for 10 seconds, followed by heat treatment at 100 ° C. for 8 minutes, followed by well mixing for 10 seconds. The mixture was centrifuged at 12,000 rpm for 3 minutes and the supernatant was used as template DNA for the PCR reaction.

Clostridium difficile The specificity of the primers and probes of the present invention was investigated in standard strains and standard pathogenic microbial standard strains. Standard strains used in this example are shown in Table 1.

Number Standard strain name One Clostridium difficile (ATCC 9689) 2 Branhamella catarrhalis (ATCC 8193) 3 Citrobacter freundii (ATCC 6750) 4 Enterobacter aerogenes (ATCC 13048) 5 Enterobacter cloacae ( ATCC 29003) 6 Escherichia coli (ATCC 23499) 7 Plesiomonas shigelloides (ATCC 14029 ) 8 Proteus vulgaris ( ATCC 6059) 9 Providencia rettgeri (ATCC 9250) 10 Proteus mirabilis (ATCC 4630) 11 Pseudomonas aeruginosa (ATCC 15682) 12 Rhanella aquatilis (ATCC 33071) 13 Shigella sonnei (ATCC 9290) 14 Streptococcus agalactiae (ATCC 8058) 15 Streptococcus oralis (ATCC 35037) 16 Streptococcus bovis (ATCC 9809) 17 Streptococcus intermedius (ATCC 27335) 18 Streptococcus pyogenes (ATCC 14918) 19 Streptococcus pneumoniae (ATCC 6301) 20 Streptococcus equisimilis (ATCC 9542) 21 Streptococcus equinus ( ATCC 9812) 22 Staphylococcus aureus (ATCC 27695) 23 Staphylococcus epidermidis ( ATCC 146) 24 Staphylococcus saphrophyticus (ATCC 12162) 25 Vibrio parahemolyticus (ATCC 17802) 26 Vibrio vulnificus (ATCC 27562)

Example 2 DNA Extraction from Fecal Patients

1. Pretreatment of feces

(1) 0.05 to 0.1 g of fecal diarrhea without preservatives and dilution without dilution was placed in a 1.5 ml tube and sterilized with phosphate-saline buffer (PBS, 0.05 M, pH: 7.4) 1 ml was added and mixed.

(2) Centrifuged at 200xg for 5 minutes.

(3) The supernatant was transferred to a new 1.5 ml tube.

(4) (2) and (3) were repeated twice.

(5) Centrifuged at 9,000xg for 10 minutes.

(6) 1 ml of PBS was added to precipitate the precipitate, and then centrifuged at 9,000 × g for 10 minutes.

(7) (6) was repeated once.

(8) The sterilized tertiary distilled water was added to precipitate the precipitate, and then centrifuged at 9,000xg for 10 minutes. The upper layer was then discarded and the precipitate was treated with an InstaGene matrix (Bio-Rad C0.).

2. Isolation of DNA from Pretreated Feces

DNA was isolated by the same method as genomic DNA isolation of the standard strain of Clostridium difficile of Example 1. This isolated DNA (hereinafter also referred to simply as a clinical sample) was used as a template for the PCR reaction.

Example 3: Preparation of primers and probes for PCR

1. A toxin gene specific primers and probes of Clostridium difficile Produce

A primer was designed that amplifies only the A toxin gene of Clostridium difficile without reacting to other pathogenic microorganisms. Primers are summarized in Tables 2 and 3.

Clostridium difficile A toxin gene specific translocation primer Identification gene Primer Name Sequence A location in toxin * SEQ ID NO: A toxin of Clostridium difficile CDA1F 5′-atatctaaagaagagttaataaaactcgcat-3 ′ 10-40 One CDA2F 5′-cgcatatagcattagaccaa-3 ′ 36-55 2 CDA3F 5′-aaactatactaactaatttagacgaatata-3 ′ 71-100 3 CDA4F 5′-ataagttaactacaaacaataatgaaaata-3 ′ 101-130 4 CDA5F 5′-aatatttacaattaaaaaaactaaatgaat-3 ′ 131-160 5 CDA6F 5′-caattgatgtttttatgaataaatataaaa-3 ′ 161-190 6 CDA7F 5′-ttcaagcagaaatagagcac-3 ′ 192-211 7 CDA8F 5′-ttattaaaaattccaatacaagccctgtag-3 ′ ′ 251-280 8 CDA9F 5′-aaaaaaatttacattttgtatggataggtg-3 ′ 281-310 9 CDA10F 5′-cttgaatacataaaacaatgggctgatatt-3 ′ 311-340 10 CDA11F 5′-aatgcagaatataatattaaactgtggtat-3 ′ 361-390 11 CDA12F 5′-actgtggtatgatagtgaagcattc-3 381-405 12 CDA13F 5′-accactgaagcattacagct-3 ′ 445-464 13 CDA14F 5′-ttattataaatctcaaatcaataaa-3 ′ 561-585 14 CDA15F 5′-atagatgatattataaagtctcatctagta-3 ′ 601-630 15 CDA16F 5′-tctgaatataatagagatgaaactg-3 ′ 631-655 16 CDA17F 5′-cgtggaaatttagctgcagcatctgacata-3 ′ 781-810 17 CDA18F 5′-gtaagattattagccctaaaaaattttggc-3 ′ 811-840 18 CDA19F 5′-tcagaaaactttgataaacttgatcaacaa-3 ′ 991-1020 19 CDA20F 5′-ttaaaagataattttaaactcattatagaa-3 ′ 1021-1050 20 CDA21F 5′-aaattagaaaatttaaatgtatctgatctt-3 ′ 1081-1110 21 CDA22F 5′-aaaaaatagatatcaatttttaaac-3 ′ 1206-1230 22 CDA23F 5′-taaaccaacaccttaacccagccat-3 ′ 1226-1250 23 CDA24F 5′-agccatagagtctgataataacttca-3 ′ 1246-1271 24 CDA25F 5′-aagtaggttttatgccagaagctcg-3 ′ 1361-1385 25 CDA26F 5′-ctccacaataagtttaagtggtcca-3 ′ 1386-1410 26 CDA27F 5′-gtgatgatggagaatctattttaga-3 ′ 1871-1895 27 CDA28F 5′-attaaataaatataggatacctgaaataaatataggatacctgaa-3 ′ 1896-1940 28 CDA29F 5′-agattaaaaaataaggaaaaagtaa-3 ′ 1921-1945 29 CDA30F 5′-ggacatggtaaagatgaattcaaca-3 ′ 1960-1984 30

