KR20110119611A - Detection chip for performing virulence analysis of clostridium difficile, detection kit, and detection method using the same - Google Patents

Abstract

PURPOSE: A test chip and a kit for analyzing pathogenecity of Clostridium difficile are provided to accurately test risk of Clostridium difficile and quickly block Clostridium difficile infection. CONSTITUTION: A test chip for analyzing pathogenecity of Clostridium difficile contains a probe having oligonucleotides of sequence numbers 36-37 or complementary oligonucleotides thereof. A real-time PCR kit for early analyzing pathogenecity of Clostridium difficile contains a primer pair of sequence numbers 5 and 6 for amplifying toxin B gene, DNA polymerase, dNTPs, PCR buffer solution, and PCR product marker. The marker is labeled at one end of a probe with an oligonucleotide of sequence number 74 or complementary oligonucleotide thereof.

Description

Detection chip for performing virulence analysis of Clostridium difficile, detection kit, and detection method using the same}

The present invention relates to a test probe capable of simultaneously performing epidemiologic analysis and pathogenic analysis (toxicity analysis) for Clostridium difficile, and a Clostridium difficile test chip, test kit, and test method using the same, and in more detail The following relates to a test probe capable of simultaneously performing genotyping of a Clostridium difficile epidemiologic analysis gene and a toxin gene, and a test chip, a kit, and a test method using the same.

Specifically, the present invention rapidly blocks the spread of infection by simultaneously performing epidemiological analysis and pathogenic analysis on Clostridium difficile, which is a problem in hospital infection, with a microarray chip, and confirming the type of toxin of the infected person. It relates to providing an inspection probe capable of quickly and accurately inspecting the dangers of lithium difficile, and an inspection chip, a kit, and an inspection method using the same.

In addition, in addition to simultaneously performing epidemiological analysis and pathogenicity analysis on pathogens causing intestinal diseases such as food poisoning with a microarray chip, the present invention additionally uses a real-time PCR method to identify the type of toxin of the infected person and to determine the virulence load. load) to quantify the risk of pathogens early and accurately.

The bacteria of the genus Clostridia (Clostridia) are bacillus that form an absolute anaerobic spore, and there are four pathogens that are clinically important to humans.

Clostridium perfringens ( Clostridium perfringens) infects the skin, soft tissues, muscles, etc. It causes a variety of infections, from food poisoning to enteritis and, in severe cases, gas gangrene. C. tetani is a tetanus-causing bacterium that is widely distributed in soil and forms spores with large anaerobic gram-positive bacillus.

In addition, Clostridium botulinum ( C. botulinum) is an anaerobic Gram-positive bacillus that forms spores and has motility, causing bacterial food poisoning, infant botulinum, and wound botulinum. In addition, C. difficile is an absolute anaerobic Gram-positive bacterium, which produces spores with high resistance.

And, Clostridium difficile ( C. difficile ) is a bacterium that causes antimicrobial-induced diarrhea. In severe cases, it is a very dangerous bacterium caused by pseudomembranous colitis, and is one of the bacteria of the genus Clostridium that is clinically very important.

Among the bacteria of the genus Clostridium as described above, Clostridium difficile is reported as an intestinal bacterium that settles and exists in the intestinal tract of about 2-5% of normal adults [ Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 322-4, 2004], antibiotics, especially antibiotics that exhibit a wide range of effects, can cause many problems due to excessive proliferation of antibiotic-resistant Clostridium difficile due to the disruption of normal bacteria in the intestine.

Certain strains of these Clostridium difficile can produce toxins, causing severe diarrhea, toxic giant colon, perforation, sepsis, and pseudomembranous colitis. The toxins produced by the pathogenic Clostridium difficile are known as intestinal toxin [toxin A], cytotoxin [toxin B], and binary toxin, and have diarrhea and inflammatory reactions. Can cause. In particular, toxin A causes secretory and hemorrhagic diarrhea, and toxin B is a cytotoxin and exhibits a destructive cytopathic effect in tissue cultured cells.

The pathogenic Clostridium difficile was first reported to cause antibiotic-related diarrhea in 1978, followed by the outbreak of highly pathogenic Clostridium difficile in Montreal and Calgary, Canada on June 4, 2003. So far, about 1,400 cases of infection have been reported. In March 2005, an additional highly pathogenic Clostridium difficile was reported in Toronto, and similar cases were reported in 2003 and 2005 at Stoke Mandeville Hospital in the UK.

This highly pathogenic strain was named as NAP1/027 or BI/NAP1/027, aka Quebec strain. These highly pathogenic strains are known to produce higher levels of toxins than existing pathogenic strains, are resistant to fluoroquinolone, and produce binary toxins. In addition, the NAP1/027 strain is known to be associated with the production of more toxin A and toxin B because a portion of the toxin C gene, known as a negative regulator for toxin A and toxin B, is deleted ( INT J ANTIMICROB AG 33, S1, 2009, S13-S18).

Various antibiotics have been shown to be associated with Clostridium difficile-related disease (CDAD). It has been found that the intestinal acquisition of Clostridium difficile occurs in about 10-25% of hospitalized patients, and the acquisition of Clostridium difficile increases with the length of hospital stay ( Surgery 116:768-775, 1994, J. Infect Dis 166:561-567, 1992). The pathogenic Clostridium difficile can survive for a long time in vitro by forming spores after Clostridium difficile is excreted from the diarrhea of an infected patient. Due to these physiological characteristics, Clostridium difficile may be contaminated with the environment for a long period of time, and clostridial difficile may occur in hospitals as spores from these contaminants cause additional infections through the oral cavity of a normal person. Therefore, early identification of the pathogenicity of Clostridium difficile and the transmission route of the infectious agent is required to prevent the outbreak of the in-hospital outbreak caused by such Clostridium difficile.

The pathogenicity (toxicity) of Clostridium difficile can be determined by the toxin of the strain isolated from CDAD patients, and the determination of such toxins is performed by cytotoxicity test and enzymatic immunization ( Korean J Lab Med 2009; 29: 122- It is possible with 6). However, such a test method requires a long time to confirm the result, and particularly, in the case of cytotoxicity test, a continuous culture process and a high level of difficulty technology are required. In order to compensate for these shortcomings, a molecular genetic method has recently been developed and applied to quickly and accurately perform a toxin test.

The molecular genetic method has the advantage of being able to amplify the toxin A gene and the toxin B gene, which are genes encoding toxins, to determine the existence or not, and to determine the pathogenic Clostridium difficile within a short time. .

In this regard, Korean Patent Application Publication No. 10-2004-0032588 (published on Apr. 17, 2004) describes a method for detecting Clostridium difficile using an oligonucleotide primer specific to Clostridium difficile. After designing primers specific for the Clostridium difficile toxin A gene and the primers for the Clostridium difficile toxin B gene, a method for detecting Clostridium difficile using the PCR method using these was disclosed. have. In addition, Korean Patent Registration No. 10-0920971 (announced on Oct. 09, 2009) designed primers for detecting 10 types of food-hazardous microorganisms including Clostridium perfringens, and conventional PCR or real Disclosed is a method for detecting 10 kinds of food hazard microorganisms using the time PCR method.

However, in the method of detecting Clostridium difficile using such a PCR method, careful attention and time are required when synthesizing a primer specific for a toxin gene, and there is a problem that a false-positive product is generated by non-specific hybridization of the primer. Since Clostridium difficile strains are detected using primers for known toxin genes (eg, toxin A, toxin B, binary toxin, etc.), there is a high possibility of test errors when new strains appear. In addition, for the epidemiologic analysis to quickly block the spread of infection, there is an aspect that a PCR detection method using a primer for a toxin gene is not appropriate.

In other words, recently, a Clostridium difficile strain in which a portion of the toxin A gene was deleted was discovered, and an error occurred in the method of detecting Clostridium difficile pathogenicity according to the determination of the presence or absence of the toxin gene by the conventional PCR method. It has been confirmed that it may be possible, and this problem has raised the inconvenience of additional confirmation of the partially deleted toxin gene ( Korean J Lab Med 2006; 26: 27-31). This suggests that the PCR detection method through toxin gene identification is an ineffective method to prevent the widespread spread of Clostridium difficile in hospitals.

On the other hand, the epidemiological investigation of Clostridium difficile includes phenotypic analysis methods such as serotyping and immunoblotting, PCR-ribotyping, Restriction Endonuclease Analysis (REA), and AP- Various genotyping methods such as arbitrarily primed PCR (PCR), pulse-field gel electrophoresis (PFGE), and toxinotyping are widely used.

