CN111344419A - Kit for tuberculosis diagnosis and method for diagnosing tuberculosis by using the same - Google Patents
Kit for tuberculosis diagnosis and method for diagnosing tuberculosis by using the same Download PDFInfo
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Abstract
The present invention relates to a kit for tuberculosis diagnosis and a method for detecting tuberculosis using the same, and particularly, to a kit for simultaneously diagnosing mycobacterium tuberculosis and nontuberculous mycobacteria, the kit comprising: (a) primers capable of simultaneously and specifically amplifying a specific gene of Mycobacterium tuberculosis and a gene commonly present in Mycobacterium; and (b) a probe capable of identifying the amplified product; and to a method for simultaneously diagnosing Mycobacterium tuberculosis and nontuberculous mycobacteria using the kit.
Description
[ technical field ] A method for producing a semiconductor device
The present invention relates to a kit for diagnosis of tuberculosis and a method for detecting tuberculosis using the same, and more particularly, to a kit for simultaneously diagnosing mycobacterium tuberculosis (mycobacterium tuberculosis) and mycobacterium non-tuberculosis (mycobacterium), the kit comprising: (a) (i) primers capable of simultaneously and specifically amplifying a specific gene of Mycobacterium tuberculosis and a common gene of Mycobacterium; and (b) a probe capable of identifying the amplified product; and a method for simultaneously diagnosing Mycobacterium tuberculosis and non-tuberculosis mycobacteria using the kit.
[ background of the invention ]
Tuberculosis afflicts not only the lungs, but also the lymph glands, kidneys, spine, peritoneum, intestines and skin. Tuberculosis is a disease caused by mycobacterium tuberculosis, and shows various symptoms such as fever, fatigue, and weight loss. Despite advances in various medical technologies, nowadays, infection caused by mycobacterium tuberculosis still frequently occurs, and particularly, korea has the highest annual incidence of tuberculosis in OECD countries.
It is estimated that about 20 million people corresponding to one third of the world's population are infected with mycobacterium tuberculosis, and it is reported that 900 million people are newly diagnosed with tuberculosis every year, and 300 million people die from Tuberculosis (TB). In korea, the statistical data in 2006 shows that the number of patients newly diagnosed with tuberculosis and receiving treatment in 242 health centers and private hospitals and clinics across the country is 35,000, that is, 67.2 patients per 10 ten thousand. Meanwhile, according to the statistical data of the causes of death from the national statistical agency (national statistical Office), 2,948 people died of tuberculosis in 2006, which means that the mortality rate of tuberculosis reaches 6.7 out of every 100,000 people, accounting for 55.7% of all deaths caused by infectious diseases, and thus tuberculosis is an extremely serious disease that is the first place among 30 OECD member countries in korea. Why tuberculosis, which has been regarded as a disease occurring in undeveloped countries, has recently emerged as a global health problem, mainly because mycobacterium tuberculosis causes opportunistic infections in patients with Acquired Immune Deficiency Syndrome (AIDS), and countries frequently make contact with each other in international society due to globalization.
To prevent the spread of tuberculosis, various methods are required, including early diagnosis, administration of appropriate antibiotics, isolation of patients, periodic screening of populations with high levels of tuberculosis exposure, etc., especially early and rapid diagnosis of HIV-infected patients, which is extremely important in preventing death due to tuberculosis. The most common methods for diagnosing tuberculosis include X-ray chest examination and acid-fast bacillus smear, but cannot distinguish between mycobacterium tuberculosis and nontuberculous mycobacteria, and thus the accuracy of diagnosis is low, i.e., 50% to 75%, resulting in sensitivity and reliability problems. Lung disease caused by non-tubercular mycobacteria exhibits symptoms and findings similar to tuberculosis, such as cough and sputum, but does not spread to others and has a completely different treatment than tuberculosis, and tuberculosis is ultimately determined by additional screening in the us and europe, even if the initial sputum smear test is positive. However, korea is classified as non-tubercular mycobacteria as a rare area, and thus patients infected with non-tubercular mycobacteria may be erroneously diagnosed as having tuberculosis and receive tuberculosis treatment due to negligence in secondary examination of the non-tubercular mycobacteria, resulting in adverse circumstances such as restriction due to infectivity, and the like. Also in an increasing number of cases, some patients are incorrectly diagnosed as drug-resistant tuberculosis, and drug-resistant combined diseases cannot be cured by primary drugs, so that taking secondary anti-tuberculosis drugs for many years causes economic and mental damage. Therefore, even if the primary sputum smear test is positive, additional tests such as a nucleic acid amplification test or a culture test are required, but in the case of the culture test, it takes 4 to 8 weeks to culture only Mycobacterium tuberculosis, and thus it is difficult to perform rapid diagnosis and appropriate early treatment for Mycobacterium tuberculosis. In view of this, for early screening of tuberculosis, it is desirable that a combined examination of an Acid Fast Bacilli (AFB) smear and a nucleic acid amplification test is performed, and it is being certified in developed countries including the united states.
