KR102132965B1 - Kit For Detecting Tuberculosis and Method of Detecting Tuberculosis Using the Same - Google Patents

Abstract

The present invention relates to a tuberculosis diagnosis kit and a method for detecting tuberculosis using the same, specifically, (a) (i) Mycobacterium tuberculosis gene and a gene commonly present in mycobacteria can be specifically amplified at the same time. The present invention relates to a kit for simultaneous diagnosis of tuberculosis and mycobacteria comprising a primer capable of identifying the amplified product, and (b) the amplified product, and a method for simultaneously diagnosing tuberculosis and non-tuberculosis antibacterialosis using the same.

Description

Kit for tuberculosis diagnosis and diagnosis method of tuberculosis using the same {Kit For Detecting Tuberculosis and Method of Detecting Tuberculosis Using the Same}

The present invention relates to a tuberculosis diagnosis kit and a method for detecting tuberculosis using the same, specifically (a) (i) Mycobacterium tuberculosis specific gene and a gene that is commonly present in mycobacteria are simultaneously specifically amplified A kit for simultaneously diagnosing Mycobacterium tuberculosis and non-tuberculosis mycobacteria comprising a primer capable of identifying, and (b) a probe capable of identifying the amplified product, and a method for simultaneously diagnosing Mycobacterium tuberculosis and non-tuberculosis mycobacteria using the same will be.

Tuberculosis is found not only in the lungs, but also in the lymph glands, kidneys, spine, peritoneum, intestines, and skin. It is a disease caused by Mycobacterium tuberculosis. Symptoms vary with fever, fatigue, and weight loss. Even today, with the development of various medical technologies, infections caused by Mycobacterium tuberculosis are frequent, and in Korea, the incidence of tuberculosis is ranked first among OECD countries.

It is estimated that about 2 billion people, one third of the world's population, are infected with tuberculosis, and 9 million people are diagnosed with new tuberculosis each year and 3 million are reported to die of tuberculosis (TB). According to statistics in 2006, 35,000 patients were diagnosed with tuberculosis at 242 public health centers and private hospitals and clinics nationwide, reaching 67.2 per 100,000 population, and according to the National Statistical Office's cause of death, tuberculosis The number of people who died in 2006 was 2,948, and the mortality rate of tuberculosis reached 6.7 per 100,000 people, accounting for 55.7% of the total deaths from infectious diseases, ranking first among the 30 members of the Organization for Economic Cooperation and Development (OECD). It is a serious disease. The main reason why tuberculosis, which has been regarded as a backward disease, has recently emerged as a global health problem, is that the tuberculosis bacteria cause opportunistic infections in patients with acquired immunodeficiency syndrome (ADIS) and frequent exchanges among the international community due to globalization of the global village. Can be lifted.

In order to prevent the spread of tuberculosis, various approaches such as early diagnosis, administration of appropriate antibiotics, isolation of patients, and regular screening of groups with high risk of tuberculosis exposure are required. Particularly, early and rapid diagnosis of HIV-infected patients prevents death due to tuberculosis. The most common method for diagnosis of tuberculosis is X-ray chest imaging and sputum smear, but the accuracy is 50~75% because tuberculosis and non-tuberculosis antibacterials cannot be distinguished. As it is very low, there is a problem in sensitivity and reliability. Pulmonary disease caused by non-tuberculous mycobacteria shows symptoms similar to tuberculosis such as cough and sputum, and test results, but it is not transmitted to others and treatment is completely different. In the United States or Europe, the first sputum smear test is positive. Even if it appears, tuberculosis is finally determined through additional precision tests, but in Korea, non-TB antibacterials are classified as rare areas, and the secondary test for this is neglected. In some cases, the number of cases that suffer from economic and psychological damage has increased, such as receiving anti-tuberculosis drugs for some years, and some patients are mistaken for drug-resistant tuberculosis, which is not the first drug treatment. Therefore, even if the first sputum smear test shows a positive response, additional tests such as nucleic acid amplification test or culture test are necessary, but in the case of culture test, only the culture of tuberculosis bacteria takes 4 to 8 weeks, so rapid diagnosis and appropriate early treatment are difficult. Considering the facts, for the early detection of tuberculosis, the diagnosis that combined acid-fast bacillus (AFB) smear and nucleic acid amplification test is the most ideal, and it is a trend that the United States and other developed countries are enacting it.