However, in the investigation of the epidemiology of Clostridium difficile, the phenotypic analysis method has a problem in that the experimental process is complicated and the resolution is low. In addition, AP-PCR and PFGE have high resolution but are not suitable for long-term genotyping because of problems in reproducibility. In addition, PCR-ribotyping is judged to be more appropriate than AP-PCR and PFGE because of its simple experimental process and high resolution (Infection Vol. 34, No. 3, 2002 Vol. 34, No. 3, 167). -175), because standardization is difficult and comparison with results of other laboratories is difficult, so that the development of a new dynamic analysis method that has good resolution and reproducibility and can be standardized is urgently required in the technical field to which the present invention belongs.

Genotyping for identification of pathogenicity and epidemiologic investigation of Clostridium difficile is essential in the management of pathogenic Clostridium difficile infection. The development of new and diverse molecular genetic analysis methods made it possible to quickly and accurately investigate the pathogenicity of Clostridium difficile and the epidemiological investigation through genotyping, but the Clostridium difficile pathogenicity analysis and epidemiologic analysis developed to date have been developed using various methods, respectively. The provision of a new technology that enables pathogenic analysis and epidemiological analysis of Clostridium difficile through a single unified analysis method is provided in the technical field to which the present invention pertains. It is in desperate need.

In addition, the provision of a new base technology capable of early and objectively discriminating the risk of pathogens by quantifying the toxic load while simultaneously performing efficient toxicity analysis and epidemiological analysis on pathogens that cause intestinal diseases such as food poisoning are simultaneously performed. need.

Republic of Korea Patent Publication No. 10-2004-0032588 (2004. 04. 17) Republic of Korea Patent Publication No. 10-0920971 (2009. 10. 09)

The present invention relates to a test probe capable of simultaneously performing epidemiologic analysis and pathogenic analysis (toxicity analysis) for Clostridium difficile, and a Clostridium difficile test chip, test kit, and test method using the same, and in more detail It is an object of the present invention to provide a test probe capable of simultaneously performing genotyping of a Clostridium difficile epidemiologic analysis gene and a toxin gene, and a test chip, a kit, and a test method using the same.

Specifically, the present invention rapidly blocks the spread of infection by simultaneously performing epidemiological analysis and pathogenic analysis on Clostridium difficile, which is a problem in hospital infection, with a microarray chip, and confirming the type of toxin of the infected person. An object of the present invention is to provide an inspection probe capable of quickly and accurately inspecting the risk of lithium difficile, and an inspection chip, a kit, and an inspection method using the same.

In addition, in addition to simultaneously performing epidemiological analysis and pathogenicity analysis on pathogens causing intestinal diseases such as food poisoning with a microarray chip, the present invention additionally uses a real-time PCR method to identify the type of toxin of the infected person and to determine the virulence load. load) to quantify the risk of pathogens early and accurately.

As a result of resolving the technical problems of the above-described invention and repeating intensive research to meet the object of the above-described invention, the inventors of the present invention provide simplicity and high resolution of the experiment, while confirming the exact pathogenicity and conducting epidemiological investigations at the same time, Microarray method for toxin genes and genes for epidemiological analysis was applied to confirm pathogenicity and epidemiological investigation of pathogens that cause intestinal diseases such as food poisoning, and selectively conduct real-time PCR method to induce intestinal diseases such as food poisoning. A test method that can simultaneously perform pathogenic analysis and epidemiological analysis of the pathogens that are being tested has been completed.

Such a test method of the present invention is not a technology applicable only to Clostridium difficile, which is described as an example in the Examples of the present invention, but has a toxic gene causing intestinal diseases such as food poisoning and a target gene for epidemiologic analysis. It should be understood as a basic technology that can be applied to the identification and epidemiological investigation of pathogens that cause various intestinal diseases.

Meanwhile, the term virulence gene used in the specification of the present invention refers to a gene encoding a toxin of a pathogen, and in the case of Clostridium difficile, it may be a toxin A gene, a toxin B gene, or a binary toxin gene. However, it goes without saying that the base technology of the test method of the present invention is not limited to such toxin genes, but is applicable to toxin genes of various pathogens.

In addition, the term gene for epidemiologic analysis used in the specification of the present invention means a marker gene suitable for species identification and strain identification of a pathogen, for example, a single gene between different species. It is preferable that variations are present in the microarray chip analysis. For example, in the case of Clostridium difficile, it may be a slpA gene or a cwp66 gene. However, it goes without saying that the base technology of the test method of the present invention is not limited to such marker genes, and is applicable to marker genes of various pathogens.

In one embodiment of the present invention, the test probe for the epidemiological analysis and pathogenic analysis of Clostridium difficile specifically binds to the toxin gene of Clostridium difficile, and the base sequence of SEQ ID NO: 35 to SEQ ID NO: 47 At least one first probe group selected from the group consisting of oligonucleotides having a base sequence and oligonucleotides having a base sequence complementary to these oligonucleotides, and a gene for epidemiologic analysis for Clostridium difficile It includes at least one second probe group selected from the group consisting of oligonucleotides that bind specifically and have a nucleotide sequence of SEQ ID NO: 49 to SEQ ID NO: 71, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides. .

In the test probe of one embodiment of the present invention, the first probe group is specifically with at least one toxin gene selected from the group consisting of toxin A gene, toxin B gene, toxin C partial deletion gene, and binary toxin gene. It is characterized by combining.

Specifically, the probe of an oligonucleotide having SEQ ID NO: 35 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide includes toxin A gene, oligonucleotides having SEQ ID NOs: 36 to 37, and complementary oligonucleotides to these oligonucleotides. Probes of oligonucleotides having a typical nucleotide sequence include toxin B gene, oligonucleotides having SEQ ID NOs: 38 to 45, and oligonucleotides having a base sequence complementary to these oligonucleotides. , Oligonucleotides having SEQ ID NOs: 46 to 47 and probes of oligonucleotides having a base sequence complementary to these oligonucleotides are characterized in that they specifically bind to a binary toxin gene.

In the test probe of an embodiment of the present invention, the second probe group is characterized in that it specifically binds to at least one gene for epidemiologic analysis selected from the group consisting of slpA gene and cwp66 gene. Specifically, the probes of oligonucleotides having SEQ ID NO: 49 to SEQ ID NO: 65 and oligonucleotides having a base sequence complementary to these oligonucleotides include the slpA gene, oligonucleotides having SEQ ID NO: 66 to SEQ ID NO: 71, and The probe of oligonucleotides having a base sequence complementary to the oligonucleotides is characterized by specifically binding to the cwp66 gene.

The test chip for epidemiologic analysis and pathogenicity analysis of Clostridium difficile according to an embodiment of the present invention specifically binds to a toxin gene of Clostridium difficile, and the base sequence of SEQ ID NO: 35 to SEQ ID NO: 47 At least one first probe group selected from the group consisting of oligonucleotides having a sequence and oligonucleotides having a base sequence complementary to these oligonucleotides, and specific for a gene for epidemiologic analysis for Clostridium difficile And at least one second probe group selected from the group consisting of oligonucleotides having a base sequence of SEQ ID NO: 49 to SEQ ID NO: 71 and oligonucleotides having a base sequence complementary to these oligonucleotides. Preferably, the first probe group and the second probe group are microarrayed on the same substrate. It is preferable that the substrate is a glass substrate or a plastic substrate generally used for DNA chip fabrication.

The gene PCR amplification kit for epidemiological analysis and pathogenic analysis for Clostridium difficile according to an embodiment of the present invention is a first primer group for amplifying one or more toxin genes selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 14 , SEQ ID NO: 17 to SEQ ID NO: 32 comprises a second primer group for amplification of one or more kinematic analysis genes selected from the group consisting of, DNA polymerase, dNTPs, PCR buffer, and a labeling material of the PCR amplification product. Preferably, the primer may be provided as a combination of at least one forward primer and a reverse primer from the first primer group, and may be provided as a combination of at least one forward primer and a reverse primer from the second primer group. As an example, the labeling material of the PCR amplification product may be directly bound to the 5'end of the primer.

The test kit for epidemiologic analysis and pathogenicity analysis for Clostridium difficile according to an embodiment of the present invention amplifies a test chip defined as described above and a gene for toxin gene and epidemiological analysis of Clostridium difficile. And a labeling means for detecting the amplified gene hybridized with the test chip.