Since tuberculosis has problems such as side effects and easy spread of therapy against tuberculosis, early diagnosis of infectious bacteria is an extremely important factor in treating diseases. Therefore, various diagnostic techniques are being actively developed and applied to early diagnosis of tuberculosis.
For said diagnosis of tuberculosis, smear microscopy using acid-fast bacilli or culture tests may be considered. The smear microscopy method for detecting acid-fast bacilli is a simple method that can quickly obtain the result of identification, but its sensitivity is low, and it cannot distinguish between Mycobacterium tuberculosis and nontuberculous mycobacteria. The culture test can detect even trace amounts of Mycobacterium tuberculosis and has high specificity, but requires a long time to obtain results. In addition, immunodiagnostic methods such as simple ELISA or rapid methods can be used, but it is difficult to detect trace amounts of mycobacterium tuberculosis.
Recently, Polymerase Chain Reaction (PCR) has been used for diagnosis of Mycobacterium tuberculosis, and PCR-electrophoresis or real-time PCR is also considered. The PCR method is rapid and highly sensitive, and thus can detect even trace amounts of Mycobacterium tuberculosis, but the PCR electrophoresis method must require an agarose gel preparation to identify amplified genes like conventional PCR, and real-time PCR requires expensive equipment, and thus is economically inefficient.
Under such a technical background, the inventors of the present application confirmed to provide a kit capable of simultaneously diagnosing genes of mycobacterium tuberculosis and mycobacterium, which is capable of rapidly, accurately and qualitatively diagnosing tuberculosis and is capable of diagnosing tuberculosis by distinguishing mycobacterium tuberculosis from nontuberculous mycobacterium using only a trace amount of sample genes, and a method for simultaneously diagnosing mycobacterium tuberculosis and nontuberculous mycobacterium using the kit, thereby completing the present invention. Further, they confirmed that the diagnostic method using the two genes IS6110 and mtp40 can be used for the diagnosis of Mycobacterium tuberculosis, and that Mycobacterium tuberculosis can be accurately diagnosed even if one of the two genes IS defective, thereby completing the present invention.
The above information described in the background section is provided only for the purpose of promoting an understanding of the background of the present invention, and thus, may not include information on the related art that has been known to those of ordinary skill in the art to which the present invention pertains.
[ disclosure ] to provide a novel composition
Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a kit for simultaneously diagnosing tuberculosis and nontuberculous mycosis, which is more accurate than conventional kits and can accurately and simply diagnose tuberculosis by distinguishing it from nontuberculous mycosis; and a method for simultaneously diagnosing tuberculosis and nontuberculous mycobacteriosis using the kit.
According to an aspect of the present invention, the above and other objects can be accomplished by the provision of a kit for simultaneous diagnosis of tuberculosis and nontuberculous mycobacteriosis, comprising: (a) (ii) a primer comprising a complementary binding region that specifically binds to a Mycobacterium tuberculosis (Mycobacterium tuberculosis) -specific gene and a nucleic acid oligomer comprising nucleotides that are not complementary to the Mycobacterium tuberculosis-specific gene, and (ii) a primer comprising a complementary binding region that specifically binds to a Mycobacterium consensus gene and a nucleic acid oligomer comprising nucleotides that are not complementary to the gene in common with mycobacteria; and (b) a membrane on which probes complementarily binding to the respective nucleic acid oligomers are immobilized.
According to another aspect of the present invention, there is provided a method for simultaneously diagnosing tuberculosis and nontuberculous mycobacteriosis in vitro using the above kit, the method comprising: amplifying nucleic acid extracted from a sample using (i) primers comprising complementary binding regions that specifically bind to a Mycobacterium tuberculosis (Mycobacterium tuberculosis) specific gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium tuberculosis specific gene and (ii) primers comprising complementary binding regions that specifically bind to a Mycobacterium shared gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium shared gene; and loading the amplified products onto a membrane having immobilized thereon probes that complementarily bind to the respective nucleic acid oligomers.
