KR20190017698A - Kit For Detecting Tuberculosis and Method of Detecting Tuberculosis Using the Same - Google Patents

Abstract

The present invention relates to a kit for diagnosing tuberculosis and a method for detecting tuberculosis by using the same and, specifically, to a kit for simultaneously diagnosing tuberculosis and mycobacteria, which comprises: (a) (i) a primer capable of simultaneously and specifically amplifying a Mycobacterium tuberculosis gene and a gene commonly present in mycobacteria; and (b) a probe capable of identifying the amplified product, and to a method for simultaneously diagnosing tuberculosis and nontuberculous mycobacteriosis by using the kit.

Description

TECHNICAL FIELD The present invention relates to a kit for diagnosing tuberculosis and a method of diagnosing tuberculosis using the kit,

The present invention relates to a kit for diagnosing tuberculosis and a method for detecting tuberculosis using the same, and more particularly, to a tuberculosis diagnostic kit comprising (a) (i) specifically amplifying Mycobacterium tuberculosis- specific genes and genes commonly present in mycobacteria (B) a kit for simultaneously diagnosing Mycobacterium tuberculosis and non-tuberculosis mycobacteria including a probe capable of confirming the amplified product, and a method for simultaneously diagnosing Mycobacterium tuberculosis and non-tuberculosis mycobacteria using the same will be.

Tuberculosis is not only found in the lungs but also in the lymphatic system, kidneys, vertebrae, peritoneum, intestines and skin. It is a disease caused by Mycobacterium tuberculosis. Symptoms vary with fever, fatigue, and weight loss. Even today, many medical technologies are developing, Mycobacterium tuberculosis infections are frequent. In Korea, the incidence of tuberculosis is the highest among OECD countries.

About two-thirds of the world's population, estimated to be infected with tuberculosis, is estimated to be infected with 9 million new tuberculosis patients each year, and 3 million TB people die from tuberculosis (TB). According to the statistical data of 2006, there are 35,000 patients diagnosed as tuberculosis in 242 public health centers and civilian diseases / clinics nationwide, and 67.2 per 100,000 population, The number of deaths in 2006 was 2,948, with 6.7 deaths per 100,000 population, accounting for 55.7% of all deaths due to infectious diseases, making it one of the top 30 OECD member economies. It is a serious disease. Tuberculosis, which has been regarded as a disease of the backward country, has emerged as a global health problem in recent years, largely due to the possibility of TB infection in patients with acquired immunodeficiency syndrome (ADIS) and the frequent exchange of international society by globalization of the global community. .

In order to prevent the spread of tuberculosis, various approaches such as early diagnosis, appropriate antibiotic administration, isolation of patients, and regular screening for the groups with a high possibility of exposure to tuberculosis are needed. In particular, early and rapid diagnosis of HIV infection prevent tuberculosis death The most common method for the diagnosis of tuberculosis is X-ray chest radiography and acid-fast bacilli smear test, but the accuracy is 50-75% Which is very low and has a problem of sensitivity and reliability. Atypical mycobacterial pulmonary disease is similar to tuberculosis symptoms such as coughing and sputum, but it is not transmitted to other people and treatment method is totally different. In the USA and Europe, Of the patients with tuberculosis who were infected with non-tuberculous mycobacterium were diagnosed as tuberculosis because they were classified as rare. In addition, some patients suffer from economic and psychological injuries, such as drug-resistant tuberculosis, which is not treated as the first-line drug, and have received a second anti-tuberculosis drug for several years. Therefore, additional tests such as nucleic acid amplification test or culture test are necessary even if the primary sputum smear test is positive. However, in case of culture test, it takes 4 ~ 8 weeks to cultivate tuberculosis, Considering this fact, diagnosis of AFB (acid-fast bacillus) smear test and nucleic acid amplification test is the most ideal for early screening of tuberculosis, and this trend is being advanced in developed countries including USA.