In the test kit of an embodiment of the present invention, the primer is a first primer group for amplifying one or more toxin genes selected from the group consisting of SEQ ID NOs: 3 to 14, and SEQ ID NOs: 17 to 32. It includes a second primer group for amplifying genes for at least one epidemiologic analysis selected from the group. Preferably, the primer may be provided as a combination of at least one forward primer and a reverse primer from the first primer group, and may be provided as a combination of at least one forward primer and a reverse primer from the second primer group. As an example, the labeling means may be directly bonded to the 5'end of the primer.

Alternatively, the labeling means is directly bound to the 5'end of the primer and labeled on the amplified gene, and is not complementary to the probes of the test chip and is not complementary to the probes of the test chip, and is specific for each of the amplified genes. It may be at least one of those labeled on a detection helper nucleotide sequence having a nucleotide sequence complementary to the sequence.

Optionally, the labeling means may be directly labeled on each detection helper nucleotide sequence for each of the amplified genes, or a non-homologous nucleotide sequence having no homology for each of the amplified genes is used as the detection helper nucleotide sequence. After tagging, the labeling means may be indirectly labeled by preparing a nucleotide sequence having a sequence complementary to the non-homologous nucleotide sequence and to which the labeling means is bound and hybridizing to the tagged non-homologous nucleotide sequence.

In another embodiment of the present invention, a test method for early analysis of the pathogenicity of Clostridium difficile,

Preparing a gene sample from the subject, and

Using the prepared gene sample as a template, a primer pair of SEQ ID NOs: 3 and 4 for Clostridium difficile toxin A gene or a primer pair of SEQ ID NOs: 5 and 6 for Clostridium difficile toxin B gene, and amplification Amplifying the toxin A gene or the toxin B gene in the test subject's gene sample by real-time PCR using a labeling material indicating the amount of gene amplification in relation to the toxin A gene or the toxin B gene.

After amplification of the toxin A gene or the toxin B gene, calculating a toxic load for Clostridium difficile toxin A or toxin B based on the amplification amount of the toxin A gene or the toxin B gene calculated using the labeling material, and

And detecting the pathogenicity of Clostridium difficile based on the calculated toxic loading.

In the test method for early analysis of the pathogenicity of Clostridium difficile according to another embodiment of the present invention, the labeling material is a fluorescent material, and the toxin A gene or toxin is measured by measuring the intensity of fluorescence from the labeling material. It is characterized in that the amount of amplification of the B gene is calculated.

In another embodiment of the present invention, in the test method for early analysis of the pathogenicity of Clostridium difficile,

Before amplification, a positive standard, which is a gene sample of which the copy number of Clostridium difficile toxin A gene or toxin B gene is known, is applied to the primer pairs of SEQ ID NOs: 3 and 4 for the toxin A gene or Clostridium difficile toxin B gene. Amplifying by real-time PCR using a primer pair of SEQ ID NOs: 5 and 6 and a label indicating the amount of gene amplification in relation to the toxin A gene or toxin B gene to be amplified;

Measuring the change in fluorescence intensity of the labeling material with respect to the entire PCR amplification period of the positive standard, and analyzing the number of PCR reactions (Cp value or Ct value) reaching a constant fluorescence intensity; and

Correlating the copy number and Cp value or Ct value of the positive standard character from a standard curve obtained by measuring the change in fluorescence intensity of the labeling material,

The change in the fluorescence intensity of the labeling material for the entire PCR amplification period of the toxin A gene or the toxin B gene in the gene sample of the subject is measured, and the number of PCR reactions (Cp value or Ct value) reaching a certain fluorescence intensity is analyzed. And the steps to do it,

Clostridium difficile toxin A or toxin B in the gene sample of the subject by matching the Cp value or Ct value obtained by PCR amplification of the toxin A gene or the toxin B gene in the test subject's gene sample to the copy number of the positive standard It further comprises the step of quantitatively measuring the toxic load on the.

In the present invention, the labeling material is a probe that specifically binds to Clostridium difficile toxin A gene and is composed of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 35 or an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide. An oligonucleotide having the nucleotide sequence of SEQ ID NO: 74 as a probe that may be labeled at one end of the probe, for example, at the 5'end, specifically binds to the Clostridium difficile toxin B gene, or complementary to such an oligonucleotide. It may be labeled at one end, for example, at the 5'end of a probe composed of an oligonucleotide having a base sequence.

For reference, in the present invention, the combination of the forward primer and reverse primer of SEQ ID NO: 3 to SEQ ID NO: 4 is used to amplify the toxin A gene nucleic acid, and at least one forward primer and reverse primer selected from SEQ ID NO: 5 to SEQ ID NO: 8 The combination is For the amplification of the toxin B gene nucleic acid, the combination of the forward primer and the reverse primer of SEQ ID NO: 9 to SEQ ID NO: 10 is for amplification of the toxin C deletion portion gene nucleic acid, at least one forward primer and reverse primer selected from SEQ ID NO: 11 to SEQ ID NO: 14 The combination of the two-component toxin gene nucleic acid amplification, the combination of at least one forward primer and reverse primer selected from SEQ ID NO: 17 to SEQ ID NO: 25 is to amplify the slpA gene nucleic acid, at least one selected from SEQ ID NO: 26 to SEQ ID NO: 32 The combination of the forward primer and the reverse primer of is characterized in that it is used for amplification of the cwp66 gene nucleic acid.

Meanwhile, in the present invention, the labeling material is Cy5, Cy3, biotin-binding material, EDANS (5-(2'-aminoethyl) amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), tetramethylrhodamine It may be an isocyanate (TMRITC), x-rhodamine, or Texas red, and it is preferable that it is a fluorescent substance such as Cy3 or Cy5. In addition, the labeling material may be SYBR green I, VIC, or FAM, but the present invention is not limited thereto, and various labeling materials such as radioactive material or luminescent material known in the art may be used.

According to the present invention, it is possible to simultaneously proceed with the epidemiologic analysis and pathogenicity analysis of Clostridium difficile through genotyping of the Clostridium difficile epidemiologic analysis gene and the toxin gene, thereby quickly blocking the spread of infection and By identifying the type, you can quickly and accurately test the dangers of Clostridium difficile.

That is, the present invention can simultaneously analyze and manage the degree of toxicity and transmission of Clostridium difficile through rapid and accurate toxicity analysis for Clostridium difficile and epidemiological analysis necessary to prevent the outbreak of a population within a specific area. In addition, intestinal diseases such as diarrhea can be quickly and easily tested with high specificity and sensitivity.

In addition, in addition to simultaneously performing epidemiologic analysis and pathogenicity analysis for Clostridium difficile with a microarray chip, the present invention additionally uses a real-time PCR method to identify the type of toxin of an infected person and quantify the virulence load. Thus, there is an advantage in that the danger of Clostridium difficile can be accurately read at an early stage.

In particular, if the base technology of the test method of the present invention is used, a toxin gene encoding a toxin as well as Clostridium difficile and a gene for epidemiologic analysis suitable for the determination of the pathogen endogenous (e.g., single between different species) Efficient toxicity analysis and epidemiologic analysis for various pathogens (e.g., pathogens causing intestinal diseases such as food poisoning) with mutations in genes, which are suitable for microarray chip analysis, while quickly quantifying their toxic load Thus, the risk of pathogens can be determined objectively at an early stage.