According to another aspect of the present invention, there is provided a method for providing information for the simultaneous diagnosis of tuberculosis and nontuberculous mycosis in vitro using the above kit, the method comprising: amplifying nucleic acid extracted from a sample using (i) primers comprising complementary binding regions that specifically bind to a Mycobacterium tuberculosis (Mycobacterium tuberculosis) specific gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium tuberculosis specific gene and (ii) primers comprising complementary binding regions that specifically bind to a Mycobacterium shared gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium shared gene; and
the amplified products are loaded onto a membrane on which probes that complementarily bind to the respective nucleic acid oligomers are immobilized.
[ description of the drawings ]
FIG. 1 shows a method of selecting genomic target sites based on tuberculosis-causing bacteria and designing primers based on the genomic target sites.
FIG. 2 shows a band for identifying an amplification product in a kit according to an embodiment of the present invention.
FIG. 3 shows a schematic diagram of a reaction performed on a membrane included in a kit according to an embodiment of the present invention, the membrane having immobilized thereon a probe that complementarily binds to a nucleic acid oligomer.
FIG. 4 shows the change in the color location according to the presence or absence of a tuberculosis causative gene.
FIG. 5 shows the results of tests based on negative or positive determination criteria for tuberculosis and nontuberculous mycobacteriosis.
Detailed description and exemplary embodiments
Unless defined otherwise, 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. Generally, the nomenclature used herein is well known and commonly employed in the art.
Among molecular diagnostic products, products using Polymerase Chain Reaction (PCR) can be broadly classified into two types. One is conventional PCR, in which nucleic acids are extracted from a sample, and target nucleic acids are amplified and subjected to electrophoresis, thereby identifying the results of the PCR. When this method is used, inexpensive kits are commercially available, but electrophoresis requires much labor and time. Real-time PCR is an alternative to electrophoresis, which enables real-time monitoring of nucleic acid amplification of pathogens, but kits for real-time PCR are expensive and it is difficult to identify the amplification results of 5 or more nucleic acids. Therefore, since it is required to develop a molecular diagnostic product that is inexpensive and convenient to use, the inventors of the present application developed an in vitro diagnostic kit for qualitative analysis.
In this regard, the present invention relates to a kit for the simultaneous diagnosis of tuberculosis and nontuberculous mycobacteria disease, said kit comprising: (a) (ii) a primer comprising a complementary binding region that specifically binds to a mycobacterium tuberculosis (mycobacterium tuberculosis) -specific gene and a nucleic acid oligomer that includes nucleotides that are not complementary to the mycobacterium tuberculosis-specific gene, and (ii) a primer comprising a complementary binding region that specifically binds to a mycobacterium consensus gene and a nucleic acid oligomer that includes nucleotides that are not complementary to the gene shared by mycobacteria; and (b) a membrane on which probes complementarily binding to the respective nucleic acid oligomers are immobilized.
The kit of the invention comprises primers, each primer comprising a complementary binding region and a nucleic acid oligomer, and said primers comprising a complementary binding region that specifically binds to a mycobacterium tuberculosis-specific gene and a complementary binding region of a mycobacterium consensus gene, and a nucleic acid oligomer comprising a nucleic acid oligomer that is not complementary to a nucleotide of said mycobacterium tuberculosis-specific gene and a nucleic acid oligomer of a nucleotide of said gene that is shared by mycobacteria. According to the present invention, a mycobacterium tuberculosis infection and a non-tuberculosis mycobacterium infection can be diagnosed simultaneously.
In one embodiment, the mycobacterium tuberculosis-specific gene may be one or more genes specifically present in mycobacterium tuberculosis, and preferably may include two or more mycobacterium tuberculosis-specific genes. Conventional diagnostic products for Mycobacterium tuberculosis use only a single specific gene for Mycobacterium tuberculosis. However, the present invention can provide a diagnostic method using two or more types of genes for diagnosing Mycobacterium tuberculosis at the same time, and Mycobacterium tuberculosis can be accurately diagnosed even if any one of the two types of genes is deleted in a subject to be diagnosed. The Mycobacterium tuberculosis-specific gene may be, for example, IS6110(SEQ ID NO:14) and/or mtp40(SEQ ID NO:15), and by using IS6110 and mtp40 simultaneously, Mycobacterium tuberculosis can be accurately diagnosed even in the absence of one of the two types of genes.