*: Location in base sequence was based on GenBank Accession Number X92982.

Clostridium difficile A toxin gene specific inversion primer Identification gene Primer Name Sequence A location in toxin * SEQ ID NO: A toxin of Clostridium difficile CDA1 5′-atgcgagttttattaactcttctttagatat-3 ′ 40-10 31 CDA2 5′-ttggtctaatgctatatgcg-3 ′ 55-36 32 CDA3 5′-tatattcgtctaaattagttagtatagttt-3 ′ 100-71 33 CDA4 5′-tattttcattattgtttgtagttaacttat-3 ′ 130-101 34 CDA5 5′-attcatttagtttttttaattgtaaatatt-3 ′ 160-131 35 CDA6 5′-ttttatatttattcataaaaacatcaattg-3 ′ 190-161 36 CDA7 5′-gtgctctatttctgcttgaa-3 ′ 211-192 37 CDA8 5′-ctacagggcttgtattggaatttttaataa-3 ′ 280-152 38 CDA9 5′-cacctatccatacaaaatgtaaattttttt-3 ′ 310-281 39 CDA10 5′-aatatcagcccattgttttatgtattcaag-3 ′ 340-311 40 CDA11 5′-ataccacagtttaatattatattctgcatt-3 ′ 390-361 41 CDA12 5′-gaatgcttcactatcataccacagt-3 ′ 405-381 42 CDA13 5′-agctgtaatgcttcagtggt-3 ′ 464-445 43 CDA14 5′-tttattgatttgagatttataataa-3 ′ 585-561 44 CDA15 5′-tactagatgagactttataatatcatctat-3 ′ 630-601 45 CDA16 5′-cagtttcatctctattatattcaga-3 ′ 655-631 46 CDA17 5′-tatgtcagatgctgcagctaaatttccacg-3 ′ 810-781 47 CDA18 5′-gccaaaattttttagggctaataatcttac-3 ′ 840-811 48 CDA19 5′-ttgttgatcaagtttatcaaagttttctga-3 ′ 1020-991 49 CDA20 5′-ttctataatgagtttaaaattatcttttaa-3 ′ 1050-1021 50 CDA21 5′-aagatcagatacatttaaattttctaattt-3 ′ 1110-1081 51 CDA22 5′-gtttaaaaattgatatctatttttt-3 ′ 1230-1206 52 CDA23 5′-atggctgggttaaggtgttggttta-3 ′ 1250-1226 53 CDA24 5′-tgaagttattatcagactctatggct-3 ′ 1271-1246 54 CDA25 5′-cgagcttctggcataaaacctactt-3 ′ 1385-1361 55 CDA26 5′-tggaccacttaaacttattgtggag-3 ′ 1410-1386 56 CDA27 5′-tctaaaatagattctccatcatcac-3 ′ 1895-1871 57 CDA28 5′-ttcaggtatcctatatttatttcaggtatcctatatttatttaat-3 ′ 1940-1896 58 CDA29 5′-ttactttttccttattttttaatct-3 ′ 1945-1921 59 CDA30 5′-tgttgaattcatctttaccatgtcc-3 ′ 1984-1960 60

*: Location in base sequence was based on GenBank Accession Number X92982.

2. Preparation of B Toxin Gene Specific Primers and Probes of Clostridium difficile

A primer was designed that amplifies only the B toxin gene of Clostridium difficile without reacting to other pathogenic microorganisms. Primers are summarized in Tables 4 and 5.