1 is a diagram showing sequence information of Clostridium difficile toxin A gene clone.
2 is a diagram showing sequence information of Clostridium difficile toxin B gene clones.
3 is a diagram showing the results of homology analysis of Clostridium difficile toxin C gene clones.
4 is a diagram showing the results of homology analysis of Clostridium difficile SlpA gene clones.
5 is a diagram showing the results of homology analysis of Clostridium tpi (Triosephosphate isomerase) gene clones.
6 is a photograph of the result of electrophoresis of the PCR amplification product of the Cwp66 gene, which is a colonization factor of Clostridium difficile.
7 is a diagram showing the results of homology analysis of Clostridium difficile cwp66 gene clone.
8 is a graph showing the analysis results of Clostridium difficile toxin A gene and toxin B gene using real-time PCR in the method for testing Clostridium difficile according to an embodiment of the present invention.
9 is a photograph of an electrophoresis result of a PCR amplification product of a Clostridium difficile toxin C gene in the test method for Clostridium difficile according to an embodiment of the present invention, showing a sample having a partial deletion of the toxin C gene. This is a picture that confirms.
10A to 10F are a microarray of probes for a tpi gene, a toxin A gene, a toxin B gene, and a binary toxin gene in the test method for Clostridium difficile according to an embodiment of the present invention. It is a graph showing the experimental result of the analysis by scanning fluorescence after hybridization reaction of the gene amplification product of the specimen to the DNA chip of the example.
11 is a test method for Clostridium difficile according to an embodiment of the present invention, in which a gene amplification product of a specimen is used for partial deletion of the tpi gene, the toxin A gene, the toxin B gene, the binary toxin gene, and the toxin C gene. This is a fluorescence image of the DNA chip confirmed after hybridization to the DNA chip of the embodiment of the present invention in which the probe is microarrayed.
12A to 12C are hybridization of a gene amplification product of a specimen to a DNA chip of an embodiment of the present invention in which a probe for the slpA gene is microarrayed in the method for testing Clostridium difficile according to an embodiment of the present invention. It is a graph showing the experimental results analyzed by scanning fluorescence after reacting.
13A to 13B are hybridization of a gene amplification product of a specimen to a DNA chip of an embodiment of the present invention in which a probe for the Cwp66 gene is microarrayed in a method for testing Clostridium difficile according to an embodiment of the present invention. It is a graph showing the experimental results analyzed by scanning fluorescence after reacting.
14 is a method for testing a gene for toxin gene and epidemiologic analysis for Clostridium difficile according to an embodiment of the present invention, wherein the gene amplification product of a specimen is a tpi gene, a toxin A gene, a toxin B gene, a two-component toxin gene, This is a fluorescence image of a DNA chip confirmed after hybridization to a DNA chip of an embodiment of the present invention in which probes for the partial deletion of the toxin C gene, the slpA gene, and the cwp66 gene are microarrayed.
FIG. 15 is a result of analysis of the detection limit concentration and detection limit copy number of the Clostridium difficile toxin A gene and the toxin B gene using real-time PCR in the method for testing Clostridium difficile according to an embodiment of the present invention. It is a graph showing.

Specifically, in one embodiment of the present invention, the presence and deletion of toxin A, toxin B, and binary toxin genes, and toxin C, known as a negative regulator of toxin A and toxin B genes, are specific in the gene of the present invention. A specific probe capable of analyzing sequence mutations and deletions was designed, and at the same time, the cwp66 gene associated with colonization of Clostridium difficile and the surface protein SlpA (S-layer protein A) gene were used for dynamic analysis through genotyping. Through mutation analysis, specific sequences for each genotype were designed as probes (see Table 2).

In addition, the two groups of probes designed as described above were bound to one area on the chip and integrated on the fixed surface, and the toxin gene and cwp66 and SlpA genes derived from the isolated strain were obtained using the primers in Table 1 below. After amplification, the amplification products were simultaneously hybridized with probes integrated on the chip.

By analyzing the hybridization pattern with a specific probe by this method and determining the pathogenicity and genotype of Clostridium difficile, it was possible to confirm the pathogenicity and genotype with a single application of the chip, and the analysis of highly pathogenic strains and the pattern of toxin expression. It was possible to analyze the serotype of the pathogenic Clostridium difficile using the analysis of and the association with the serotype of the surface protein SlpA (S-layer protein A) gene. Therefore, the present invention can significantly improve the shortcomings of the existing analysis method by enabling the epidemiologic investigation and the presence of pathogenic Clostridium difficile through this process.

Table 1. Clostridium difficile toxin gene and primer sequences for amplification of slpA and cwp66 genes

Table 2. Probe sequences for genotyping of Clostridium difficile toxin genes and slpA and cwp66 genes

Hereinafter, the present invention will be described in more detail based on the following examples. It goes without saying that the following examples of the present invention are intended to embodi the present invention and do not limit or limit the scope of the present invention. What can be easily inferred by experts in the technical field to which the present invention pertains from the detailed description and examples of the present invention is interpreted as belonging to the scope of the present invention. The references cited in the present invention are incorporated herein by reference.

Example 1: Acquisition of clones of toxin A and toxin B genes encoding enterotoxins of Clostridium difficile and confirmation of sequence information

NK9(5'-CCACCAGCTGCAGCCATA-3') primer and NK11(5'-TGATGCTAATAATGAATCAAAATGGTAAC-3) with reference to a previous study (Korean J Lab Med 2006; 26: 27-31) for Toxin A gene analysis. ') was amplified using primers. Toxin A gene was amplified using the positive standard strain Clostridium difficile genomic DNA (ATCC9689D-5) as a template, and the negative standard strain Clostridium perfringens as a negative control. (ATCC 13124D-5) was amplified as a template.

The presence or absence of the toxin A gene was determined by the presence or absence of a 1200bp amplification product, and the amplification product was purified using a GeneAll ^R Expin ^TM Gel SV (GeneAll, KOREA) gel extraction kit, and then pGEM-T easy vector (Promega). , USA) and transformed into DH5-alpha (RBC, Taiwan) E. coli. The prepared clone was incubated for 16 hours in LB medium, and the plasmid was isolated using the GeneAll plasmid prep kit, and then sequence analysis was requested (Solgent, KOREA).As a result, the sequence information of the gene obtained through the cloning process was blasted. It was confirmed that it was the toxin A gene through a blast search (FIG. 1).

The toxin B gene analysis process was performed in the same manner as the toxin A gene analysis process, and as primers for amplification, CDTB1 (5'-GGATCATTCTGGTGAATGGATAA-3') primer and CDTB2 (5'-GCTCTTTGATTGCTGCACCT-3') primer were used. I did. The presence or absence of the toxin B gene was determined by the presence or absence of a 878 bp product, and sequence analysis was performed in the same manner as the toxin A gene sequencing process (FIG. 2).

Example 2: Confirmation of sequence information of toxin C gene and securing of clones

In order to confirm the nucleotide sequence variation and partial deletion of the Toxin C gene, the previous study results ( INT J ANTIMICROB AG 33, S1, 2009, S13-S18) were used as a template using genomic DNA extracted from a clinically isolated strain. For reference, the toxin C gene was amplified with a C1 (5'-TTA ATT AAT TTT CTC TAC AGC TAT CC -3') primer and a C2 (5'-TCT AAT AAA AGG GAG ATT GTA TTA T -3') primer.

As for the amplified product, a clone was obtained through the same experimental method as in Example 1, and the gene sequence was confirmed through sequencing to obtain a toxin C gene clone (FIG. 3).

Example 3: Confirmation of slpA gene sequence information and securing of clones

To confirm the nucleotide sequence mutation of the SlpA (Surface layer protein A) gene, the genomic DNA extracted from the clinically isolated strain was used as a template, slpAcom19 (5'-GTT GGG AGG AAT TTA AGR AAT G -3') primer and slpAcom22 (5' -GCW GTY TCT ATT CTA TCD TYW -3') After amplifying the slpA gene with a primer (INT J ANTIMICROB AG 33, S1, 2009, S13-S18), the amplified product was cloned through the same experimental method as in Example 1. To secure the slpA gene clone was obtained by confirming the gene sequence through sequencing analysis (Fig. 4).

Example 4: Clostridium tpi (Triosephophate isomerase) gene amplification and sequence information confirmation for securing a positive control

In order to secure positive clones to secure primers and probes containing the Clostridium difficile-specific Clostridium tpi (Triosephophate isomerase) gene sequence for selective confirmation of Clostridium difficile, the previous study was conducted. Based on the primers selected, the Clostridium tpi (Triosephophate isomerase) gene was amplified ( J CLIN MICROBIOL, Dec. 2004, p.5710-5714). For the gene sequence information, the conservative sequence confirmed as a result of obtaining the tpi (triosephophate isomerase) gene sequence of Clostridium difficile from NCBI's nucleotide database, and analyzing homology using the MEGA 4.1 program, is used as a primer sequence (sequence). No. 1 and SEQ ID No. 2) and probe sequences (SEQ ID No. 33 and SEQ ID No. 34) were selected (Fig. 5).

Example 5: Acquisition of cwp66 gene clone, a colonization factor of Clostridium difficile, confirmation of sequence information, selection of specific probes and primers

In order to amplify the cwp66 gene, one of the colonization factors of Clostridium difficile, primers (SEQ ID NO: 72 and SEQ ID NO: 73) were used based on sequence information (accession no. NC_009089) from the nucleotide database. After selection, the amplification product was cloned by performing a PCR process using the clinically isolated strain as a template (see FIG. 6: PCR amplification products confirmed in lanes 2, 3, 4, 6, 9, 12, 13, 14, 15, 17) .