Early diagnosis of infectious disease is a very important factor in the treatment of the disease because tuberculosis has problems such as side effects and infection. Therefore, various diagnostic techniques have been actively developed and applied for early diagnosis of tuberculosis.

With regard to the diagnosis of tuberculosis, an AFB staining test or a culture test may be considered. The AFB staining method can detect rapid results with a simple method, but it has a low sensitivity and it is impossible to distinguish between mycobacteria and non - tuberculous mycobacteria. The culture can be detected with a very small amount of Mycobacterium tuberculosis and the specificity is high. Immunodiagnostic methods such as ELISA and rapid method (Rapid method) are used in addition to simple and convenient method, but it is somewhat difficult to detect a very small amount of Mycobacterium tuberculosis.

Recently, polymerase chain reaction (PCR) such as PCR method has been used for the diagnosis of tubercle bacilli, and PCR-electrophoresis or real-time PCR is considered. PCR method is fast and sensitive to detect a small amount of Mycobacterium tuberculosis. PCR-electrophoresis, however, requires agarose gel preparation to confirm the amplified gene as in conventional PCR, and real-time PCR requires expensive equipment There is a disadvantage that economical efficiency is low.

Under these technical backgrounds, the inventors of the present application can simultaneously diagnose tuberculosis rapidly and accurately by diagnosing the genes for Mycobacterium tuberculosis and mycobacteria, and can diagnose tuberculosis by distinguishing it from non-tuberculous acid bacteria by only a small amount of sample gene Kits, and a method for simultaneous diagnosis of tuberculosis and non-tuberculosis antibiotics using the kit. The present invention has been completed based on this finding. In addition, by using two genes used for diagnosis of Mycobacterium tuberculosis, that is, IS6110 and mtp40 simultaneously, it was confirmed that even if one gene of two genes is deficient, it is possible to provide a diagnostic method that can accurately diagnose Mycobacterium tuberculosis. Completed.

The above-described information described in the background section is for improving the understanding of the background of the present invention only, and it is not intended to include information on the prior art already known to those skilled in the art .

It is an object of the present invention to provide a kit for simultaneous diagnosis of tuberculosis and non-tuberculous mycorrhizal fungi which can accurately and simply diagnose tuberculosis differently from non-tuberculous acid bacteria, And to provide a method for simultaneously diagnosing tuberculosis and non-TB disease.

(A) a nucleic acid oligomer comprising (i) a complementary binding site specifically binding to Mycobacterium tuberculosis-specific gene and a nucleic acid oligomer containing an M. tuberculosis-specific gene and a non-complementary base Primer; (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 commonly present in mycobacteria and an uncharacterized base; And (b) a kit for simultaneous diagnosis of tuberculosis and non-tuberculous mycobacteria comprising a membrane in which a probe that binds complementarily to the nucleic acid oligomer is immobilized.

The present invention also relates to a method for producing a nucleic acid comprising the steps of: (i) introducing a nucleic acid extracted from a specimen into a cell, which comprises (i) a nucleic acid oligomer containing a complementary binding site specifically binding to Mycobacterium tuberculosis- 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 commonly present in mycobacteria and a non-complementary base Amplifying; And a step of loading the amplification product into a fixed membrane that binds the probe complementarily binding the nucleic acid oligomer to the tubing and non-tuberculosis in vitro using the kit according to any one of claims 1 to 16. The present invention relates to a method for simultaneously diagnosing an autoimmune disease.

The present invention further relates to a method for producing a nucleic acid comprising the steps of: (i) introducing a nucleic acid extracted from a sample into a sample containing (i) a nucleic acid oligomer containing a complementary binding site specifically binding to Mycobacterium tuberculosis- 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 commonly present in mycobacteria and a non-complementary base Amplifying; And

The kit according to any one of claims 1 to 16, comprising a step of loading the amplification product with a probe that binds complementarily to the nucleic acid oligomer on a fixed membrane, The present invention relates to a method for providing information for simultaneous diagnosis of mycobacteria.