Clostridium difficile B toxin gene specific translocation primer Identification gene Primer Name Sequence Location in B toxin * SEQ ID NO: B toxin of Clostridium difficile CDB1F 5′-gaagatgaatatgttgcaatattgg-3 ′ 61-85 61 CDB2F 5′-atgctttagaagaatatcataatat-3 ′ 86-110 62 CDB3F 5′-gtttaacagatatttatatagatac-3 ′ 161-185 63 CDB4F 5′-atataaaaaatctggtagaaataaa-3 ′ 186-210 64 CDB5F 5′-tagttacagaagtattagagctaaa-3 ′ 236-260 65 CDB6F 5′-gaataataatttaactccagttgag-3 ′ 261-285 66 CDB7F 5′-gaagttcaaagtagttttgaatctg-3 ′ 1021-1045 67 CDB8F 5′-tcatcacttggtgatatggaggcat-3 ′ 1081-1105 68 CDB9F 5′-ctagaagttaaaattgcatttaata-3 ′ 1111-1135 69 CDB10F 5′-tagtttaaatccagctattagcgag-3 ′ 1236-1260 70 CDB11F 5′-gcagataatggtagatttatgatgg-3 ′ 1321-1345 71 CDB12F 5′-gatgttaaaactactattaacttaa-3 ′ 1381-1405 72 CDB13F 5′-agcatatgcggcagcttatcaagat-3 ′ 1416-1440 73 CDB14F 5′-actccttatgatagtgtactgtttc-3 ′ 1801-1825 74 CDB15F 5′-tataatcctggagatggtgaaatac-3 ′ 1861-1885 75 CDB16F 5′-tctgatagacctaagattaaattaa-3 ′ 1921-1945 76 CDB17F 5′-gatatatttgcaggttttgatgtag-3 ′ 1981-2005 77 CDB18F 5′-ttagctaaagaggatatttctccta-3 ′ 2046-2070 78 CDB19F 5′-agacttatcctggaaaattattact-3 ′ 2126-2140 79 CDB20F 5′-agaattattggatcattctggtgaa-3 ′ 2256-2280 80 CDB21F 5′-ttacaagaaattagaaataattcta-3 ′ 2401-2425 81 CDB22F 5′-attgaagaagctaagaatttaactt-3 ′ 2521-2545 82 CDB23F 5′-acgtattggaagtacaaaaagaaga-3 ′ 3790-3814 83 CDB24F 5′-gcaggaggaacttatgcatt-3 ′ 3956-3975 84 CDB25F 5′-atctacactaagtattgaagagaat-3 ′ 4295-4319 85 CDB26F 5′-ggattcaatagcgaattaca-3 ′ 4351-4370 86

*: Location in base sequence was based on GenBank Accession Number X92982.

Table 4 (continued)

Identification gene Primer Name Sequence Location of B toxin * SEQ ID NO: Clostridium difficile B toxin CDB27F 5′-tggtgaggtaaatggaagtaatgga-3 ′ 4365-4389 87 CDB28F 5′-aaatgcaattatagaagttgattta-3 ′ 4415-4439 88 CDB29F 5′-tacctgatgtagttcttata-3 ′ 4463-4482 89 CDB30F 5′-catatagctttgtagatagtgaag-3 ′ 4561-4584 90 CDB31F 5′-gttcatgaatagaaaaggtaataca-3 ′ 4701-4725 91 CDB32F 5′-caccaaatatttatacagatgaaa-3 ′ 5080-5103 92 CDB33F 5′-ttaatgagctttttagaaagtatga-3 ′ 5120-5144 93 CDB34F 5′-gtggtactacttctattggccaa-3 ′ 5178-5200 94 CDB35F 5′-taagtggtactacttctattggccaa-3 ′ 5233-5258 95 CDB36F 5′-cttcatattatgaggatggattga-3 ′ 5741-5764 96 CDB37F 5′-catcataatgaagatttaggaaatg-3 ′ 6201-6225 97 CDB38F 5′-agactggatggatatatgat-3 ′ 6623-6642 98 CDB39F 5′-ctatactggttggttagattt-3 ′ 6963-6983 99 CDB40F 5′-ctgtatcagctcaattagtg-3 ′ 7070-7089 100 CDB41F 5′-ttgcagcaactggttcagtt-3 ′ 7019-7038 101

*: Location in base sequence was based on GenBank Accession Number X92982.

Clostridium difficile B toxin gene specific inversion primer Identification gene Primer Name Sequence Location in B toxin * Sequence number B toxin of Clostridium difficile CDB1R 5′-ccaatattgcaacatattcatcttc-3 ′ 85-61 102 CDB2R 5′-atattatgatattcttctaaagcat-3 ′ 110-86 103 CDB3R 5′-gtatctatataaatatctgttaaac-3 ′ 185-161 104 CDB4R 5′-tttatttctaccagattttttatat-3 ′ 210-186 105 CDB5R 5′-tttagctctaatacttctgtaacta-3 ′ 260-236 106 CDB6R 5′-ctcaactggagttaaattattattc-3 ′ 285-261 107 CDB7R 5′-ctgattcaaaactactttgaacttc-3 ′ 1045-1021 108 CDB8R 5′-atgcctccatatcaccaagtgatga-3 ′ 1105-1081 109 CDB9R 5′-tattaaatgcaattttaacttctag-3 ′ 1135-1111 110 CDB10R 5′-ctcgctaatagctggatttaaacta-3 ′ 1260-1236 111 CDB11R 5′-ccatcataaatctaccattatctgc-3 ′ 1345-1321 112 CDB12R 5′-ttaagttaatagtagttttaacatc-3 ′ 1405-1381 113 CDB13R 5′-atcttgataagctgccgcatatgct-3 ′ 1440-1416 114 CDB14R 5′-gaaacagtacactatcataaggagt-3 ′ 1825-1801 115 CDB15R 5′-gtatttcaccatctccaggattata-3 ′ 1885-1861 116 CDB16R 5′-ttaatttaatcttaggtctatcaga-3 ′ 1945-1921 117 CDB17R 5′-ctacatcaaaacctgcaaatatatc-3 ′ 2005-1981 118 CDB18R 5′-taggagaaatatcctctttagctaa-3 ′ 2070-2046 119 CDB19R 5′-agtaataattttccaggataagtct-3 ′ 2140-2126 120 CDB20R 5′-ttcaccagaatgatccaataattct-3 ′ 2280-2256 121 CDB21R 5′-tagaattatttctaatttcttgtaa-3 ′ 2425-2401 122 CDB22R 5′-aagttaaattcttagcttcttcaat-3 ′ 2545-2521 123 CDB23R 5′-tcttctttttgtacttccaatacgt-3 ′ 3814-3756 124 CDB24R 5′-aatgcataagttcctcctgc-3 ′ 3975-3956 125