Cwp66F (SEQ ID NO: 72): 5'-TAGGCTACTTTCTGGTGTCGTTGAA-3'

Cwp66R (SEQ ID NO: 73): 5'-TGGAATCCTGAAAGTGGACATTATGC-3'

The cloning process was carried out in the same manner as in Example 1, and after sequencing was performed (see Fig. 7) to confirm specific nucleotide mutations, primers were selected to selectively amplify each of them (SEQ ID NOs: 26 to 32). . After additionally selecting specific nucleotide sequences in the amplified product with the selected primers, they were synthesized with probes (SEQ ID NOs: 66 to 71) to obtain microarray probe contents.

Example 6: Preparation of primers and probes for identification of toxin A gene using real-time PCR

A primer sequence capable of identifying the toxin A gene was selected based on the sequence information of FIG. 1 (SEQ ID NO: 3 and SEQ ID NO: 4). Primer selection was made by analyzing and selecting an appropriate Tm (melting temperature: the temperature at which 50% of the DNA is separated into a single strand) using OligoAnalyzer 3.1 (http://eu.idtdna.com/).

As a PCR amplification composition, commercially available HS Prime Taq Premix (GeNet Bio, KOREA) was used, and dUTP was added to the amplification composition instead of dTTP to use the UNG enzyme. In addition, 0.01 unit of HK™-UNG thermolabile Uracil N-Glycosylase (EPICENTRE, UK) was added to prevent cross-contamination, and the amplification process was 50 After the reaction was carried out at °C for 2 minutes and at 94°C for 10 minutes, the reaction cycle of 94°C for 15 seconds and 60°C for 1 minute was repeated 40 times.

The presence or absence of amplification was confirmed by the presence or absence of a 116 bp amplification product, and a probe specific for the toxin A gene (SEQ ID NO: 35) was used for more accurate identification. To measure the fluorescence value, label VIC in the 5'direction of the probe of SEQ ID NO: 35, and MGB and NFQ in the 3'direction, and then use the SLAN Real Time PCR system (LG Life Science, Korea). Then, the amplification product was confirmed by the fluorescence value. To read the experimental results, the fluorescence value emitted at a wavelength of 530 nm was measured to determine whether the toxin A gene was amplified or not.

In addition, in order to verify the inhibitory factor of the RCR reaction, internal control gene primers (SEQ ID NO: 15 and SEQ ID NO: 16) were added together and simultaneously proceeded in one tube. The reading of the amplification of the internal control gene was determined by measuring the fluorescence value emitted at a wavelength of 630 nm, and the internal control gene was CPN60A (NC_003071), and a probe for the internal control gene (SEQ ID NO: 48). Was labeled with cy5 in the 5'direction and BHQ2 in the 3'direction (FIG. 8).

Example 7: Preparation of primers and probes for identification of toxin B gene using real-time PCR

The primer sequence for identifying the toxin B gene was selected using OligoAnalyzer 3.1 (http://eu.idtdna.com/) in substantially the same manner as the selection process of the primer sequence for the toxin A gene (SEQ ID NO: 5 and sequence. Number 6).

As a PCR amplification composition, commercially available HS Prime Taq Premix (GeNet Bio, KOREA) was used, and dUTP was added to the amplification composition instead of dTTP to use the UNG enzyme. In addition, 0.01 unit of HK™-UNG thermolabile Uracil N-Glycosylase (EPICENTRE, UK) was added to prevent cross-contamination, and the amplification process was 50 After the reaction was carried out at °C for 2 minutes and at 94°C for 10 minutes, the reaction cycle of 94°C for 15 seconds and 60°C for 1 minute was repeated 40 times.

The presence or absence of amplification was confirmed by the presence or absence of a 107bp amplification product.After designing a probe specific to the toxin B gene (SEQ ID NO: 74) for more accurate identification, FAM was labeled at the 5'end and MGB, NFQ at the 3'end. Was labeled.

tcdBP3 (SEQ ID NO: 74): FAM-5'-ATGCAGCCAAAGTTG-3'-MGB-NFQ

The amplified product was confirmed by fluorescence value using the SLAN Real Time PCR system (LG Life Science, Korea) with the probe labeled as described above. To read the experimental results, the fluorescence value emitted at a wavelength of 470 nm was measured to determine the amplification of the toxin B gene.

In addition, in order to verify the inhibitory factor of the RCR reaction, the internal control gene primers used in Example 6 were added together and simultaneously proceeded in one tube. The reading of the amplification of the internal control gene was determined by measuring the fluorescence value emitted at a wavelength of 630 nm (FIG. 8).

Example 8: Quantification of toxic loads of toxin A gene and toxin B gene using real-time PCR

Real-time PCR method is an application of a fluorescent material to the PCR technique. During the reaction, the degree of emission of the fluorescent material is detected and quantitatively analyzed in real time along with amplification of the target gene present in the sample. This is a method that can quickly and accurately analyze the amplification of the target gene and its pattern. Real-time PCR methods are largely divided into a method using a TaqMan probe and a method using a SYBR Green I (SYBR Green I) method. In the above embodiments of the present invention, real-time PCR was performed using the TaqMan probe, but the present invention is not limited thereto.

The basic principle of real-time PCR is the detection and quantification of fluorescence performed in real time at every cycle of PCR by the principle of polymerase and fluorescence resonance energy transfer (FRET). Quantification in real-time PCR is a method of measuring the amplification product in real time within the exponential phase range by monitoring the progress of each cycle during PCR, and the important concept at this time is Ct (threshold cycle) or Cp (crossing). point). This value is the number of cycles at which the amount of fluorescence generated by separation of the newsletter exceeds the baseline level and increases significantly so as to be detectable, and this is the initial template amount (in the case of the present invention, the number of copies of the toxic gene) Is reflected relatively accurately.

That is, if real-time PCR is used, PCR amplifies a positive standard whose number of copies of a toxic gene is known before amplification, measures the change in fluorescence intensity of the labeling material for the entire PCR amplification period of the positive standard, and reaches a constant fluorescence intensity. After analyzing the number of reactions (Cp value or Ct value), the number of copies of the positive standard can be correlated with the Cp value or Ct value from the standard curve obtained by measuring the change in fluorescence intensity. And, using the correspondence between the copy number and Cp value or Ct value of the positive standard person (toxic gene whose copy number is known) thus obtained, the fluorescence intensity of the labeling material with respect to the entire PCR amplification period of the toxic gene of the subject The Cp value or Ct value obtained by measuring the change is correlated with the number of copies of the positive standard, so that the toxic load of the subject can be measured.

Therefore, in this example, the prepared Clostridium difficile genomic DNA was gradually diluted to confirm the detection limits of the toxin A gene and the toxin B gene, and used as a DNA template (positive standard character) for real-time PCR. When genomic DNA was applied, detection of both toxin A gene and toxin B gene ^{was possible up to 5×10 -7} ng, and 100 copies of toxin A gene were detected using the prepared clone as a template to confirm the exact number of detected copies. (copy), in the case of the toxin B gene, it was confirmed that up to 50 copies can be detected (FIG. 15).

Example 9: Confirmation of partial deletion of toxin C gene by PCR

In order to confirm the partial deletion of the Toxin C gene, the primers of SEQ ID NO: 9 and SEQ ID NO: 10 were used to react at 94°C for 10 minutes, followed by 15 seconds at 94°C, 30 seconds at 55°C, and 30 at 72°C. The reaction cycle of the second was repeated 35 times. The amplification product was subjected to electrophoresis on a 3% agarose gel for 30 minutes, and then a partial deletion of 39 bp was confirmed (see FIG. 9: partial deletion of the toxin C gene in lanes 4-8).

Example 10: Analysis of Clostridium difficile toxin genes through microarray chip fabrication and hybridization process

Microarray by selecting probes specific for partial deletion of toxin A gene, toxin B gene, binary toxin, and toxin C gene for the overall analysis of Clostridium difficile toxin genes. A DNA chip was prepared.

The microarray (oligonucleotide microarray) DNA chip was manufactured according to a manufacturing method well known in the art. That is, probes specific for partial deletion of toxin A gene, toxin B gene, binary toxin and toxin C gene are microarrayed on a microarray chip substrate made of glass or plastic with aldehyde attached to the surface of the substrate. A DNA chip was produced by taking a picture using the equipment. The fabrication of the microarrayed DNA chip is carried out using methods well known to those skilled in the art.