In the present specification, non-tuberculous mycosis may refer to a disease in which symptoms such as cough, sputum and various inflammations of the lung are caused by the retention of mycobacteria, which are not tuberculous mycobacteria but are similar to them, in the bronchi. The Mycobacterium consensus gene may be one or more genes specifically present in Mycobacterium including Mycobacterium tuberculosis, and may be, for example, rpoB (SEQ ID NO: 16).
In a specific embodiment, the kit of the present invention can be used to simultaneously amplify two types of genes existing in Mycobacterium tuberculosis, i.e., IS6110 and mtp40, and rpoB gene common to Mycobacterium including Mycobacterium tuberculosis, thereby enabling to distinguish Mycobacterium tuberculosis from non-tuberculous mycobacteria. As a result of the diagnosis, the case where the mycobacterium tuberculosis-specific gene IS6110 and/or mtp40 IS amplified can be diagnosed as tuberculosis, and the case where only the rpoB gene IS amplified can be determined as nontuberculous mycobacteriosis. According to the present invention, tuberculosis can be accurately diagnosed even in the absence of one of two genes of Mycobacterium tuberculosis.
In the present specification, the term "primer" refers to a single-stranded oligonucleotide capable of serving as a starting point for template-directed DNA synthesis in a buffer (i.e., four types of different nucleoside triphosphates and a polymerase) at an appropriate temperature and under appropriate conditions.
The primers of the invention may comprise fragments of a mycobacterium tuberculosis-specific gene or a mycobacterium consensus gene. The fragment may refer to a certain portion of a mycobacterium tuberculosis-specific gene or a mycobacterium consensus gene, and may have a certain length, and may be, for example, IS6110 and/or mtp40 and rpoB gene or portions thereof, having a length of 5bp to 50bp, particularly 10bp to 30 bp.
In the present specification, "complementary binding" means that a primer hybridizes to a corresponding nucleic acid strand under polymerization reaction conditions and can form a duplex structure. Complementary binding may occur even when complementarity between the paired nucleotide sequences forms Watson-Crick (Watson-Crick) pairs, or even in the presence of some non-Watson-Crick base pairing.
The primer may be a forward primer and may include, for example, a complementary binding region that specifically binds to mycobacterium tuberculosis-specific gene IS6110 and/or mtp40 and the rpoB gene, which IS a common gene of mycobacteria. When IS6110 and/or mtp40 and the rpoB gene that IS a common gene of mycobacteria are used as templates, forward primers can be combined with the strand complementary thereto to specifically amplify the sequence of the rpoB gene complementary to IS6110 and/or mtp40 and the common gene of mycobacteria, for example, the primers can be selected from the group consisting of SEQ ID NOS: 1 to 6.
The primers include nucleic acid oligomers comprising nucleotide oligomers that are not complementary to a mycobacterium tuberculosis-specific gene and a non-mycobacterium tuberculosis gene or fragment thereof. When PCR IS used to amplify the target gene, i.e. IS6110 and/or mtp40, and the mycobacterium consensus gene rpoB gene, the primers may comprise nucleic acid oligomers comprising nucleotides that are not complementary to IS6110 and/or mtp40 and the mycobacterium consensus gene rpoB gene, i.e. genes unrelated to IS6110 and/or mtp40 and the mycobacterium consensus gene rpoB, said nucleic acid oligomers having a length of about 20bp to about 60bp, in particular about 20bp to about 40bp, and preferably about 20bp to about 30 bp. Whether the resulting product, i.e., the nucleic acid, is amplified can be easily confirmed by reacting the nucleic acid oligomer with a corresponding probe complementary to the nucleic acid oligomer immobilized on the membrane. The nucleic acid oligomer can be selected from, for example, SEQ ID NOs 9 to 11.
To react the nucleic acid oligomers with probes that complementarily bind to the respective nucleic acid oligomers immobilized on the membrane, a non-nucleotide spacer may further be included between a region of nucleotides that is not complementary to a tuberculosis-specific gene, a non-tuberculosis mycobacterium gene, or a fragment thereof, and nucleotides for binding to the probes that complementarily bind to the nucleic acid oligomers immobilized on the membrane. The non-nucleotide spacer may be an aliphatic C3, C6, C9, or C12 spacer, wherein the number of C represents the number of carbon atoms in the structure of the spacer. The spacer may be alkyl, alkenyl, or alkynyl.