*: Location in base sequence was based on GenBank Accession Number X92982.

Table 5 (continued)

Identification gene Primer Name Sequence Location in B toxin * Sequence number B toxin of Clostridium difficile CDB25R 5′-attctcttcaatacttagtgtagat-3 ′ 4319-4295 126 CDB26R 5′-tgtaattcgctattgaatcc-3 ′ 4370-4351 127 CDB27R 5′-tccattacttccatttacctcacca-3 ′ 4389-4365 128 CDB28R 5′-taaatcaacttctataattgcattt-3 ′ 4439-4415 129 CDB29R 5′-tataagaactacatcaggta-3 ′ 4482-4463 130 CDB30R 5′-cttcactatctacaaagctatatg-3 ′ 4584-4561 131 CDB31R 5′-tgtattaccttttctattcatgaac-3 ′ 4725-4701 132 CDB32R 5′-tttcatctgtataaatatttggtg-3 ′ 5103-5080 133 CDB33R 5′-tcatactttctaaaaagctcattaa-3 ′ 5144-5120 134 CDB34R 5′-ttggccaatagaagtagatccac-3 ′ 5200-5178 135 CDB35R 5′-ttggccaatagaagtagtaccactta-3 ′ 5258-5233 136 CDB36R 5′-tcaatccatcctcataatatgaag-3 ′ 5764-5741 137 CDB37R 5′-catttcctaaatcttcattatgatg-3 ′ 6225-6201 138 CDB38R 5′-atcatatatccatccagtct-3 ′ 6642-6623 139 CDB39R 5′-aaatctaaccaaccagtatag-3 ′ 6983-6963 140 CDB40R 5′-cactaattgagctgatacag-3 ′ 7089-7070 141 CDB41R 5′-aactgaaccagttgctgcaa-3 ′ 7038-7019 142

*: Location in base sequence was based on GenBank Accession Number X92982.

Example 4 Specificity Test of Prepared Primers

In order to determine the specificity of the primers of the present invention, PCR was performed on standard strains and control strains. Primers used are shown in Tables 6 and 7 below.

Clostridium difficile A toxin amplification primers Potential primer Inversion primer Expected size CDA1F CDA8R 270 bp CDA2F CDA12R 369 bp CDA3F CDA11R 319 bp CDA4F CDA18R 639 bp CDA5F CDA14R 454 bp CDA6F CDA15R 469 bp CDA7F CDA13R 273 bp CDA9F CDA17R 529 bp CDA10F CDA13R 154 bp CDA15F CDA19R 419 bp CDA16F CDA23R 619 bp CDA20F CDA29R 924 bp CDA21F CDA30R 903 bp CDA22F CDA26R 204 bp CDA24F CDA28R 694 bp CDA25F CDA27R 534 bp

Clostridium difficile B toxin amplification primers Potential primer Inversion primer Expected size CDB1F CDB6R 224 bp CDB2F CDB4R 124 bp CDB3F CDB5R 99 bp CDB7F CDB9R 114 bp CDB8F CDB10R 179 bp CDB11F CDB15R 564 bp CDB12F CDB16R 564 bp CDB13F CDB17R 589 bp CDB14F CDB18R 289 bp CDB19F CDB22R 419 bp CDB20F CDB21R 169 bp CDB23F CDB27R 599 bp CDB24F CDB26R 414 bp CDB25F CDB28R 144 bp CDB29F CDB33R 681 bp CDB30F CDB32R 543 bp CDB31F CDB34R 500 bp CDB35F CDB36R 484 bp CDB37F CDB41R 837 bp CDB38F CDB39R 360 bp CDB38F CDB40R 468 bp

Clostridium difficile using the above and a primer PCR was performed using standard strains and standard pathogenic microbial standard strains as samples. The reaction conditions were heat treated at 95 ° C. for 3 minutes, and then reacted 30 times at 95 ° C. for 20 seconds, 57 ° C. for 1 minute, and 72 ° C. for 30 seconds, and finally extended at 72 ° C. for 10 minutes. After the reaction, the result was confirmed by electrophoresis using 2% agarose gel.

The results are shown in Figures 2A, 2B and Table 8. FIG. 2A is a photograph of electrophoresis after PCR using primary primers CDA7F and CDA13R specific to Clostridium difficile A toxin gene and secondary primers CDA10F and CDA13R.