The kit according to the present invention makes it possible to diagnose tuberculosis with only a small amount of sample genes and diagnose by simultaneously amplifying the genes common to the Mycobacterium tuberculosis-specific gene and the mycobacteria to thereby diagnose the tubercle bacillus and the non- Make the diagnosis possible. In addition, since the results are confirmed through a lateral flow assay after gene amplification, expensive equipment such as real-time PCR is not required, and an agarose gel for electrophoresis to identify amplified genes as in conventional PCR Because the manufacturing process is not required, results can be confirmed in a shorter time than the PCR-electrophoresis method.

FIG. 1 is a diagram showing a process of selecting a target region for a genome of a tuberculosis causative organism and a primer design process.
2 is a schematic diagram of a strip for identifying an amplification product in a kit according to an embodiment of the present invention.
FIG. 3 is a schematic diagram of a reaction in a membrane to which a probe complementary to a nucleic acid oligomer contained in a kit according to an embodiment of the present invention is immobilized.
Fig. 4 shows the change in coloring position depending on the presence or absence of a tuberculosis causative gene.
FIG. 5 shows test results according to the negative or positive criteria for tuberculosis and non-TB.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Polymerase Chain Reaction (PCR) of molecular diagnostic products can be categorized into two types. One is the conventional PCR, which extracts the nucleic acid from the specimen, amplifies the target nucleic acid, and confirms the result through electrophoresis. If you use this method, you can buy an inexpensive kit, but the electrophoresis takes a lot of labor and time. Real-time PCR (Real-time PCR), which is used instead of electrophoresis, can monitor nucleic acid amplification of pathogens in real time, but the kit is expensive and it is difficult to confirm the result of amplification of more than 5 multiple nucleic acids.

Accordingly, it is required to develop a low-cost molecular diagnostic product while maintaining the convenience of the user. Therefore, the inventors of the present application have developed an in vitro diagnostic kit for qualitative analysis.

(A) a primer comprising (i) a complementary binding site which binds to a Mycobacterium tuberculosis- specific gene, and (ii) a nucleic acid oligomer containing a non-complementary base and a M. tuberculosis-specific gene; (ii) a primer comprising a complementary binding site which binds to a gene commonly present in mycobacteria, and a nucleic acid oligomer containing a gene and a non-complementary base which are common to mycobacteria; And (b) a membrane in which a probe that binds complementarily to the nucleic acid oligomer is immobilized, and a kit for simultaneous diagnosis of tuberculosis and non-TB disease.

The kit according to the present invention comprises a complementary binding site and a primer comprising a nucleic acid oligomer, wherein the primer comprises a complementary binding site specifically binding to a M. tuberculosis-specific gene and a gene commonly present in mycobacteria , A nucleic acid oligomer containing the M. tuberculosis-specific gene and a gene commonly present in the mycobacteria and a non-complementary base. According to the present invention, it is possible to simultaneously diagnose the infection of Mycobacterium tuberculosis and non-tuberculous mycobacteria.

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

In the present specification, non-tuberculous acute acidosis is not a tubercle bacillus, but a mycobacteria other than mycobacterium tuberculosis, which is similar to the tuberculosis bacteria, may remain in the bronchi and may cause symptoms such as cough and sputum and various inflammation in the lung. The gene commonly present in the mycobacteria may be one or more genes specifically present in mycobacteria including Mycobacterium tuberculosis, for example, rpoB (SEQ ID NO: 16).

In a specific example, the kit according to the present invention is used to simultaneously amplify the two genes IS6110 and mtp40 present in Mycobacterium tuberculosis and the rpoB gene commonly present in mycobacteria including Mycobacterium bacillus to simultaneously isolate Mycobacterium tuberculosis and non-tuberculous mycobacteria . As a result of diagnosis, tuberculosis can be diagnosed by amplification of one or more of mtp40 and mtp40 and the rpoB gene. If only rpoB gene is amplified, it can be determined as non-tuberculous bacillus. According to the present invention, it is possible to accurately diagnose the presence or absence of tuberculosis even in a mycobacteria lacking one gene.