As such, early diagnosis of infectious bacteria is a very important factor in the treatment of diseases because tuberculosis has problems such as side effects and transmission. Therefore, various diagnostic techniques have been actively developed and applied to early diagnosis of tuberculosis.

In connection with the diagnosis of tuberculosis, an antibacterial smear staining method or a culture test may be considered. The anti-bacterial smear staining method can confirm a quick result by a simple method, but has a disadvantage in that it is not sensitive and cannot distinguish between tuberculosis bacteria and non-tuberculosis antibacterial bacteria. The culture test can detect even a small amount of Mycobacterium tuberculosis, and the specificity is high, but it takes a long time to derive the results. In addition, although the method is simple and convenient, an immunodiagnostic method such as an ELISA or a rapid method (Rapid method) is used, but it is somewhat unreasonable to detect a small amount of Mycobacterium tuberculosis.

Recently, polymerase chain reaction (PCR), such as PCR, is used for the diagnosis of Mycobacterium tuberculosis, and PCR-electrophoresis or real-time PCR is considered. The PCR method is fast and highly sensitive and can detect even a small amount of tuberculosis bacteria, but the PCR-electrophoresis method requires the preparation of an agarose gel to identify the amplified gene as in conventional PCR, and real-time PCR requires expensive equipment. There is a disadvantage of low economic efficiency.

Under this technical background, the inventors of the present application can quickly and qualitatively and accurately diagnose tuberculosis by simultaneously diagnosing the genes for Mycobacterium tuberculosis and Mycobacterium, and diagnosis of tuberculosis by distinguishing it from non-TB antibacterial bacteria with only a small amount of sample gene It has been confirmed that a kit and a method for simultaneously diagnosing tuberculosis and non-tuberculosis antibacterials using the same can be provided, and the present invention has been completed. In addition, by using two types of genes used for diagnosing Mycobacterium tuberculosis, i.e., IS6110 and mtp40 at the same time, it is confirmed that a diagnostic method capable of accurately diagnosing Mycobacterium tuberculosis can be provided even if one of the two genes is deficient. Completed.

The above information described in the background section is only for improving the understanding of the background of the present invention, and therefore does not include information about prior art already known to those skilled in the art to which the present invention pertains. Can.

The object of the present invention is to recognize the problems of the above-mentioned prior art, and to accurately and simply diagnose tuberculosis and non-tuberculosis anti-tuberculosis, which are accurate and simple to distinguish from tuberculosis anti-tuberculosis compared to the prior art. It provides a method of simultaneously diagnosing tuberculosis and non-tuberculosis antibacterialosis.

In order to achieve the above object, the present invention includes (a) (i) a complementary binding site that specifically binds Mycobacterium tuberculosis specific gene and a nucleic acid oligomer containing a non-complementary base with Mycobacterium tuberculosis specific gene. Primer to do; (ii) a primer comprising a complementary binding site that specifically binds to a gene common to mycobacteria and a nucleic acid oligomer containing a base common to a gene common to mycobacteria and a non-complementary base; And (b) a kit for simultaneous diagnosis of tuberculosis and non-tuberculosis anti-acidosis, comprising a membrane to which a probe that complementarily binds the nucleic acid oligomer is immobilized.

The present invention also includes a nucleic acid oligomer containing a complementary binding site specifically binding the nucleic acid extracted from the sample with (i) Mycobacterium tuberculosis specific gene and a tuberculosis specific gene and a non-complementary base. Primer and (ii) a primer comprising a complementary binding site that specifically binds to a gene commonly present in mycobacteria and a nucleic acid oligomer containing a gene common to mycobacteria and a non-complementary base Amplifying; And loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is fixed, tuberculosis and non-tuberculosis in vitro using the kit according to any one of claims 1 to 16. It relates to a method of simultaneously diagnosing anti-acidosis.

The present invention further comprises a nucleic acid oligomer containing a complementary binding site that specifically binds (i) Mycobacterium tuberculosis-specific gene and a non-complementary base with Mycobacterium tuberculosis specific gene. Primer and (ii) a primer comprising a complementary binding site that specifically binds to a gene commonly present in mycobacteria and a nucleic acid oligomer containing a gene common to mycobacteria and a non-complementary base Amplifying; And

Tuberculosis and non-tuberculosis anti-tuberculosis in vitro using the kit according to any one of claims 1 to 16, comprising loading the amplification product on a membrane fixed with a probe complementary to the nucleic acid oligomer. It relates to a method of providing information to the simultaneous diagnosis of mycosis.