In Figure 2A, M; 100 bp molecular weight label, C; Distilled water (negative control); Clostridium difficile (ATCC 9689) (positive control) was used as a sample and amplified by A toxin specific primers CDA10F and CDA13R. Enterobacter croaker (Enterobacter cloacaeATCC 29003) as a sample and amplified by A toxin specific primers CDA7F and CDA13R, 2; Enterobacter croaker (Enterobacter cloacaeATCC 29003) as a sample and amplified by A toxin specific primers CDA10F and CDA13R, 3; Pseudomonas aeruginosaPseudomonas aeruginosaATCC 15682) as a sample and amplified by A toxin specific primers CDA7F and CDA13R, 4; Pseudomonas aeruginosaPseudomonas aeruginosaATCC 15682) as a sample and amplified by A toxin specific primers CDA10F and CDA13R, 5; Star pyrocoke piogenesStreptococcus. pyogenesATCC 14918) as a sample and amplified by A toxin specific primers CDA7F and CDA13R, 6; Star pyrocoke piogenesStreptococcus. pyogenesATCC 14918) as a sample and amplified by A toxin specific primers CDA10F and CDA13R, 7; Vibrio Bulnipicus (Vibrio vulnificusATCC 27562), resulting from amplification with A toxin specific primers CDA7F and CDA13R, 8; Vibrio Bulnipicus (Vibrio vulnificusATCC 27562) as a sample and amplified by A toxin specific primers CDA10F and CDA13R.

FIG. 2B is an electrophoresis photograph after PCR using primary primers CDB38F and CDB40R and secondary primers CDB38F and CDB39R specific for Clostridium difficile B toxin gene. FIG. The picture order is shown in Figure 2A.

As shown in Figures 2A and 2B, PCR was performed using primers CDA7F and CDA13R, CDA10F and CDA13R, CDB38F and CDB40R and CDB38F and CDB39R, which resulted in specific amplification only in Clostridium difficile and in general pathogenic microorganisms. Not amplified. Therefore, the primer of the present invention was found to be specific for the A or B toxin gene of Clostridium difficile.

Table 7 shows the results of PCR using the primers of the present invention for samples containing general pathogenic microorganisms. As shown in Table 7, therefore, the primers of the present invention were specific only to the A or B toxin gene of Clostridium difficile.

Strain name Primer, amplification product size and amplification CDA7F / CDA13R CDA10F / CDA13R CDB38F / CDB40R CDB38F / CDB39R CDB30F / CDB32R CDB35F / CDB36R 273 bp 154 bp 468 bp 360 bp 543 bp 484bp Clostridium difficile (ATCC 9689) + + + + + + Aeromonas hydrophilia － － － － － － Branhamella catarrhalis (ATCC 8193) － － － － － － Burkholderia cepacia － － － － － － Candida glabrata － － － － － － Candida tropicalis － － － － － － Citrobacter freundii (ATCC 6750) － － － － － － Enterobacter aerogenes (ATCC 13048) － － － － － － Enterobacter cloacae (ATCC 29003) － － － － － － Escherichia coli (ATCC 23499) － － － － － － Enterococcus facium － － － － － － Enterobacter faecalis － － － － － － Plesiomonas shigelloides (ATCC 14029) － － － － － － Proteus vulgaris (ATCC 6059) － － － － － － Providencia rettgeri (ATCC 9250) － － － － － － Proteus mirabilis (ATCC 4630) － － － － － － Pseudomonas aeruginosa (ATCC 15682) － － － － － －

Table 8 (continued)

Strain name Primer, amplification product size and amplification CDA7F / CDA13R CDA10F / CDA13R CDB38F / CDB40R CDB38F / CDB39R CDB30F / CDB32R CDB35F / CDB36R 273 bp 154 bp 468 bp 360 bp 543 bp 484bp Rhanella aquatilis (ATCC 33071) － － － － － － Shewanella putrefaciens － － － － － － Shigella sonnei (ATCC 9290) － － － － － － Streptococcus agalactiae (ATCC 8058) － － － － － － Streptococcus oralis (ATCC 35037) － － － － － － Streptococcus bovis (ATCC 9809) － － － － － － Streptococcus intermedius (ATCC 27335) － － － － － － Streptococcus pneumoniae (ATCC 6301) － － － － － － Streptococcus equisimilis (ATCC 9542) － － － － － － Streptococcus equinus (ATCC 9812) － － － － － － Streptococcus mitis － － － － － － Streptococcus intermidius － － － － － － Streptococcus. pyogenes (ATCC 14918) － － － － － － Staphylococcus aureus (ATCC 27695) － － － － － － Staphylococcus haemolyticus － － － － － － Staphylococcus epidermidis (ATCC 146) － － － － － － Salmonella typhi － － － － － － Salmonella paratyphi A － － － － － － Vibrio parahemolyticus (ATCC 17802) － － － － － － Vibrio vulnificus (ATCC 27562) － － － － － －

+: Positive, PCR result target DNA amplified

-: Negative, PCR Result Purpose DNA Not amplified.