In one embodiment, when a non-nucleotide spacer such as a C3 spacer (propyl spacer) is positioned between a region of nucleotides that is not complementary to a tuberculosis-specific gene, a non-tuberculosis mycobacterium gene, or a fragment thereof, and nucleotides for binding to a probe immobilized on a membrane that complementarily binds to a nucleic acid oligomer, and the gene is amplified using Taq polymerase, complementary amplification is not allowed by introgression of the portion of nucleotides bound to the probe, so that a portion of the single-stranded nucleic acid is the result, and the portion is allowed to bind to the probe.
The primer may further comprise a primer comprising a label. The primer containing the label IS a reverse primer, and when IS6110 and/or mtp40 and the mycobacterial consensus gene rpoB gene are used as templates, the reverse primer can bind to the template strand and amplify the template, and for example, the primer can be selected from the group consisting of SEQ ID NOS: 1 to 6.
In one embodiment, the label included in the primer may be biotin, Cy5, Cy3, FITC, 5- (2' -aminoethyl) amino-1-naphthalenesulfonic acid (EDANS), Tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), x-rhodamine, DIG, or an antibody or nanoparticle bound thereto, but the present invention is not limited to the label.
In this regard, whether or not color development or fluorescent expression occurs can be confirmed by reaction with a binding agent for inducing color development of a label, and amplification of a target nucleic acid can be detected. In this case, the binder may be streptavidin (streptavidin), but the present invention is not limited to streptavidin.
In a specific embodiment, when amplification is performed using a reverse primer to which biotin is attached, biotin binds to streptavidin on a membrane, and amplification can be confirmed by color development in the case of gold particles or can be confirmed using fluorescent signals having various wavelengths according to the tendency of particles conjugated to biotin, i.e., nanoparticles, and thus, whether several to tens of target nucleic acids are amplified can be confirmed simultaneously depending on the type of probe attached to the membrane.
The kit of the present invention comprises (b) a membrane on which probes complementarily binding to the respective nucleic acid oligomers are immobilized. When the product amplified by the primer is injected into the side of the membrane, the amplified product is transferred, thereby complementarily hybridizing the probe immobilized on the membrane with the nucleic acid oligomer included in the amplified product.
The primers of the present invention can be used for multiplex PCR, thereby enabling simultaneous diagnosis of Mycobacterium tuberculosis and various non-tuberculosis mycobacteria. That is, when multiplex PCR is performed using several to tens of additional primers having different oligomer sequences, followed by reacting the primers with oligomer probes capable of complementarily binding the primers, several to tens of amplification products can be identified using a single lateral flow membrane.
Therefore, the primer of the present invention can also be used in various multiplex PCR, thereby enabling mass production of a lateral flow membrane. This will use a single lateral flow membrane to identify various resultants, and since amplification products can be identified using the same membrane regardless of the type of PCR amplification product, it is possible to reduce the production cost of product production and to facilitate quality management, thereby improving production efficiency.
The probe that complementarily binds to each 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 immobilized on the membrane, and may further include an oligomer that is 20bp to 60bp in length, particularly 20bp to 40bp in length, and has a repetitive nucleotide sequence. In this regard, the further added nucleic acid oligomers may comprise, for example, the sequence of SEQ ID NO 13.
In some cases, the kits of the invention may optionally further comprise an internal control primer, such internal control primer being used to confirm the false negative problem, i.e., whether the PCR is performed correctly, and may optionally be selected from genes normally expressed regardless of the presence or absence of Mycobacterium tuberculosis or mycobacteria in the sample.
In some cases, the kit of the present invention may optionally include reagents required to perform nucleic acid amplification PCR, such as polymerase, buffer, and deoxyribonucleotide-5-triphosphate. The kits of the invention may further comprise various polynucleotide molecules as well as various buffers and reagents.
The optimal amount of reagents, buffers or reactants for a specific reaction in a kit can be determined by one of ordinary skill in the art, and the above-mentioned primers comprising complementary binding regions that specifically bind to a mycobacterium tuberculosis specific gene and a mycobacterium consensus gene (forward) and/or labeled primers comprising a label (reverse) and a membrane to which the probe is immobilized can be manufactured in separate respective packages or compartments.
Another embodiment of the present invention relates to a method for the simultaneous diagnosis of tuberculosis and non-tubercular mycosis in vitro using a kit, said method comprising: amplifying nucleic acid extracted from a sample using (i) primers comprising complementary binding regions that specifically bind to a Mycobacterium tuberculosis (Mycobacterium tuberculosis) specific gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium tuberculosis specific gene and (ii) primers comprising complementary binding regions that specifically bind to a Mycobacterium shared gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium shared gene; and loading the amplified products onto a membrane having immobilized thereon probes that complementarily bind to the respective nucleic acid oligomers.