For example, in an oligonucleotide probe imprinted on a glass substrate using a microarray device, an amine group at the 5'end causes an aldehyde group on the glass substrate to react with Schiff's base and is fixed on the glass substrate. In addition, it may include a step of reducing the aldehyde remaining unreacted. Although the conditions for reducing the aldehyde are not particularly limited thereto, it is preferable to use a reducing agent such as _{NaBH 4.}

The fabricated microarray chip was used to confirm the presence or absence of the toxic gene product and partial deletion of the toxic gene with a fluorescence pattern that was hybridized with the amplification product of each toxic gene.

Specifically, for the probes used in the microarray chip fabrication, an amino linker and 9 thymines were added to the 5'end of each probe sequence, and then specific sequences were sequentially synthesized. The synthesis of an oligonucleotide probe was commissioned by IDT of the United States, and the synthesized probe was dissolved in nuclease free water at a concentration of 100 μM, and an equivalent amount of 2X printing solution (Genocheck, Korea). ) And then dispensed into a 394-well plate by 5 µl each.

The prepared probe was repeated three times using a robotic microarryer (Cartesian, USA), and was divided into four areas with four microarray pins and integrated.

The integrated microarray chip maintained a humidity of 60% at room temperature so that the slide and the probe reacted for 16 hours. To immobilize the probe, it was washed in 0.1% SDS solution for 3 minutes and then washed twice with distilled water for 3 minutes.

The washed microarray chips were reacted for 5 minutes in a reducing solution [sodium borohydride 0.625g + absolute ethanol 62.5ml + 1X PBS 187.5ml)], washed twice with distilled water, and dried for the experiment. Kept until.

In the hybridization experiment, a perfusion chamber was adhered to the prepared microarray chip so that the reaction could proceed independently, and a hybridization solution (Genocheck, Korea) and 10 A solution in which µl of the amplification product was mixed was applied to the microarray chip and reacted.

After the hybridization reaction, the microarray chip was washed in 1X SSC solution for 3 minutes at room temperature, and then washed in 1X SSC solution and 0.1X SSC solution for 3 minutes, respectively, and the microarray chip was dried using a centrifuge. After storing the fluorescence images by scanning with PMT 500, laser power 100, and 5 μm resolution, the positive and negative values of each probe were determined as fluorescence values.

In order to determine the positive reactions for partial deletion of toxin A gene, toxin B gene, binary toxin gene, and toxin C gene (36bp), an experiment was conducted using a clinical isolate and a standard strain (ATCC13124D-5) as PCR templates. The PCR process was carried out by repeating the reaction cycle at 94°C for 10 minutes, followed by 15 seconds at 94°C, 30 seconds at 55°C, and 30 seconds at 72°C.

The primers used in the PCR are the primers shown in Table 1 (tpi gene primers are SEQ ID NO: 1 to SEQ ID NO: 2; toxin A gene primers are SEQ ID NO: 3 to SEQ ID NO: 4; toxin B gene primers are SEQ ID NO: 5 to SEQ ID NO: 8; Toxin C gene partial deletion primers are SEQ ID NOs: 9 to 10; Two-component toxin gene primers are SEQ ID NOs: 11 to SEQ ID NO: 14), and multiplex PCR (multiplex PCR) was performed to amplify five gene products. Each primer concentration was added to a final 200 nM.

The amplification product was treated with lambda-exonuclease to enable hybridization with a microarray chip, and a phosphate functional group was added to the 5'direction of the forward primer to select lambda-exonuclease. Only the product in the forward direction was decomposed. Cy3 was labeled on the reverse primer so as to confirm the fluorescence value, and probes as shown in Table 2 (tpi gene probe: SEQ ID NO: 33 to SEQ ID NO: 34; toxin A gene probe: SEQ ID NO: 35; toxin B gene probe) SEQ ID NO: 36 to SEQ ID NO: 37; Toxin C gene partial deletion probe is SEQ ID NO: 38 to SEQ ID NO: 45; two-component toxin gene probe is a microarray chip in which SEQ ID NO: 46 to SEQ ID NO: 47) is integrated and hybridization reaction of the amplification product Was carried out (see Figs. 10A to 10F, Fig. 11). Meanwhile, in order to confirm the hybridization result of the two-component toxin gene, a clinically isolated strain was used as a template and amplification was performed in the same manner as above, and the result was confirmed.

In this example, as a single-chain method of PCR amplification products, a method of selectively single-chaining by labeling phosphate in one direction of the primer and treating lambda-exonuclease was used, but the present invention is limited thereto. Of course not. For example, the present invention relates to a method of denaturing a double strand of an amplification product using a chemical substance such as NaOH to form a single chain, a method of denaturing a double strand by applying heat to the amplification product to form a single chain, and use during the amplification process. A method of performing asymmetric amplification by adding one of the primer pairs at a low concentration, and selectively single-chaining by labeling a functional group to prevent the exonuclease from acting in the 5'direction of one of the primer pairs used during the amplification process. It will be readily understood by those skilled in the art that the method (PTO synthesis) or the like can be used. And this method can be applied not only to the present embodiment, but also to Embodiments 11 to 13 to be described later.

In addition, although not described in detail in the present embodiment, in the present invention, when the fluorescence is detected by hybridizing the fluorescence-labeled single-chain amplification product with the microarray chip integrated with the above-described probes, the fluorescence value is low and accurate negative or negative Korean Patent No. 10 developed by the present applicant to improve the problem of difficult positive determination and to improve the economical problem of having to label each of the corresponding gene amplification primers when performing multiple PCR amplifying multiple gene products at the same time. Additional technologies in -0939454 are available.

That is, if the technology disclosed in Korean Patent Registration No. 10-0939454 is used, a plurality of genes (five genes in this example) amplification products are labeled separately from the primer-binding fluorescent labeling material (eg, Cy3, etc.). Alternatively, a helper nucleotide sequence (or auxiliary probe) for fluorescence detection having an auxiliary fluorescent substance (eg, Cy3, etc.) is prepared and hybridized with a complementary portion in the nucleotide sequence of each of the five gene amplification products, thereby microarray. It is possible to increase the detection sensitivity of toxic genes and genes for epidemiologic analysis using a chip, and additionally solve the troublesome and uneconomic problem of fluorescent labeling of primers for amplifying toxic genes and genes for epidemiologic analysis.

In addition, those skilled in the art to which the present invention pertains will readily understand that the technology disclosed in Korean Patent Registration No. 10-0939454 can be applied to Examples 10 to 13 of the present invention.

For example, the helper sequence for fluorescence detection is not complementary to a plurality of microarrayed gene probes (5 in Example 10 and 7 in Example 13), but amplification of multiple genes For each product, a specific partial or total nucleotide sequence and a complementary sequence are determined and designed. In addition, a fluorescent substance such as Cy3 is directly labeled on each of the fluorescence detection helper nucleotide sequences for each of the gene amplification products designed as described above, or a non-homologous nucleotide sequence (universal sequence) having no homology with the plurality of gene sequences is used. A nucleotide sequence (complementary universal sequence) labeled with a fluorescent substance having a tag (tag) to the prepared fluorescence detection helper nucleotide sequence and a sequence complementary to this non-homologous nucleotide sequence is prepared, and hybridized to the tagged non-homologous nucleotide sequence. By doing so, it is possible to indirectly label a fluorescent substance such as Cy3. This indirect labeling method has the advantage of solving the inconvenience and uneconomical problem of labeling a fluorescent substance for each of the fluorescence detection helper sequences for a plurality of gene amplification products. Such an indirect labeling method can be implemented by applying the technology disclosed in Korean Patent Application No. 10-2009-0098838 (filing date: 2009. 10. 16.) of the present applicant.

Example 11: Analysis of slpA genotype using a microarray chip

In order to analyze the slpA genotype, which is a gene for epidemiologic analysis of Clostridium difficile, a microarray DNA chip with probes of SEQ ID NO: 49 to SEQ ID NO: 65 was prepared in the same manner as in Example 10.

PCR amplification by repeating the reaction cycle of 15 seconds at 94°C, 30 seconds at 50°C, and 30 seconds at 72°C after reacting the sample gene at 94°C for 10 minutes using the primers of SEQ ID NOs: 17 to 25 Thus, a target product was obtained, which was hybridized with a microarray DNA chip in the same manner as in Example 10 to analyze slpA genotype (FIGS. 12A to 12C ). As shown in FIGS. 12A to 12C, as a result of conducting an experiment using the clinical isolates in this Example, each slpA genotype could be clearly identified.