Strain name Primer, amplification product size and amplification CDB39F / CDA7R CDB41F / CDA2R CDA2F / CDA12R CDA22F / CDA26R CDB14F / CDB18R CDB24F / CDB26R 1.68kb 1.48kb 369 bp 204 bp 289 bp 414 bp Clostridium difficile (ATCC 9689) + + + + + + Aeromonas hydrophilia － － － － － － Branhamella catarrhalis (ATCC 8193) － － － － － － Burkholderia cepacia － － － － － － Candida glabrata － － － － － － Candida tropicalis － － － － － － Citrobacter freundii (ATCC 6750) － － － － － － Enterobacter aerogenes (ATCC 13048) － － － － － － Enterobacter cloacae (ATCC 29003) － － － － － － Escherichia coli (ATCC 23499) － － － － － － Enterococcus facium － － － － － － Enterobacter faecalis － － － － － － Plesiomonas shigelloides (ATCC 14029) － － － － － － Proteus vulgaris (ATCC 6059) － － － － － － Providencia rettgeri (ATCC 9250) － － － － － － Proteus mirabilis (ATCC 4630) － － － － － － Pseudomonas aeruginosa (ATCC 15682) － － － － － －

Table 9 (continued)

Strain name Primer, amplification product size and amplification CDB39F / CDA7R CDB41F / CDA2R CDA2F / CDA12R CDA22F / CDA26R CDB14F / CDB18R CDB24F / CDB26R 1.68kb 1.48kb 369 bp 204 bp 289 bp 414 bp Rhanella aquatilis (ATCC 33071) － － － － － － Shewanella putrefaciens － － － － － － Shigella sonnei (ATCC 9290) － － － － － － Streptococcus agalactiae (ATCC 8058) － － － － － － Streptococcus oralis (ATCC 35037) － － － － － － Streptococcus bovis (ATCC 9809) － － － － － － Streptococcus intermedius (ATCC 27335) － － － － － － Streptococcus pneumoniae (ATCC 6301) － － － － － － Streptococcus equisimilis (ATCC 9542) － － － － － － Streptococcus equinus (ATCC 9812) － － － － － － Streptococcus mitis － － － － － － Streptococcus intermidius － － － － － － Streptococcus. pyogenes (ATCC 14918) － － － － － － Staphylococcus aureus (ATCC 27695) － － － － － － Staphylococcus haemolyticus － － － － － － Staphylococcus epidermidis (ATCC 146) － － － － － － Salmonella typhi － － － － － － Salmonella paratyphi A － － － － － － Vibrio parahemolyticus (ATCC 17802) － － － － － － Vibrio vulnificus (ATCC 27562) － － － － － －

+: Positive, PCR result target DNA amplified

-: Negative, PCR Result Purpose DNA Not amplified.

Example 5 Nested PCR Test Using Clinical Specimens

1. PCR using a clinical sample as a template

In this example, CDA7F and CDA13R and CDB38F and CDB40R were used as primary primers, and CDA10F and CDA13R and CDB38F and CDB39R were used as secondary primers, respectively, and PCR was performed using a clinical sample as a template. Among the primers, CDA7F and CDA13R and CDA10F and CDA13R are examples of primer pairs specific for the Clostridium difficile A toxin gene. In addition, CDB38F and CDB40R and CDB38F and CDB39R are examples of primer pairs specific for the Clostridium difficile A toxin gene.

The composition of the PCR reaction is as follows. 500 mM KCl, 100 mM HCl, pH 9.0, 1% Triton X-100, 2.5 mM dNTP (dATP, dGTP, dTTP, dCTP), 1.5 mM MgCl ₂ , 1U Taq DNA Polymerase, PCR Enhancer and Toxins Gene-specific primers were used at 10 pmol each. The first PCR reaction was heat-treated at 95 ° C. for 3 minutes, followed by 25 reactions at 95 ° C. for 20 seconds, 57 ° C. for 1 minute, and 72 ° C. for 30 seconds, and finally extended at 72 ° C. for 10 minutes. The primary PCR product was used as template DNA in the secondary PCR reaction mixture and reacted 30 times under the same PCR conditions as the primary. After the reaction, electrophoresis was performed using 2% agarose gel.

The results are shown in FIGS. 3A and 3B. Figure 3A is a template for the clinical specimen, using CDA7F and CDA13R specific to the Clostridium difficile A toxin gene as a primary primer, after performing nested PCR using CDA10F and CDA13R as a secondary primer, electrophoresis One picture. In FIG. 3A, lane M is 100 bp molecular weight label, lane C is distilled water (negative control), lane P is Clostridium difficile (ATCC 9689) (positive control), and lanes 1-5 are Clostridium difficile Clinical sample.

FIG. 3B shows a clinical sample as a template, followed by nested PCR using CDB38F and CDB40R specific to the Clostridium difficile B toxin gene as a primary primer, and a second primer using CDB38 and CDB39. One picture. The order of lanes is the same as in FIG. 3A. As shown in FIG. 3B, it was confirmed that Clostridial difficile A toxin and B toxin genes were amplified in clinical samples in lanes 1 to 5.