The kit according to the present invention enables the diagnosis of tuberculosis with only a small amount of sample gene, and simultaneously amplifies and diagnoses tuberculosis specific genes and genes common to mycobacteria, thereby quickly and accurately qualitative molecules of tuberculosis bacteria and non-tuberculosis antibiotics Make diagnosis possible. In addition, since the result is verified through a side-flow assay after gene amplification, it is economical because expensive equipment such as in real-time PCR is not required, and agarose gel for electrophoresis to confirm the amplified gene as in conventional PCR Since the manufacturing process is not required, the results can be confirmed in a shorter time than PCR-electrophoresis.

1 shows a process of selecting a target region and designing a primer for each genomic tuberculosis causative agent.
2 is a schematic diagram of a strip for identifying amplification products in a kit according to an embodiment of the present invention.
3 is a schematic diagram of a reaction in a membrane in which a probe complementarily binding to a nucleic acid oligomer included in a kit according to an embodiment of the present invention is fixed.
Figure 4 shows the change in the color development position according to the presence or absence of the tuberculosis causative gene.
Figure 5 shows the test results according to the negative or positive criteria for tuberculosis and non-tuberculosis always.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

Among the molecular diagnostic products, products using polymerase chain reaction (PCR) can be broadly classified into two types. One is a conventional PCR, extracting a nucleic acid from a sample, amplifying a target nucleic acid, and confirming the result through electrophoresis. Using this method, you can purchase a low-cost kit, but electrophoresis takes a lot of labor and time. The real-time PCR (Real-time PCR) technique used in place of electrophoresis can monitor the nucleic acid amplification of pathogens in real time, but the kit is expensive and it is difficult to check the results of amplification of multiple nucleic acids of 5 or more.

Accordingly, as maintaining the user's convenience and developing a low-cost molecular diagnostic product is required, the inventors of the present application have developed an in vitro diagnostic kit for qualitative analysis.

In this respect, the present invention provides a primer comprising (a) (i) a complementary binding site binding to a Mycobacterium tuberculosis specific gene and a nucleic acid oligomer containing a base complementary to a TB specific gene; (ii) a primer comprising a complementary binding site binding to a gene common to mycobacteria and a nucleic acid oligomer containing a base common to a gene common to mycobacteria and a non-complementary base; And (b) a membrane in which a probe complementarily binding to the nucleic acid oligomer is immobilized, and relates to a kit for simultaneous diagnosis of tuberculosis and non-tuberculosis antibacterialosis.

The kit according to the present invention includes a primer comprising a complementary binding site and a nucleic acid oligomer, wherein the primer includes a complementary binding site that specifically binds a tuberculosis specific gene and a gene commonly present in Mycobacteria. , A nucleic acid oligomer containing a non-complementary base and a gene commonly present in the Mycobacterium tuberculosis specific gene. According to the present invention, it is possible to simultaneously diagnose the presence of infection with tuberculosis bacteria and non-tuberculosis antibacterial bacteria.

In one embodiment, the Mycobacterium tuberculosis specific gene may be one or more or two or more genes specifically present in Mycobacterium tuberculosis, and preferably may include two or more Mycobacterium tuberculosis specific genes. Conventional TB germ diagnostic products use only one type of Mycobacterium tuberculosis specific gene. However, the present invention can provide a diagnostic method capable of accurately diagnosing Mycobacterium tuberculosis even if one or more of the two or more genes in a diagnosis object is deficient by simultaneously using two or more genes used to diagnose Mycobacterium tuberculosis. The tuberculosis specific gene may be, for example, IS6110 (SEQ ID NO: 14) and/or mtp40 (SEQ ID NO: 15) days. By using IS6110 and mtp40 simultaneously, it is possible to accurately diagnose Mycobacterium tuberculosis even if one of the two genes is deficient.

In the present specification, non-tuberculosis anti-acidosis is not a tuberculosis bacterium, but it can mean a disease that causes symptoms such as coughing, sputum, and various inflammations in the lungs, while mycobacteria, except for tuberculosis, which is a similar bacterium, stay in the bronchus. The gene commonly present in the mycobacteria may be one or more genes specifically present in the mycobacteria including tuberculosis bacteria, and may be, for example, rpoB (SEQ ID NO: 16).