As used herein, the term "primer" refers to a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerization enzymes) within a suitable temperature and buffer .

The primer according to the present invention may include a Mycobacterium tuberculosis-specific gene or a fragment of a gene commonly present in mycobacteria. The fragment may refer to any part of the M. tuberculosis-specific gene and the gene commonly present in the mycobacteria, and the fragment may have any length, for example, IS6110 and / or mtp40 and the rpoB gene, May refer to a region having a length of 5 bp to 50 bp, particularly 10 bp to 30 bp, for any site in the sequence.

As used herein, 'complementary binding' means that a primer hybridizes with a corresponding nucleic acid strand under the conditions of performing a polymerization reaction, and can form a duplex structure. Complementarity can also be formed if the complementarity between the paired nucleotide sequences results in a Watson-Crick pair or in the presence of some non-Watson-Crick base pair.

The primer may be a forward primer, for example, a complementary binding site that specifically binds to the M. tuberculosis-specific genes IS6110 and / or mtp40 and the rpoB gene, which is a gene commonly present in mycobacteria. The forward primer is IS6110, and / or if the mtp40 and M. gene of the rpoB gene template that exists in common to bacterial this by binding to complementary strand IS6110 and / or the rpoB gene mtp40 and the gene present in common to mycobacterial And the complementary sequence can be specifically amplified. For example, the primer can be selected from the group consisting of SEQ ID NOS: 1 to 6.

The primer includes a nucleic acid oligomer containing a Mycobacterium tuberculosis-specific gene and a non-tuberculous antibiotic gene or fragment thereof and a non-complementary base. Using PCR target gene of IS6110 and / or mtp40 and Mycoplasma on bacteria upon amplification of gene rpoB gene present in common IS6110 and / or mtp40 the present gene rpoB gene and the emergency beam of the base to be common to mycobacterial A gene which is not related to the rpoB gene, which is a gene commonly present in mtp40 and mycobacteria, is about 20-60 bp, specifically about 20-40 bp, preferably about 20-30 bp . ≪ / RTI > By reacting such a nucleic acid oligomer with a probe complementarily synthesized to the membrane, it is possible to easily confirm whether the resultant, that is, the nucleic acid amplification is amplified. The nucleic acid oligomer may be selected from the group consisting of SEQ ID NOS: 9 to 11, for example.

Such a nucleic acid oligomer binds to a probe complementarily synthesized in the membrane with a non-envelope base moiety and a fragment of a Mycobacterium tuberculosis-specific gene and a non-TB nucleic acid, or a fragment thereof, in order to react with a probe complementarily synthesized in the membrane And a non-nucleotide spacer between the bases. The non-nucleotide spacer may be a C3, C6, C9 or C12 aliphatic spacer and the number of C means the number of carbon atoms in the non-nucleotide spacer structure. Such a non-nucleotide spacer may be an alkyl, an alkenyl, or an acinyl group.

In one embodiment, a non-nucleotide spacer is provided between the M. tuberculosis-specific gene and the non-tuberculous antibiotic gene or a fragment thereof and a base for binding a non-complementary base moiety and a probe complementarily synthesized to the membrane, When the gene is amplified using Taq polymerase by positioning C3 spacer (profile spacer), complementary amplification can be prevented by invading the base part for binding with the probe so as to obtain a partial single strand nucleic acid product And this portion was allowed to bind the probe to the membrane.

The primer may further comprise a primer comprising a marker. The primer comprising the marker may be a reverse primer, and the reverse primer may be used to amplify a template by binding to the template strand when the reverse primer is a template containing the gene rpoB , which is a gene commonly present in IS6110 and / or mtp40 and mycobacteria, The primers may be selected from the group consisting of SEQ ID NOS: 1-6.

In one embodiment, the primers included in the primers include but are not limited to biotin, Cy5, Cy3, FITC, EDANS (5- (2'- aminoethyl) amino-1-naphthalene sulfate), tetramethylrhodamine But are not limited to, rhodamine isocyanate (TMRITC), x-rhodamine, DIG or an antibody or nanoparticles associated therewith.