2. Diagnosis by PCR after Clinical Specimen and Selective Culture

Clostridium difficile was carried out by PCR using the primary primers (CDA7F / CDA13R and CDB38F / CDB40R) and the secondary primers (CDA10F / CDA13R and CDB38F / CDB39R). Toxin genes were detected. In addition, by using Cycloserine Cefoxitin Fructose Agar (CCFA) as a selection medium, Clostridium difficile was cultured and then used as a template, specific to the Clostridium difficile B toxin gene. PCR was performed using red primers (primary primer: CDB38F / CDB40R and secondary primer: CDB38F / CDB39R). These PCR results are shown in comparison with Table 10.

culture B toxin PCR after incubation Sample A Toxin PCR Sample B Toxin PCR Total Samples (102) Positive (31) Positive (29) Positive (29) Positive (29) 29 Voice (2) Voice (2) Voice (2) 2 Voice (71) Voice (71) Positive (43) Positive (43) 43 Voice (28) Voice (28) 28

As shown in Table 10, Clostridium difficile directly in clinical specimens. A method for detecting A and B toxin genes by PCR is toxin-producing clot which is detected by culturing Clostridium difficile and PCR using a specific primer to Clostridium difficile B toxin gene as a template. It was consistent with the lithium difficile positive and negative results. The culture result was negative, but when it was diagnosed by the PCR method directly from the sample, it was found that 60 (43/71)% of the positive samples existed. This means that Clostridium difficile detection rate is low as 70% due to difficulty in culture. Therefore, the culture method alone was not suitable for use in diagnosis, and the PCR method using the primer of the present invention was found to be effective.

3. Detection by PCR using primers specific for Clostridium difficile A toxin gene and primer pairs specific for Clostridium difficile B toxin gene

In this embodiment, the nested PCR is performed using a clinical sample as a template, a primer specific for the Clostridium difficile B toxin gene, and a primer specific for the Clostridium difficile A toxin gene as an inverted primer. Clostridium difficile was detected. CDB39F and CDA7R were used as primary primers, and CDB41F and CDA2R were used as secondary primers. The composition of the PCR reaction product was the same as in Example 5. The first PCR reaction was heat-treated at 95 ° C. for 3 minutes, followed by 25 reactions of 1 minute at 95 ° C., 2 minutes at 54 ° C., and 2 minutes at 72 ° C., and finally extended at 72 ° C. for 10 minutes. The primary PCR product was used as template DNA in a secondary PCR reaction mixture and reacted 30 times under the same conditions as the primary PCR reaction. After the reaction, electrophoresis was performed using 2% agarose gel. The results are shown in FIG.

Figure 4 is a photograph of the electrophoresis of the PCR product. In Figure 4, M lane is 100bp molecular weight label, C lane is distilled water (negative control), P lane is Clostridium difficile (ATCC 9689) (positive control), lanes 1-5 are Clostridium difficile Clinical sample.

As shown in Fig. 4, clinical specimens were obtained through nested PCR using primers specific for the Clostridium difficile B toxin gene and inverted primers specific for the Clostridium difficile A toxin gene. It was confirmed that Clostridium difficile in the body could be detected.

4. Detection of Clostridium difficile in clinical specimens by double PCR

In this example, duplex nested PCR was performed using primers specific for the Clostridium difficile A toxin gene and the Clostridium difficile B toxin gene of the present invention.

CDA7F and CDA13R and CDB38F and CDB40R were used as the primary primers, and CDA10F and CDA13R and CDB38F and CDB39R were used as the secondary primers, and double PCR was performed using a clinical sample as a template. That is, PCR was performed after putting the two pairs of primers in one in vitro. The composition of the PCR reaction product was the same as in Example 5. However, A toxin-specific primers were used by 7 pmol each in the first and second PCR reactions, and B toxin gene-specific primers were used by 20 pmol each in the first and second PCR reactions. The first PCR reaction was heat-treated at 95 ° C. for 3 minutes, followed by 25 reactions at 95 ° C. for 30 seconds, 57 ° C. for 1 minute, and 72 ° C. for 30 seconds, and finally extended at 72 ° C. for 10 minutes. The primary PCR product was used as template DNA and then put into the secondary PCR reaction mixture, and reacted 30 times under the conditions of the primary PCR reaction. After the reaction, electrophoresis was performed using 2% agarose gel.

The results are shown in FIG. 5 is a photograph of electrophoresis of the obtained double PCR product. In FIG. 5, M lane is 100 bp molecular weight label, C lane is distilled water (negative control), P lane is Clostridium difficile (ATCC 9689) (positive control), and lane 1 is Enterobacter croaker (Enterobacter cloacaeATCC 29003) and lane 2 is Pseudomonas aeruginosa (Pseudomonas aeruginosaATCC 15682) and lane 3 is Starpyrocoke pyogenes (Streptococcus. pyogenesATCC 14918) and lane 4 is Vibrio Bulnipicus (Vibrio vulnificusATCC 27562). 5 to 8 lanes Clinical sample of Clostridium difficile. As shown in FIG. 5, clostridial difficile could be detected from the clinical sample by double PCR using the primer pair of the present invention.

5. Dual PCR of Clinical Specimens and Clostridium difficile in selective media Detection of Clostridium difficile by PCR after incubation

As described above, double PCR was performed using the clinical specimen as a direct sample to detect Clostridium difficile. In addition, Clostridium difficile in clinical specimens was incubated with Cyserrine Cefoxitin Fructose Agar (CCFA) as a selective medium, followed by Clostridium difficile B toxin gene specific primer. PCR was performed. The PCR results are summarized in Table 11.

culture B toxin PCR after incubation Sample Duplex PCR Total Samples (37) Positive (20) Positive (19) Positive (19) 19 Voice (1) Voice (1) One Voice (17) Voice (17) Voice (17) 17

As shown in Table 11, the double PCR method using the clinical specimen as a sample, and the Clostridium difficile A or B toxin specific primer of the present invention was incubated with Clostridium difficile in the specimen, followed by Clostridium The PCR method using specific primers for the difficile B toxin gene showed that the results of detecting the toxin-producing Clostridium difficile were in agreement.