The present invention also provides a method of providing information for the simultaneous diagnosis of tuberculosis and non-tubercular mycosis using a kit, the method comprising: amplifying nucleic acid extracted from a sample using (i) primers comprising complementary binding regions that specifically bind to a Mycobacterium tuberculosis (Mycobacterium tuberculosis) specific gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium tuberculosis specific gene and (ii) primers comprising complementary binding regions that specifically bind to a Mycobacterium shared gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium shared gene; and loading the amplified products onto a membrane having immobilized thereon probes that complementarily bind to the respective nucleic acid oligomers.
The description of the configuration relating to the kit for performing the method of the invention is equally applicable to the method.
In one embodiment, the sample may include a wide range of all biological fluids, cell lines or tissue cultures obtained from suspected patients or individuals to be diagnosed or from body fluids of individual origin obtained from the diagnosis of tuberculosis and non-tubercular mycobacteriosis, and may be, for example, sputum, a culture sample, a tissue sample or bronchial lavage, although the invention is not limited thereto.
The method may further comprise extracting nucleic acids from the sample, and the extraction of the nucleic acids may be performed using, for example, various commercially available kits or extraction reagents.
Examples
Hereinafter, the present invention will be described in more detail with reference to the following examples. It will be apparent to those of ordinary skill in the art that these examples are provided for illustrative purposes only and should not be construed to limit the scope of the present invention
1. Manufacture of the kit
The kit of the present invention (hereinafter also referred to as PaxView TB/NTM MPCR-ULFA kit) includes two kits. One is a kit for amplifying a nucleic acid (hereinafter referred to as PaxView TB/NTM MPCR kit), and the other is a kit for identifying an amplified product (hereinafter referred to as PaxView ULFA kit). The primers in the primer mixture that is part of the PaxView TB/NTM MPCR kit include a forward primer (SN) that also includes a universal region of a non-complementary nucleic acid oligomer in addition to the pathogenic region and the internal control region for the three diseases; and a reverse primer (ASN) tagged with a biotin sequence (see fig. 1).
After PCR, when the amplified product was loaded on the ULFA device (e.g., strip: FIG. 2) of the PaxView ULFA kit, the product bound to gold-streptavidin conjugate (gold-SV was present in the pad formed of glass fiber), followed by binding to a probe (oligonucleotide complementary to the universal region) adsorbed on NC membrane, thereby developing color at the labeled site of each pathogen (see FIG. 3).
The principle of color development is based on the reaction between biotin and streptavidin labeled with each primer, and can be applied to confirm a DNA reaction in addition to an antigen-antibody reaction. Like avidin, streptavidin is capable of forming a complex with biotin, and thus gold particles bound to biotin can cause a macroscopic readable color development.
After development, the ULFA device was read visually. According to the result determination method described in the specification of the kit, the result is determined to be positive or negative, and since the position of the color line varies depending on the presence or absence of pathogenic bacteria of a disease, it can be confirmed that the expected band pattern matches the actual band pattern (see fig. 4).
The tuberculosis diagnostic kit (TB/NTM diagnostic kit) of the present invention is a new platform for tuberculosis diagnosis, and is a rapid and accurate qualitative molecular diagnostic kit, in which genes extracted from a sample are used as templates and amplified, the amplified genes are loaded on ULFAs, and when gold nanoparticles are displayed in the form of a band, whether infection with Mycobacterium tuberculosis and/or non-tuberculosis mycobacteria can be diagnosed.
2. Tuberculosis diagnosis
(1) Preparation of the reagent: preparation and Conditioning of PCR Master mix (Master mix)
Using the primers shown in Table 1 below, 5. mu.l of the primer mix was mixed with 10. mu.l of the 2 XPCR premix to prepare the required amount of PCR master mix (see Table 2).
[ Table 1]
[ Table 2] PCR Master mix
Mu.l of master mix was dispensed into each of the multiple PCR tubes, and 5. mu.l of extracted sample DNA or positive control (included in the kit) or negative control (TE buffer or distilled water, not included in the kit) was pipetted and mixed well with the master mix. The resulting mixture was centrifuged and placed in a gene amplification apparatus to allow a reaction therebetween (see Table 3).