Here, it is possible to react with a binding agent that induces color development of the marker, thereby detecting the color development or fluorescence expression and detecting the amplification of the target nucleic acid. At this time, the binding agent may be streptavidin, but is not limited thereto.

In a specific embodiment, biotin is attached to the reverse primer to amplify and bind to Streptavidine in the membrane. Particles conjugated to biotin, for example, nanoparticles, In the case of fluorescence, it can be confirmed by fluorescence of various wavelengths, and it is possible to distinguish whether amplification of the target nucleic acid is performed from several to dozens depending on the type of probe attached to the membrane.

The kit according to the present invention comprises (b) a membrane to which a probe that binds complementarily to the nucleic acid oligomer is immobilized. Accordingly, when the product amplified by the primer is injected into the side of the membrane, the amplified product moves, and the probe immobilized on the membrane and the nucleic acid oligomer contained in the amplified product can perform a complementary hybridization reaction.

The primers according to the present invention can be carried out by multiplex PCR, which makes it possible to simultaneously diagnose Mycobacterium tuberculosis and various kinds of non-tuberculous mycobacteria. That is, if multiple PCR reactions are carried out using probes with several to several tens of different oligomer sequences and then reacted with oligomer probes to which complementary probes can be reacted, Several hundreds of amplified products can be identified with the lateral flow membrane.

Therefore, it can be used in a variety of multiply-packed eggs to enable mass production in the production of side flow membranes. This confirms the results of various items using one side flow membrane. By using the same membrane to confirm amplification products irrespective of PCR amplification products, cost reduction and quality control Thereby making it possible to increase the productivity of the product.

The probe that binds complementarily 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.

Optionally, the probe may further comprise a nucleic acid oligomer for immobilization on the membrane, and may further comprise an oligomer of 20-60 bp, particularly 20-40 bp, having a repeated base sequence. Wherein the additional nucleic acid oligomer may comprise, for example, the sequence of SEQ ID NO: 13.

Optionally, the kit according to the invention may optionally further comprise an internal control primer. These internal control primers are used to confirm whether a false negative, that is, a PCR reaction is properly performed, and genes normally expressed can be arbitrarily selected regardless of the presence of mycobacteria or mycobacteria in the sample. In a specific embodiment according to the present invention, the internal control primer may comprise a sequence of SEQ ID NO: 7 or 8, for example a beta globin gene (SEQ ID NO: 17).

The kit according to the present invention may optionally contain a reagent 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 further comprise various polynucleotide molecules, various buffers and reagents.

The optimum amount of the reagent, buffer or reagent used for the specific reaction in the kit can be determined by those skilled in the art, and can be determined by a combination of the aforementioned Mycobacteria-specific gene and complementary binding specifically binding to a gene commonly present in mycobacteria (Reverse direction) comprising a primer (forward direction) and / or (ii) a marker comprising a nucleic acid oligomer containing a nucleotide sequence complementary to the nucleotide sequence of the M. tuberculosis-specific gene and a gene commonly present in the M. tuberculosis- And the membranes on which the probes are immobilized, may be fabricated in separate packages or compartments, each containing a separate package or compartment.

In another aspect, the present invention provides a primer comprising (i) a primer comprising a complementary binding site that specifically binds to a Mycobacterium tuberculosis gene, and a nucleic acid oligomer containing an M. tuberculosis-specific gene and an unacceptable base, and (ii) Amplifying with a primer comprising a complementary binding site that specifically binds to a gene commonly present in a mycobacteria and a nucleic acid oligomer that contains a gene commonly present in mycobacteria and an uncharacterized base; And a method for simultaneously diagnosing tuberculosis and non-tuberculosis acute mycobacteria in vitro using the kit, comprising loading the amplification product with a probe, which binds complementarily to the nucleic acid oligomer, on a fixed membrane.