In a specific embodiment, using the kit according to the present invention, the two genes IS6110 and mtp40 present in Mycobacterium tuberculosis and the rpoB gene commonly present in the genus Mycobacterium containing Mycobacterium tuberculosis are simultaneously amplified to distinguish Mycobacterium tuberculosis and nontuberculous Mycobacterium bacteria. Can. As a result of diagnosis, when one or more of the tuberculosis specific genes IS6110 and/or mtp40 and the rpoB gene are amplified, it can be diagnosed as tuberculosis, and when only the rpoB gene is amplified, it can be determined as non-tuberculous mycosis. According to the present invention, it is possible to accurately diagnose the presence of tuberculosis even in tuberculosis bacteria lacking one gene.

A'primer' herein is a single-stranded oligonucleotide that can serve as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable temperature and buffer. Means

The primer according to the present invention may include a tuberculosis specific gene or a fragment of a gene commonly present in mycobacteria. The fragment may refer to any region of a tuberculosis specific gene and a gene common to mycobacteria, and the fragment may have any length, for example, IS6110 and/or mtp40 and rpoB genes or these It may mean a site having a length of 5 bp to 50 bp, specifically 10 bp to 30 bp, for any site.

In the present specification,'complementary binding' means that the primer is hybridized with a corresponding nucleic acid strand under conditions of performing a polymerization reaction, and may form a duplex structure. Complementary binding may be formed even if complementarity between paired nucleotide sequences forms a Watson-Crick pair or some non-Watson-Crick base pair is present.

The primer may be a forward primer, for example, may include a complementary binding site that specifically binds to the tuberculosis specific gene IS6110 and/or mtp40 and the rpoB gene, a gene common to mycobacteria. The forward primer binds to the complementary strand when the rpoB gene, which is a gene common to IS6110 and/or mtp40 and mycobacteria, is used as a template, and the rpoB gene, which is a gene common to IS6110 and/or mtp40 and mycobacteria And complementary sequences can be specifically amplified, for example, the primers can be selected from the group consisting of SEQ ID NOs: 1-6.

The primer includes a nucleic acid oligomer containing a base complementary to a tuberculosis specific gene and a non-tuberculous antibacterial gene or a fragment thereof. When amplifying the rpoB gene, which is a gene common to the target genes IS6110 and/or mtp40 and mycobacteria using PCR, the base is not complementary to the rpoB gene, a gene common to IS6110 and/or mtp40 and mycobacteria A nucleic acid oligomer containing, that is, a gene not related to the rpoB gene, which is a gene commonly present in IS6110 and/or mtp40 and mycobacteria, is about 20-60 bp, specifically 20-40 bp, preferably about 20-30 bp It may include. By reacting the nucleic acid oligomer with a probe complementarily synthesized on the membrane, it is possible to easily check whether the result, that is, whether to amplify the nucleic acid. The nucleic acid oligomer may be selected from the group consisting of SEQ ID NOs: 9 to 11, for example.

In order to react with the probe complementarily synthesized on the membrane, the nucleic acid oligomer is combined with a probe complementarily synthesized on the membrane and a base portion not complementary to a tuberculosis specific gene and a non-tuberculous antibacterial gene or a fragment thereof. A non-nucleotide spacer may be additionally included between the bases. The non-nucleotide spacer may be a C3, C6, C9 or C12 aliphatic spacer, and the number C means the number of carbon atoms in the non-nucleotide spacer structure. The non-nucleotide spacer may be an alkyl, alkenyl, or alkynyl group.

In one embodiment, a non-nucleotide spacer, e.g., between a tubercle-specific gene and a non-tuberculosis antibacterial gene or fragment thereof and a base for binding to a non-complementary base moiety and a probe complementarily synthesized to the membrane When a gene is amplified using Taq polymerase by locating the C3 spacer (propyl spacer), complementary amplification does not invade the base portion for binding to the probe, thereby ensuring partial single-stranded nucleic acid results And this portion was allowed to bind the probe to the membrane.

The primer may further include a primer containing a marker. The primer containing the marker is a reverse primer, and the reverse primer can bind to the template strand and amplify the template when the rpoB gene, which is a gene common to IS6110 and/or mtp40 and mycobacteria, is used as a template, e.g. For example, the primer may be selected from the group consisting of SEQ ID NOs: 1-6.

In one embodiment, the markers included in the primers are biotin, Cy5, Cy3, FITC, EDANS (5-(2'-aminoethyl)amino-1-naphthalene sulfate), tetramethylrodamine (TMR), tetramethyl Rhodamine isocyanate (TMRITC), x-rhodamine, DIG or an antibody, or a nanoparticle bound thereto, but may not be limited thereto.