[ Table 3] PCR conditions
(2) Confirmation of PCR results
After the PCR process was completed, the PCR tube was removed from the PCR apparatus, followed by gentle centrifugation. The ULFA device in the PaxView ULFA Kit corresponding to the reaction amount was removed, and the developing solution was removed. Mu.l of PCR reaction solution was dropped into the square inlet at the lower end of each apparatus, and 50. mu.l of developing solution was slowly dropped at once. After 10 minutes, 50. mu.l of the washing solution was slowly dropped into the square inlet. Results were read within 20 minutes after the examination began.
(3) Result determination
The sequences of probes binding to PCR amplification products in the ULFA apparatus are shown in table 4 below, and the band results obtained due to the reaction between them are confirmed with naked eyes, thereby determining the results as negative or positive (see tables 5 and 6).
[ Table 4]
| Probe needle | Nucleotide sequence (5' ->3') | SEQ ID NO: |
| IS6110(01 line) | TAGAGTTATCCGTTGTTAC | 9 |
| mtp40(02 line) | ACATCTTCAGGTCACACTT | 10 |
| rpoB (03 line) | GACTTGGTATCTGGTGGA | 11 |
| IC(04 line) | TTTGGTGCTATTGCTTAC | 12 |
| HC (05 line) | CATTGTCAGTTAGCCTTAG | 13 |
IC: internal control
HC: hybridization controls
[ Table 5]
[ Table 6]
In tables 5 and 6, samples 5 to 7 show cases where a plurality of infections were present according to the samples, and sample 8 shows a case where a PCR amplified band of at least one of TB bacteria and NTM bacteria was identified, but a PCR amplified band of an Internal Control (IC) was not shown, which indicates a case where a large amount of PCR amplification was caused due to a large amount of DNA. In this case, since the PCR band of the internal control was not well observed, the result was determined to be positive for TB or NTM microorganisms for which PCR amplification was confirmed. Sample 10 is the case where the internal control is not amplified, which corresponds to the case where a PCR inhibitor is present in the corresponding sample, at which time DNA extraction should be performed again.
[ INDUSTRIAL APPLICABILITY ]
The kit of the present invention enables diagnosis of tuberculosis using only trace amounts of sample genes and simultaneously amplifies specific genes of Mycobacterium tuberculosis and common genes of Mycobacterium, thereby enabling rapid and accurate qualitative molecular diagnosis of Mycobacterium tuberculosis and nontuberculous mycobacteria. Furthermore, after gene amplification, the result is confirmed by lateral flow assay, and therefore, it is not necessary to use expensive equipment as in real-time PCR, which is economical, nor a process of preparing agarose gel for electrophoresis to confirm the amplified gene in conventional PCR, and therefore, it is possible to identify the result in a shorter time as compared with PCR-electrophoresis.
Particular embodiments of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these particular embodiments are merely exemplary embodiments, and are not intended to limit the scope of the present invention. Accordingly, the substantial scope of the present invention should be defined by the appended claims and equivalents thereof.
Sequence Listing free text
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Claims (18)
1. A kit for the simultaneous diagnosis of tuberculosis and non-tubercular mycosis, said kit comprising:
(a) (i) a primer comprising a complementary binding region that specifically binds to a Mycobacterium tuberculosis (Mycobacterium tuberculosis) -specific gene and a nucleic acid oligomer comprising a nucleotide that is not complementary to the Mycobacterium tuberculosis-specific gene, and
(ii) a primer comprising a complementary binding region that specifically binds to a mycobacterial consensus gene and a nucleic acid oligomer comprising nucleotides that are not complementary to said gene shared by mycobacteria; and
(b) a membrane having immobilized thereon probes that complementarily bind to the respective nucleic acid oligomers.
2. The kit of claim 1, wherein the kit simultaneously diagnoses two or more mycobacterium tuberculosis-specific genes.
3. The kit according to claim 1, wherein the Mycobacterium tuberculosis-specific genes include IS6110 and mtp 40.
4. The kit of claim 1, wherein the gene shared by mycobacteria is rpoB.
5. The kit of claim 1, wherein the nucleic acid oligomer has a length of 20bp to 60 bp.
6. The kit of claim 1, wherein a non-nucleotide spacer is included between the complementary binding region and the nucleic acid oligomer comprising non-complementary nucleotides.