(I) a primer comprising a complementary binding site that specifically binds to a Mycobacterium tuberculosis gene, and a nucleic acid oligomer containing an M. tuberculosis-specific gene and a non-complementary base; and (ii) amplifying with a primer comprising a complementary binding site specifically binding to a gene commonly present in mycobacteria, and a nucleic acid oligomer containing a non-TBase antibiotic gene and an unreacted base; And loading the amplified product with a probe that binds complementarily to the nucleic acid oligomer on a fixed membrane. The present invention also relates to a method for providing information for simultaneous diagnosis of tuberculosis and non-TB disease.

The description of the configuration relating to the kit used in carrying out the method according to the present invention can be equally applied to the method.

In one embodiment, the specimen may comprise a wide range of all biological fluids obtained from body fluids, cell lines, tissue cultures, etc., depending on the diagnosis of tuberculosis and non-TB disease, But are not limited to, sputum, cultured specimens, tissue specimens, or bronchial washing solutions.

The step of extracting the nucleic acid from the specimen may further include the step of extracting the nucleic acid, for example, by using various kits or extracting reagents which are commercially available.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

1. Production of kit

The kit according to the present invention (hereinafter also referred to as PaxView TB / NTM MPCR-ULFA Kit) comprises two kits. One is a kit for amplifying nucleic acid (hereinafter referred to as PaxView TB / NTM MPCR Kit), and the other is a kit for confirming amplification products (hereinafter referred to as PaxView ULFA Kit). The primers contained in the primer mixture of the PaxView TB / NTM MPCR Kit include three regions for disease causative bacteria and regions for internal control, and a forward primer (SN) has a universal region (non-complementary nucleic acid oligomer) ), And a reverse primer (reverse primer = ASN) is tagged with a biotin sequence (Fig. 1).

After PCR, the amplified product is loaded on a ULFA device (e.g., strip: Figure 2) of the PaxView ULFA Kit to generate a gold-streptavidin conjugate (Gold-SV, (probe = universal region complementary to oligo) adsorbed on the NC membrane after binding with the probe (present in the pad) (FIG. 3).

The principle of color development is based on the reaction between biotin and streptavidin tagged on each primer, and can be used for confirmation of DNA reaction as well as antigen-antibody reaction which is widely used. Streptavidin, similar to avidin, can form a complex with biotin, resulting in visible readability by gold particles bound to biotin.

Visualize the ULFA device after color development. The negative / positive result is judged according to the result of the determination described in the use method of the kit, and it can be confirmed that the expected band pattern matches the actual band pattern because the position of the color line is changed depending on the presence or absence of disease causative bacteria (FIG.

The tuberculosis diagnostic kit (TB / NTM diagnostic kit) according to the present invention is a new tuberculosis diagnostic platform, which amplifies a gene using a gene extracted from a sample as a rapid and accurate qualitative molecular diagnostic kit, If the particles appear in the form of bands, they can be diagnosed as having TB and / or non-tuberculous antibiotics.

2. Diagnosis of tuberculosis

(1) Preparation of reagents: Preparation and conditions of PCR Master Mixture

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

[Table 1]

 [Table 2] PCR Master Mixture

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

[Table 3] PCR condition

(2) Confirmation of PCR result

Upon completion of the PCR procedure, the PCR tubes were removed from the PCR instrument and lightly spun down. The ULFA device in the PaxView ULFA Kit was withdrawn as much as the reaction quantity, and the solution was taken out together. 5 μl of the PCR reaction solution was dropped onto the square injection port at the bottom of the device, and immediately 50 μl of the developing solution was slowly dropped. After 10 minutes, 50 μl of wash solution was slowly dropped. The results were read within 20 minutes after the start of the test.

(3) Judgment of results

      The sequence of the probe binding to the PCR amplification product in the ULFA device is shown in Table 4, and the results of the bands in which the reaction occurs are visually confirmed by combining with the result, and the positive result is determined (see the examples of Tables 5 and 6).