Here, by reacting with a binding agent that induces the color development of the marker, it is possible to check whether color development or fluorescence is expressed, and amplification of the target nucleic acid can be detected. At this time, the binding agent may be streptavidin, but is not limited thereto.

In a specific embodiment, by attaching biotin to the reverse primer and amplifying it, it is bound to streptavidine in the membrane, and particles conjugated to biotin, for example, gold particles according to the propensity of nanoparticles In the case of color, it can be confirmed by color development, and in the case of fluorescence, it can be confirmed by fluorescence of various wavelengths, so depending on the type of probe attached to the membrane, it is possible to distinguish whether several to tens of target nucleic acids are simultaneously amplified.

The kit according to the present invention includes (b) a membrane fixed with a probe complementarily binding to the nucleic acid oligomer. Through this, when the product amplified by the primer is injected to the side of the membrane, the amplified product moves, and the probe fixed to the membrane and the nucleic acid oligomer contained in the amplified product may react complementarily.

The primer according to the present invention can be performed by multiplex-PCR, which makes it possible to simultaneously diagnose Mycobacterium tuberculosis and several types of non-TB antibacterial bacteria. That is, when multiple PCs are performed with probes having several to several tens of different oligomer sequences added to each other, when a probe capable of reacting with this is used to react with the combined oligomer probe, a single Side-stream membranes can be used to identify several to dozens of amplified results.

Therefore, it can be used jointly for various multiple PCs, so that mass production is possible during the production of the side flow membrane. This is to check the results of various items using one side flow memple. Since the amplified products can be identified using the same membrane regardless of the type of PCR amplification, cost reduction and quality control of product production can be confirmed. It is easy to increase the productivity of the product.

The probe that complementarily binds to the nucleic acid oligomer may include, for example, one or more sequences selected from the group consisting of SEQ ID NOs: 9 to 12.

In some cases, the probe may further include a nucleic acid oligomer for immobilization on a membrane, and may further include an oligomer having a repeating nucleotide sequence of 20-60 bp, specifically 20-40 bp. In this case, the additional nucleic acid oligomer may include, for example, the sequence of SEQ ID NO: 13.

In some cases, the kit according to the present invention may optionally further include an internal control primer. These internal control primers are intended to confirm whether or not a false negative problem, that is, a PCR reaction was properly performed, a gene that is normally expressed regardless of the presence of Mycobacterium tuberculosis or Mycobacterium in the sample can be arbitrarily selected. In a specific embodiment according to the present invention, the internal control primer may be a beta globin gene (SEQ ID NO: 17), for example, may include the sequence of SEQ ID NO: 7 or 8.

The kit according to the present invention may optionally include reagents necessary for conducting a nucleic acid amplification PCR reaction, such as a polymerase, a buffer, and deoxyribonucleotide-5-triphosphate. The kit according to the present invention may also further include various polynucleotide molecules, various buffers and reagents.

The optimum amount of reagents, buffers or reactants used for a specific reaction in the kit can be determined by those skilled in the art, and complementary binding specifically binding to the above-mentioned Mycobacterium tuberculosis specific gene and the gene common to Mycobacteria is mentioned. A primer (forward) and/or (ii) a labeled primer comprising a marker (reverse), comprising a nucleic acid oligomer containing a base and a gene common to the Mycobacterium tuberculosis specific gene and a site and a complementary base, And membranes on which the probe is immobilized, may be manufactured in a separate package or a compartment including each.

In another aspect, the present invention provides a primer comprising (i) a complementary binding site that specifically binds Mycobacterium tuberculosis gene and a nucleic acid oligomer containing a non-complementary base with Mycobacterium tuberculosis specific gene and (ii) Myco. Amplifying with a primer comprising a nucleic acid oligomer containing a complementary binding site that specifically binds to a gene commonly present in bacteria (mycobacteria) and a gene common to mycobacteria and a non-complementary base; And loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is immobilized, wherein the kit simultaneously diagnoses tuberculosis and non-tuberculosis antibacterial activity in vitro.