As described above, the use of the A toxin and B toxin gene-specific primers according to the present invention, it was found that toxin-producing Clostridium difficile can be identified by one PCR reaction.

Example 6: Sensitivity Test of Designed PCR Kits

Samples obtained by serially diluting the Clostridium difficile strain producing the toxin were used as templates, and double PCR using the primer of the present invention was performed to measure the sensitivity of the PCR kit including the primer of the present invention.

CDA7F and CDA13R and CDB38F and CDB40R are used as the primary primers, and CDA10F and CDA13R and CDB38F and CDB39R are used as the secondary primers. CDA7F and CDA13R and CDA10F and CDA13R are primers specific for the Clostridium difficile A toxin gene, and CDB38F and CDB40R and CDB38F and CDB39R are primers specific for the Clostridium difficile A toxin gene.

The composition and reaction conditions of the PCR reactants were the same as in Example 5. PCR results are shown in FIG. 7 is a photograph of the toxin produced Clostridium difficile diluted to 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ CFU / ml, and subjected to PCR using the template as a template, followed by electrophoresis. In Figure 7, lane M is 100bp molecular weight label, lane C is distilled water (negative control), lane P is Clostridium difficile (ATCC 9689) (positive control), lanes 1-5 are Clostridium difficile Clinical sample.

As shown in Fig. 7, amplification occurred at 10 ⁷ , 10 ⁶ , 10 ⁵ and 10 ⁴ CFU / ml of toxin producing Clostridium difficile, and no amplification occurred at a concentration of 10 ³ CFU / ml. Therefore, it can be seen that the PCR kit containing the primer of the present invention can detect up to 10 ⁴ CFU / ml of Clostridium difficile.

In addition to the PCR technique described by way of example in the present embodiment, any one of the primers of the present invention is included in the scope of the present invention. In addition, a method for detecting Clostridium difficile using the nucleic acid fragment of the present invention as a probe, for example, strand displacement amplification, nucleic acid sequence-based amplification, and rotation ring amplification ( Detection methods using amplification methods such as rolling circle amplification and invaders are included.

According to the nucleic acid fragment of the present invention, it can be used as a primer or a probe for detecting toxin-producing Clostridium difficile.

According to the primer or probe of the present invention, Clostridium difficile in a sample can be detected at high speed, high sensitivity, and high specificity.

According to the method for diagnosing Clostridium difficile of the present invention, Clostridium difficile can be easily diagnosed by using the sample as a direct sample.

Claims (13)

A nucleic acid fragment comprising 6 or more consecutive nucleotides or a combination thereof derived from any one of the nucleotide sequences of SEQ ID NOs: 1-142. The nucleic acid fragment of claim 1, wherein the nucleic acid fragment is DNA, RNA or PNA. The nucleic acid fragment of claim 2, wherein the RNA has a nucleotide sequence in which T is substituted with U in the nucleotide sequences of SEQ ID NOs: 1 to 142. The nucleic acid fragment according to any one of claims 1 to 3, which is a primer or a probe. The nucleic acid fragment of claim 4, wherein the primer or probe is attached with a labeling material selected from the group consisting of radioisotopes, fluorescent materials, enzymes, chemiluminescent materials, coenzymes, and hapten. The method of claim 4, wherein the combination is 6 or more consecutive nucleotides derived from any one of the nucleotide sequence of SEQ ID NO: 1 to 30 and 6 derived from any one of the base sequence of SEQ ID NO: 31 to 60 A nucleic acid fragment comprising Clostridium difficile A toxin gene specific primer comprising at least one contiguous nucleotide each and at least 50 bp in size. According to claim 4, wherein the combination is 6 or more consecutive nucleotides derived from any one of the base sequence of SEQ ID NO: 61 to 101 and 6 derived from any one of the base sequence of SEQ ID NO: 102 to 142 A nucleic acid fragment comprising Clostridium difficile B toxin gene specific primer comprising at least one contiguous nucleotide each and at least 50 bp in size from which the PCR product is obtained. The method according to claim 4, wherein the combination is 6 or more consecutive nucleotides derived from any one of the nucleotide sequences of SEQ ID NOs: 31 to 60 and 6 derived from any one of the nucleotide sequences of SEQ ID NOs: 61 to 101 A nucleic acid fragment comprising Clostridium difficyl toxin A and B gene specific primers, each comprising one or more consecutive nucleotides, each having at least 50 bp of PCR product obtained therefrom. A method for amplifying Clostridium difficile toxins A, B or A and B gene specific regions comprising PCR using the nucleic acid fragment of any one of claims 6 to 8 as a primer. PCR using a sample obtained from the sample using the nucleic acid fragment of any one of claims 6 to 8 as a primer; And A method of diagnosing the presence of Clostridium difficile in a sample comprising analyzing the base sequence of the amplified product by the PCR. A method for diagnosing the presence of Clostridium difficile in a sample comprising hybridizing a sample obtained from the sample with the probe of claim 4. A diagnostic kit comprising the nucleic acid fragment of claim 1. A pharmaceutical composition comprising the nucleic acid fragment of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.