[Table 4]

IC: internal control

HC: hybridization control

[Table 5]

[Table 6]

In Table 5 and Table 6, samples 5 to 7 show multiple infections depending on the specimen. In sample 8, one or more PCR amplification bands of TB and NTM of Mycobacterium tuberculosis were confirmed, but internal control IC) of the PCR amplification band is invisible. In this case, the PCR band of the internal control may not be visible. Therefore, the PCR amplification is judged to be positive for TB or NTM microorganisms. In case of sample 10, the internal control is not amplified. If the PCR inhibitor is present in the sample, DNA extraction should be repeated.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual 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 tattttcccc cccttaggct gctggtggtc tacccttgga cccagaggtt 240 ctttgagtcc tttggggatc tgtccactcc tgatgctgtt atgggcaacc ctaaggtgaa 300 ggctcatggc aagaaagtgc tcggtgcctt tagtg 335

Claims (18)

(a) (i) Mycobacterium tuberculosis (Mycobacterium tuberculosis) primer containing a nucleic acid oligomers containing complementary binding site, and M. tuberculosis-specific genes and emergency beam nucleotide binding to the specific gene;
(ii) a primer comprising a complementary binding site which binds to a gene commonly present in mycobacteria, and a nucleic acid oligomer containing a gene and a non-complementary base which are common to mycobacteria; And
(b) a kit for simultaneous diagnosis of tuberculosis and non-tuberculous mycobacteria comprising a membrane in which a probe that binds complementarily to the nucleic acid oligomer is immobilized.
The kit according to claim 1, wherein the kit simultaneously diagnoses two or more Mycobacterium tuberculosis-specific genes.
The kit according to claim 1, wherein the M. tuberculosis-specific gene is IS6110 or mtp40.
The kit according to claim 1, wherein the gene commonly present in the mycobacteria is rpoB .
The kit according to claim 1, wherein the nucleic acid oligomer has a length of 20-60 bp.
The kit according to claim 1, comprising a non-nucleotide spacer between the complementary binding site and a nucleic acid oligomer containing an unreacted base.
The kit according to claim 1, wherein the primer is selected from the group consisting of SEQ ID NOS: 1-6.
The kit of claim 1, further comprising a primer comprising a marker.
8. The kit according to claim 7, wherein the primer is SEQ ID NO: 7 or 8.
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 and the probe immobilized on the membrane and the nucleic acid oligomer contained in the amplified product undergo a complementary hybridization reaction .
The kit according to claim 1, wherein the probe that binds complementarily to the nucleic acid oligomer comprises one or more sequences selected from the group consisting of SEQ ID NOS: 9 to 11.
2. The kit of claim 1, wherein the probe further comprises a nucleic acid oligomer for immobilization to the membrane.
13. The kit of claim 12, wherein the additional nucleic acid oligomer comprises the sequence of SEQ ID NO:
9. The method of claim 8,
The markers include biotin, Cy5, Cy3, FITC, EDANS (5- (2'-aminoethyl) amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC) DIG, or an antibody, or nanoparticles bonded thereto.
9. The kit according to claim 8, wherein color development is induced by the reaction of the marker and the binder.
16. The kit of claim 15, wherein the binding agent is streptavidin.
The nucleic acids extracted from the sample (i) Mycobacterium tuberculosis (Mycobacterium tuberculosis) primer containing a nucleic acid oligomers containing complementary binding site, and M. tuberculosis-specific genes and emergency beam nucleotide binding to the specific gene, and (ii) mycobacteria ( a primer comprising a complementary binding site specifically binding to a gene commonly present in the mycobacteria and a nucleic acid oligomer containing a gene common to mycobacteria and a non-complementary base; And
The kit according to any one of claims 1 to 16, comprising a step of loading the amplification product with a probe that binds complementarily to the nucleic acid oligomer on a fixed membrane, How to diagnose mycosis simultaneously.
18. The method of claim 17, wherein the sample is sputum, culture, tissue, or bronchial wash.

Images