In addition, the present invention is a primer comprising a nucleic acid oligomer containing a complementary binding site specifically binding the nucleic acid extracted from the sample (i) Mycobacterium tuberculosis gene and the tuberculosis specific gene and a non-complementary base. (ii) amplifying with a primer comprising a nucleic acid oligomer containing a complementary binding site and a non-TB antimicrobial gene and a complementary base that specifically binds to a gene commonly present in mycobacteria; And loading the amplification product onto a membrane immobilized with a probe complementary to the nucleic acid oligomer to provide information for the simultaneous diagnosis of tuberculosis and non-tuberculosis antibacterialosis using the kit.

Description of the configuration related to the kit used in carrying out the method according to the present invention can be applied to the method in the same way.

In one embodiment, the sample may include a wide range of biological fluids obtained from a bodily fluid, cell line, tissue culture, or the like, derived from a suspected patient or an individual or individual subject to diagnosis according to the diagnosis of tuberculosis and non-tuberculosis antibacterialosis, For example, it may be a sputum, cultured sample, tissue sample, or bronchial wash solution, but is not limited thereto.

The method may further include the step of extracting nucleic acid from the sample, and the extraction of nucleic acid may be performed using, for example, various kits or extraction reagents that are commercially supplied.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

1. Kit making

The kit according to the present invention (hereinafter also referred to as PaxView TB/NTM MPCR-ULFA Kit) includes two kits. One is a kit for amplifying nucleic acids (hereinafter, PaxView TB/NTM MPCR Kit), and the other is a kit for identifying amplification products (hereinafter, PaxView ULFA Kit). The primers included in the primer mixture, which is a component of the PaxView TB/NTM MPCR Kit, include a site for three types of disease-causing bacteria and a site for internal control, and a universal region, a non-complementary nucleic acid oligomer, in the forward primer (SN). ), the reverse primer (Reverse primer = ASN) is a biotin sequence is tagged (tagging) (Fig. 1).

After PCR, the amplification product is loaded onto the ULFA device (e.g., strip: FIG. 2) of the PaxView ULFA Kit, and a pad of gold-streptavidin conjugate=Gold-SV, glass fiber material ( pad), and then combined with a probe adsorbed on the NC membrane (probe = oligonucleotide complementary to the universal region) to develop color at the indicated site of each pathogen (Fig. 3).

The color development principle is based on the reaction method of biotin tagged with each primer and streptavidin, and can be utilized for DNA reaction confirmation in addition to the antigen-antibody reaction that is frequently used. Streptavidin, similar to avidin, can form a complex with biotin, resulting in human-readable color development by gold particles conjugated to biotin.

After color development, the ULFA device is read with the naked eye. The result of the kit is used to determine the yin and positivity according to the determination of the result, and the position of the color line varies depending on the presence or absence of the causative agent of the disease, so it can be confirmed that the expected band pattern and the actual band pattern match (FIG. 4).

The tuberculosis diagnostic kit according to the present invention (TB/NTM diagnostic kit) is a new tuberculosis diagnosis platform, and it is a rapid and accurate qualitative molecular diagnostic kit that amplifies the gene using the gene extracted from the sample as a template and develops it in ULFA. When the particles appear in the form of bands, it is possible to diagnose whether they are infected with Mycobacterium tuberculosis and/or Mycobacterium tuberculosis.

2. Tuberculosis diagnosis

(1) Reagent preparation: Preparation and conditions of PCR Master Mixture

Using the primers of Table 1, 5 μl of Primer mix per 10 μl of 2X PCR premix was mixed and PCR Master Mixture was prepared as needed (Table 2).

[Table 1]

[Table 2] PCR Master Mixture

15 μl of Master Mixture was dispensed into each PCR tube and mixed well by pipetting 5 μl of extracted sample DNA or positive control (included in the positive control-kit) or negative control (negative control-TE buffer or distilled water, not included in the kit). Spin-down (Spin-down) was put into a gene amplification device to react (Table 3).

[Table 3] PCR condition

(2) PCR result confirmation

When the PCR process was completed, the PCR tube was taken out from the PCR equipment, and then lightly spinned down. The ULFA device in the PaxView ULFA Kit was taken out as much as the reaction quantity and the developing solution was taken out together. 5 μl of PCR reaction solution was dropped into the square inlet at the bottom of the device, and 50 μl of the developing solution was slowly dropped immediately. After 10 minutes, 50 μl of the washing solution was slowly dropped. Results were read within 20 minutes of the start of the test.

(3) Result judgment

The sequence of the probe that binds to the PCR amplification product in the ULFA device is shown in Table 4, and the result of the band in which the reaction occurs is visually confirmed by visually confirming the result (refer to the examples in Tables 5 and 6).