Example 12: Analysis of cwp66 genotype using a microarray chip

In order to analyze the genotype of the cwp66 gene, which is a colonization factor, as a gene for epidemiologic analysis of Clostridium difficile, a microarray DNA chip with probes of SEQ ID NOs: 66 to 71 was prepared in the same manner as in Example 10.

A sample gene was amplified in the same manner as in Example 11 using primers of SEQ ID NO: 26 to SEQ ID NO: 32, and then hybridized with a microarray DNA chip in the same manner as in Example 11 to analyze the cwp66 genotype (FIG. 13A and 13b).

Example 13: Simultaneous analysis of the presence or absence of a toxin gene of Clostridium difficile and genotype of slpA gene and cwp66 gene using microarray analysis

Using the obtained clinical sample as a PCR template, analysis was performed on one microarray chip in the same manner as in Examples 10-12. The PCR process was performed in the same manner as described in Examples 10-12, and 10 μl of each amplification product was taken and a hybridization experiment was performed in the same experimental procedure as described in Example 10.

The probes integrated with the microarray were sequentially applied in the same manner as described in Example 10-12, and the positive and negative determination of each probe was determined by the presence or absence of a fluorescence value (FIG. 14).

After all, according to this embodiment, it is possible to simultaneously proceed with the epidemiologic analysis and pathogenicity analysis of Clostridium difficile through genotyping of the Clostridium difficile epidemiologic analysis gene and the toxin gene. Through the epidemiological analysis necessary to prevent the outbreak of the in-population, the degree of toxicity and transmission of Clostridium difficile can be analyzed and managed at the same time.

In particular, if the base technology of the test method of the present invention is used, not only Clostridium difficile, but also a toxin gene encoding a toxin of a pathogen causing intestinal disease and a gene for epidemiologic analysis suitable for determining the end of the pathogen (e.g., different Efficient toxicity analysis and epidemiologic analysis for various pathogens (e.g., pathogens causing intestinal diseases such as food poisoning) with mutations in a single gene between species can be performed quickly, while reducing the toxic load. By quantification, the risk of the pathogen can be determined objectively at an early stage (Fig. 15).

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. Those skilled in the art will be able to make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

<110> Genocheck Co., Ltd. <120> Detection chip for performing virulence analysis of Clostridium difficile, detection kit, and detection method using the same <130> P11-0265KR <160> 76 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> tpi-F Primer <400> 1 ggatttaaaa gaagctacta agggtacaaa 30 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> tpi-R Primer <400> 2 ccaacacata atattgggtc tattcctac 29 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> tcdAF Primer <400> 3 tgggtgcgaa tggttataaa acta 24 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> tcdAR Primer <400> 4 attagcaggt gcaaagtatt caaatc 26 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> tcdBF Primer <400> 5 caagtttacg ctcaattatt tagtactggt t 31 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> tcdBR Primer <400> 6 ggaagtaagt ctatagtttc atctaatgca gtt 33 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> tcdBF2 Primer <400> 7 agtactggtt taaatactat tcagatgca 29 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> tcdBR2 Primer <400> 8 ctgcacctaa acttacacca tct 23 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> tcdCF Primer <400> 9 gacgaaaaga aagctattga agctga 26 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> tcdCR Primer <400> 10 ctagctaatt ggtcataagt aataccagt 29 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> cdtAF Primer <400> 11 gggtaaagca aattataatg attggag 27 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> cdtAR Primer <400> 12 ttgcagtata tcctccacgc atat 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> cdtBF Primer <400> 13 gacttaattg cagttaagtg ggaa 24 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> cdtBR Primer <400> 14 tatggatctc cagcagtatt tga 23 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CPN60A204F Primer <400> 15 gggacaacca ctgcgtctat 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CPN60A204R Primer <400> 16 cacaaggaag gtcactgcaa 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> slpAF1 Primer <400> 17 tgccgttaca gtaatcggat c 21 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> slpAF2 Primer <400> 18 gcwgcwgtta cwgtwgtrgg ttc 23 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> slpAF3 Primer <400> 19 gtcaggwtta acagtdttrg cttc 24 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> slpAR1 Primer <400> 20 cmacatcwat agattcttct tttgc 25 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> slpAR2 Primer <400> 21 tctatatcca cattaacttc tttagc 26 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> slpAR3 Primer <400> 22 tctaagtcta ttgtttgctc tttagc 26 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> slpAR4 Primer <400> 23 tctatatcta ttgttgattc ttttgc 26 <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> slpAR5 Primer <400> 24 cttacatcta ttgttttttc aacagc 26 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> slpAR6 Primer <400> 25 gatattgtaa ctgtttcatc tgatgc 26 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> cwp66F2-1 Primer <400> 26 tgctataaaa agagcagatg caaatgat 28 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> cwp66F2-2 Primer <400> 27 tgctataaaa aaggcagatg caaatgat 28 <210> 28 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> cwp66F2-3 Primer <400> 28 gttgctataa aaaaatcaga tgcaaatga 29 <210> 29 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> cwp66F2-4 Primer <400> 29 gatgtcataa taaaaaactc aggaaagatt aat 33 <210> 30 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> cwp66F2-5 Primer <400> 30 aaatacttcg acaaataaaa tttctaaatg gg 32 <210> 31 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> cwp66R2-1 Primer <400> 31 caacatccat tgcataaggt ttattataaa ct 32 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> cwp66R2-2 Primer <400> 32 cctacataca tagcagaagg tttatctt 28 <210> 33 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> tpi-P1 Probe <400> 33 actgtaaaya agaaagtttt aaaagcatta ga 32 <210> 34 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> tpi-P2 Probe <400> 34 tgttaaaaga aatagatatg gactatgttg ta 32 <210> 35 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> tcdAP Probe <400> 35 actttagaaa tggtttacct cagatagga 29 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> tcdBP1 Probe <400> 36 atcaactgca ttagatgaaa ctatagactt 30 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> tcdBP2 Probe <400> 37 tacttcctac attatctgaa ggattacc 28 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> tcdCDP2 Probe <400> 38 gaacaacgma aaaaagaaga agagg 25 <210> 39 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> tcdCDP3 Probe <400> 39 aaggctgaag aacaacgmaa aaaaga 26 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> tcdC(-)RT027-1 Probe <400> 40 actggcattt attttaggcg tgtt 24 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> tcdCRT027-1 Probe <400> 41 actggcattt attttggcgt gtt 23 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> tcdCRT078-1 Probe <400> 42 actggcattt atttttggcg tgtt 24 <210> 43 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> tcdC(-)RT027-2 Probe <400> 43 gaggtcattt ctaaccaaac atcagtt 27 <210> 44 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> tcdCRT027-2 Probe <400> 44 ggaggtcatt tctaatcaaa catcagtt 28 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> tcdCRT078-2 Probe <400> 45 aggaggtcat ttctaattaa acatcagtt 29 <210> 46 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> cdtAP Probe <400> 46 acacctaatg aacttgctga tgtaaatg 28 <210> 47 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> cdtBP Probe <400> 47 ttgcagaaca aggctataag aaatatgt 28 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CPN60AP Probe <400> 48 tcacttctgg tgcgaatcc 19 <210> 49 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> slpA P1 RC Probe <400> 49 tggcactacc tcaccaattg aa 22 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> slpA P2 RC Probe <400> 50 tggtgtaact tctccaacag agt 23 <210> 51 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> slpA P3 RC Probe <400> 51 tgatatagat accccttttg gatttcctt 29 <210> 52 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> slpA P4 RC Probe <400> 52 ctaactcttc ttttgttgaa gtagttcct 29 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> slpA P5 RC Probe <400> 53 gcagaactta ttgccaaagt tgca 24 <210> 54 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> slpA P6 RC Probe <400> 54 tagcatctgc tgtagttgca ttagt 25 <210> 55 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> slpA P7 RC Probe <400> 55 cctttggtgc taatgtagta attggtt 27 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> slpA P8 RC Probe <400> 56 ttaggagcta cttcacccac aac 23 <210> 57 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> slpA P9 RC Probe <400> 57 gttccactta ttccatattc tgacacaag 29 <210> 58 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> slpA P10 RC Probe <400> 58 gctcagaacc tacttttata gtagatgct 29 <210> 59 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> slpA P11 RC Probe <400> 59 tccagttgga gttgaagaat ctttctc 27 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> slpA P12 RC Probe <400> 60 agtgtggcag ttgaagacat cttt 24 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> slpA P13 RC Probe <400> 61 gctgcgtctc tatcagtaga gt 22 <210> 62 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> slpA P14 RC Probe <400> 62 acagtaatag tagtaactgg cttacctt 28 <210> 63 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> slpA P15 RC Probe <400> 63 cctgcaacat ttacagttga aactttagt 29 <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> slpA P16 RC Probe <400> 64 agcttctttt ttcttaaccg ctga 24 <210> 65 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> slpA P17 RC Probe <400> 65 ctgcttcagc ttctaactca gatact 26 <210> 66 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> cwp66 P1 RC Probe <400> 66 ccattaggta tatccaaagt tattgtttgt c 31 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> cwp66 P2 RC Probe <400> 67 gtgtggaaca tgcctctgtt t 21 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> cwp66 P3 RC Probe <400> 68 cacttaaacc ttttgccacc tct 23 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> cwp66 P4 RC Probe <400> 69 agccttattt gtttccactt taaatgaatc 30 <210> 70 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cwp66 P5 RC Probe <400> 70 accctgtata ctgccttcat taatc 25 <210> 71 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> cwp66 P6 RC Probe <400> 71 atctatacct tgtatactac cttcattaat c 31 <210> 72 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Cwp66F Cloning Primer <400> 72 taggctactt tctggtgtcg ttgaa 25 <210> 73 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cwp66R Cloning Primer <400> 73 tggaatcctg aaagtggaca ttatgc 26 <210> 74 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> tcdBP3 Probe <400> 74 atgcagccaa agttg 15 <210> 75 <211> 352 <212> DNA <213> Clostridium difficile <220> <221> gene <222> (1)..(352) <223> toxin A clone gene <400> 75 attttggtaa taattcaaaa gcggctactg gttgggtaac tattgatggt aatagatatt 60 acttcgagcc taatacagct atgggtgcga atggttataa aactattgat aataaaaatt 120 tttactttag aaatggttta cctcagatag gagtgtttaa agggtctaat ggatttgaat 180 actttgcacc tgctaatacg gatgctaaca atatagaagg tcaagctata cgttatcaaa 240 atagattcct acatttactt ggaaaaatat attactttgg taataattca aaagcagtta 300 ctggatggca aactattaat ggtaaagtat attactttat gcctgatact gc 352 <210> 76 <211> 960 <212> DNA <213> Clostridium difficile <220> <221> gene <222> (1)..(960) <223> toxin B clone gene <400> 76 gtgaaggaag aagagaatta ttggatcatt ctggtgaatg gataaataaa gaagaaagta 60 ttataaagga tatttcatca aaagaatata tatcatttaa tcctaaagaa aataaaatta 120 cagtaaaatc taaaaattta cctgagctat ctacattatt acaagaaatt agaaataatt 180 ctaattcaag tgatattgaa ctagaagaaa aagtaatgtt aacagaatgt gagataaatg 240 ttatttcaaa tatagatacg caaattgttg aggaaaggat tgaagaagct aagaatttaa 300 cttctgactc tattaattat ataaaagatg aatttaaact aatagaatct atttctgatg 360 cactatgtga cttaaaacaa cagaatgaat tagaagattc tcattttata tcttttgagg 420 acatatcaga gactgatgag ggatttagta taagatttat taataaagaa actggagaat 480 ctatatttgt agaaactgaa aaaacaatat tctctgaata tgctaatcat ataactgaag 540 agatttctaa gataaaaggt actatatttg atactgtaaa tggtaagtta gtaaaaaaag 600 taaatttaga tactacacac gaagtaaata ctttaaatgc tgcatttttt atacaatcat 660 taatagaata taatagttct aaagaatctc ttagtaattt aagtgtagca atgaaagtcc 720 aagtttacgc tcaattattt agtactggtt taaatactat tacagatgca gccaaagttg 780 ttgaattagt atcaactgca ttagatgaaa ctatagactt acttcctaca ttatctgaag 840 gattacctat aattgcaact attatagatg gtgtaagttt aggtgcagca atcaaagagc 900 taagtgaaac gagtgaccca ttattaagac aagaaataga agctaagata ggtataatgg 960 960