7. The kit of claim 1, wherein the primer is selected from the group consisting of: 1-6 of SEQ ID NO.
8. The kit of claim 1, further comprising a primer comprising a label.
9. The kit of claim 7, wherein the primer has SEQ ID NO 7 or SEQ ID NO 8.
10. The kit according to claim 1, wherein, when a product amplified by the primer is injected into the side of the membrane, the amplified product is transferred, thereby complementarily hybridizing the probe immobilized on the membrane with the nucleic acid oligomer included in the amplified product.
11. The kit of claim 1, wherein the probe that complementarily binds each nucleic acid oligomer comprises one or more sequences selected from the group consisting of seq id nos: 9-11 of SEQ ID NO.
12. The kit of claim 1, wherein the probe further comprises a nucleic acid oligomer immobilized on the membrane.
13. The kit according to claim 12, wherein the further added nucleic acid oligomer comprises the sequence SEQ ID No. 12 or SEQ ID No. 13.
14. The kit of claim 8, wherein the label is biotin, Cy5, Cy3, FITC, 5- (2' -aminoethyl) amino-1-naphthalenesulfonic acid (EDANS), Tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), x-rhodamine, DIG, or an antibody or nanoparticle bound thereto.
15. The kit of claim 8, wherein color development is induced by a reaction between the label and a binding agent.
16. The kit of claim 15, wherein the binding agent is streptavidin.
17. A method of simultaneously diagnosing tuberculosis and non-tubercular mycosis in vitro using the kit of any one of claims 1 to 16, the method comprising:
amplifying nucleic acid extracted from a sample using (i) primers comprising complementary binding regions that specifically bind to a Mycobacterium tuberculosis (Mycobacterium tuberculosis) specific gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium tuberculosis specific gene and (ii) primers comprising complementary binding regions that specifically bind to a Mycobacterium shared gene and nucleic acid oligomers that include nucleotides that are not complementary to the Mycobacterium shared gene; and
the amplified products are loaded onto a membrane on which probes that complementarily bind to the respective nucleic acid oligomers are immobilized.
18. The method of claim 17, wherein the sample is sputum, culture, tissue, or bronchial lavage.
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| KR10-2017-0102378 | 2017-08-11 | ||
| KR20170102378 | 2017-08-11 | ||
| PCT/KR2018/009184 WO2019031920A1 (en) | 2017-08-11 | 2018-08-10 | Kit for tuberculosis diagnosis and method for diagnosing tuberculosis by using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011633A (en) * | 2020-10-14 | 2020-12-01 | 河南智泰生物科技有限公司 | Primer group, kit and method for LAMP (loop-mediated isothermal amplification) simultaneous detection of mycobacterium tuberculosis complex group and rpoB gene mutation |
| CN113122660A (en) * | 2021-05-19 | 2021-07-16 | 冯志山 | Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102334343B1 (en) * | 2020-01-29 | 2021-12-07 | (주)바이오젠텍 | Primer set for high sensitive multiplex loop-mediated isothermal amplification reaction for detection and identification of Mycobacterium tuberculosis and Nontuberculous mycobacteria |
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2018
- 2018-08-10 CN CN201880058656.5A patent/CN111344419A/en active Pending
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| KR20030075315A (en) * | 2002-03-18 | 2003-09-26 | 바이오코아 주식회사 | Method for simultaneous detection of tubercle bacillus(tb) and nonturberculous mycobacteria(ntm) by multiplex-nested polymerase chain reaction |
| KR20100031188A (en) * | 2008-09-12 | 2010-03-22 | 주식회사 엘지생명과학 | Composition for detection of m. tuberculosis complex or mycobacteria genus and simultaneous detection method for m. tuberculosis complex and mycobacteria genus with multiplex real time pcr using the same |
| CN102094077A (en) * | 2009-12-15 | 2011-06-15 | 复旦大学 | Kit for detecting genotype of mycobacterium tuberculosis clinical isolation strain quickly |
| KR20160137185A (en) * | 2015-05-22 | 2016-11-30 | 주식회사 팍스젠바이오 | Method of Detecting Target Nucleic Acid and Kit |
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| 赵自云等: "PCR 扩增 IS6110在结核病检测和流行病学中的应用评价" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011633A (en) * | 2020-10-14 | 2020-12-01 | 河南智泰生物科技有限公司 | Primer group, kit and method for LAMP (loop-mediated isothermal amplification) simultaneous detection of mycobacterium tuberculosis complex group and rpoB gene mutation |
| CN113122660A (en) * | 2021-05-19 | 2021-07-16 | 冯志山 | Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20190017698A (en) | 2019-02-20 |
| KR102132965B1 (en) | 2020-07-13 |
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