[Table 4]

IC: internal control

HC: hybridization control

[Table 5]

[Table 6]

In Tables 5 and 6, in the case of Samples 5 to 7, multiple infections were present depending on the sample, and in Sample 8, one or more PCR amplification bands of TB and NTM tuberculosis bacteria were identified, but internal control (Internal control: IC) in which the PCR amplification band is not visible, which is due to the large amount of DNA and the PCR amplification amount. In this case, the PCR band of the internal control (Internal control) may be difficult to see, and the result is determined as positive for microorganisms of TB or NTM where PCR amplification is confirmed. In the case of Sample 10, the internal control was not amplified, which corresponds to the case where the sample has a PCR inhibitor, and DNA extraction must be performed again.

The specific parts of the present invention have been described in detail above. For those of ordinary skill in the art, this specific technique is only a preferred embodiment, whereby the scope of the present invention is not limited. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> PaxGenBio Co.,Ltd <120> Kit For Detecting Tuberculosis and Method of Detecting Tuberculosis Using the Same <130> 135 <150> KR 17/102,378 <151> 2017-08-11 <160> 17 <170> KopatentIn 2.0 <210> 1 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 gtaacaacgg ataactctaa ccagcaccta accggctgtg g 41 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 cataggagct tccgaccgct ccgac 25 <210> 3 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 aagtgtgacc tgaagatgtc ccgataggga atgctcggca acg 43 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 aattcgtcac ggtgcccaac atcga 25 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 tccaccagat accaagtctg ctggacatct accgcaagct gcg 43 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 cagcgggttg ttctggtcca tgaactg 27 <210> 7 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gtaagcaata gcaccaaacc tgaggagaag tctgccgtta ctgc 44 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 cactaaaggc accgagcact ttcttgc 27 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 9 tagagttatc cgttgttac 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 10 acatcttcag gtcacactt 19 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 11 gacttggtat ctggtgga 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 12 tttggtgcta ttgcttac 18 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 13 cattgtcagt tagccttag 19 <210> 14 <211> 317 <212> DNA <213> Artificial Sequence <220> <223> IS6110 <400> 14 accagcacct aaccggctgt gggtagcaga cctcacctat gtgtcgacct gggcagggtt 60 cgcctacgtg gcctttgtca ccgacgccta cgctcgcagg atcctgggct ggcgggtcgc 120 ttccacgatg gccacctcca tggtcctcga cgcgatcgag caagccatct ggacccgcca 180 acaagaaggc gtactcgacc tgaaagacgt tatccaccat acggataggg gatctcagta 240 cacatcgatc cggttcagcg agcggctcgc cgaggcaggc atccaaccgt cggtcggagc 300 ggtcggaagc tcctatg 317 <210> 15 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> mtp40 <400> 15 cccgataggg aatgctcggc aacgcgccgt cggtggttcc caacaccacg ttagggatgc 60 actgcggcag cttcggcagc gctcccagca acgggtggct caagttgggt ctggtcgaat 120 tcggtggagt cgcaaagttg aacgctgagg tcatgtcgcc aaccacgccg tcgcgccagg 180 cggtcatgtt gggaaccggc acgccgaacc gggcgcgaat caacttcaat tgcgaggtgt 240 ggtcgaacgt gtcggagacc atcagcgggc cgcggctgta cggcgaaatg acaatgcagg 300 gaacgcgaaa acccagaccg agcggaccac gaatgccacc ggacccgggt actgcgtcga 360 tgttgggcac cgtgacgaat t 381 <210> 16 <211> 524 <212> DNA <213> Artificial Sequence <220> <223> rpoB <400> 16 tgctggacat ctaccgcaag ctgcgcccgg gcgagccgcc gaccaaggag tcggcgcagg 60 ccctgctgga gaacctgttc ttcaaggaga agcgttacga cctggcccgc gtgggtcggt 120 acaaggtgaa caagaagctg ggcctgggcg gcaccaatcc ggctcaggtg accaccacca 180 ccctcaccga ggaagacgtc gtcgccacca tcgagtacct ggtgcgcctg cacgagggcc 240 agaccacgat gaccgccccc ggtggcgtcg aggtgccggt ggatgtggac gacatcgacc 300 acttcggtaa ccgtcgcctg cgtaccgtcg gcgagctgat tcagaaccag atccgggtcg 360 gcctgtcccg tatggagcgc gtcgtgcgtg agcgcatgac cacgcaggac gtcgaggcga 420 tcaccccgca gaccctgatc aacatccgtc ccgtcgtggc ggcgatcaag gagttcttcg 480 gaaccagcca gctgtcgcag ttcatggacc agaacaaccc gctg 524 <210> 17 <211> 335 <212> DNA <213> Artificial Sequence <220> <223> Internal Control <400> 17 cctgaggaga agtctgccgt tactgccctg tggggcaagg tgaacgtgga tgaagttggt 60 ggtgaggccc tgggcaggtt ggtatcaagg ttacaagaca ggtttaagga gaccaataga 120 aactgggcat gtggagacag agaagactct tgggtttctg ataggcactg actctctctg 180 cctattggtc tattttccca cccttaggct gctggtggtc tacccttgga cccagaggtt 240 ctttgagtcc tttggggatc tgtccactcc tgatgctgtt atgggcaacc ctaaggtgaa 300 ggctcatggc aagaaagtgc tcggtgcctt tagtg 335