Claims (10)

At least one selected from the group consisting of oligonucleotides that specifically bind to the toxin B gene of Clostridium difficile and have an nucleotide sequence complementary to those oligonucleotides A test chip for the pathogenicity assay for Clostridium difficile comprising a probe. Early pathogenicity of Clostridium difficile, including primer pairs of SEQ ID NOs: 5 and 6 for amplifying the toxin B gene of Clostridium difficile, DNA polymerases, dNTPs, PCR buffers and markers of PCR amplification products Real-time PCR amplification kit for analysis. The method of claim 2,
The labeling substance is a probe that specifically binds to Clostridium difficile toxin B gene. Real-time PCR amplification kit for the early analysis of the pathogenicity of Clostridium difficile, characterized in that labeled. An inspection chip as defined in claim 1,
A primer pair of SEQ ID NOs: 5 and 6 for amplifying the toxin B gene of Clostridium difficile,
A test kit for the pathogenicity analysis of Clostridium difficile comprising a label means for detecting the amplified toxin B gene hybridized with the test chip. The method of claim 4, wherein
The labeling means is directly linked to the 5 'end of the primer labeled with the amplified toxin B gene, and a partial or full nucleotide sequence specific for the amplified toxin B gene without complementary to the probe of the test chip Test kit for the pathogenicity analysis for Clostridium difficile, characterized in that at least one of the labeled in the detection helper base sequence having a complementary base sequence. The method of claim 5,
The labeling means is directly labeled on the detection helper base sequence for the amplified toxin B gene, or a non-homologous base sequence having no homology to the amplified toxin B gene is tagged to the detection helper base sequence. And then indirectly label the labeling means by preparing a nucleotide sequence complementary to the non-homologous base sequence and hybridizing to the tagged non-homologous sequence by preparing a base sequence to which the labeling means is bound. Test kit for pathogenicity analysis for difficile. In the test method for early analysis of the pathogenicity of Clostridium difficile,
Preparing a genetic sample from the subject,
Using the prepared gene sample as a template, using the primer pairs of SEQ ID NOs: 5 and 6 for the Clostridium difficile toxin B gene, and a label indicating the amount of gene amplification in relation to the toxin B gene to be amplified, Amplifying the toxin B gene in a real sample by real-time PCR;
After amplifying the toxin B gene, calculating a toxic load for Clostridium difficile toxin B based on the toxin B gene amplification amount calculated using the labeling substance;
Detecting the pathogenicity of Clostridium difficile based on the calculated toxic load. The method of claim 7, wherein
The labeling substance is a fluorescent substance, and the test method for an early analysis of the pathogenicity of Clostridium difficile, characterized by calculating the amplification amount of the toxin B gene by measuring the intensity of fluorescence from the labeling substance. The method of claim 8,
A positive standard, which is a gene sample that knows the copy number of Clostridium difficile toxin B gene before amplification, indicates a gene amplification amount in association with the primer pairs of SEQ ID NOs: 5 and 6 for the toxin B gene and the toxin B gene to be amplified. Amplifying by real-time PCR using a labeling substance,
Measuring a change in fluorescence intensity of the labeling substance over the entire PCR amplification period of the positive standard, and analyzing the number of PCR reactions (Cp value or Ct value) attaining a constant fluorescence intensity;
Matching the copy number of the positive standard with a Cp value or a Ct value from a standard curve obtained by measuring a change in fluorescence intensity of the labeling substance;
Measuring the change in fluorescence intensity of the labeling substance over the entire PCR amplification period of the toxin B gene in the subject's gene sample, and analyzing the number of PCR reactions (Cp value or Ct value) reaching a constant fluorescence intensity;
By quantifying the Cp value or Ct value obtained by PCR amplifying the toxin B gene in the subject's gene sample to the copy number of the positive standard, the toxic load of Clostridium difficile toxin B in the subject's gene sample is quantitatively determined. The test method for the early analysis of the pathogenicity of Clostridium difficile, further comprising the step of measuring. The method according to any one of claims 7 to 9,
The labeling substance is a probe that specifically binds to Clostridium difficile toxin B gene. A test method for early analysis of the pathogenicity of Clostridium difficile, characterized in that it is labeled.

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