Claims (18)

(a) (i) a primer of SEQ ID NO: 1 or 2, comprising a nucleic acid oligomer containing a complementary binding site binding to the Mycobacterium tuberculosis specific gene IS6110 and a base complementary to the tuberculosis specific gene;
(ii) a primer of SEQ ID NO: 3 or 4, comprising a nucleic acid oligomer containing a complementary binding site binding to the Mycobacterium tuberculosis specific gene mtp40 and a base complementary to the Mycobacterium tuberculosis specific gene;
(iii) SEQ ID NO: 5 or 6 comprising a complementary binding site that binds to the rpoB gene commonly present in mycobacteria and a nucleic acid oligomer containing a non-complementary base and a gene common to mycobacteria. primer; And
(b) A kit for simultaneous diagnosis of tuberculosis and non-tuberculosis antibacterial bacterium comprising a membrane fixed with a probe selected from the group consisting of SEQ ID NOs: 9 to 11 that complementarily binds the nucleic acid oligomer.
delete delete delete delete The kit according to claim 1, comprising a non-nucleotide spacer between the complementary binding site and a nucleic acid oligomer containing a non-complementary base.
delete The kit according to claim 1, further comprising a marker.
delete The kit according to claim 1, wherein when the product amplified by the primer is injected into the side of the membrane, the amplified product moves while the probe fixed to the membrane and the nucleic acid oligomer contained in the amplified product undergo a complementary hybridization reaction. .
delete The kit of claim 1, wherein the probe further comprises a nucleic acid oligomer for immobilization on a membrane.
The kit of claim 12, wherein the additional nucleic acid oligomer comprises the sequence of SEQ ID NO: 12 or 13.
The method of claim 8,
The markers are biotin, Cy5, Cy3, FITC, EDANS (5-(2'-aminoethyl)amino-1-naphthalene sulfate), tetramethylrodamine (TMR), tetramethylrodamine isocyanate (TMRITC), x-loader Kit characterized in that it is a min, DIG or antibody or nanoparticles bound thereto.
The kit according to claim 8, wherein color development is induced by the reaction between the marker and the binder.
16. The kit of claim 15, wherein the binding agent is streptavidin.
A nucleic acid extracted from a sample comprising (i) a complementary binding site that binds Mycobacterium tuberculosis specific gene IS6110 and a nucleic acid oligomer containing a non-complementary base with a tuberculosis specific gene, SEQ ID NO: 1 or 2 primer;
(ii) a primer of SEQ ID NO: 3 or 4, comprising a nucleic acid oligomer containing a complementary binding site binding to the Mycobacterium tuberculosis specific gene mtp40 and a base complementary to the Mycobacterium tuberculosis specific gene;
(iii) SEQ ID NO: 5 comprising a complementary binding site that specifically binds gene rpoB , which is common to mycobacteria, and a nucleic acid oligomer, which contains a gene that is common to mycobacteria and a non-complementary base. Or amplifying with a primer of 6; And
Claims 1, 6, 8, 8 comprising the step of loading a probe selected from the group consisting of SEQ ID NOs: 9 to 11 complementarily binding the amplification product to the nucleic acid oligomer to a fixed membrane. A method for simultaneously detecting tuberculosis and non-tuberculosis antibacterialosis in vitro using the kit according to any one of claims 10 and 12 to 16.
The method of claim 17, wherein the sample is sputum, culture, tissue or bronchial wash.

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