KR102388060B1 - Composition for distinguishing between Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis and method for detecting Mycobacterium tuberculosis using the same - Google Patents

Abstract

The present invention relates to a composition for simultaneously detecting Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis and, more specifically, to a system or detection method capable of simultaneously detecting and discriminating between Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis by using reagent compositions required for polymerase chain reaction (PCR), such as primers and probe sets capable of discriminating between Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis. According to the present invention, the system can accurately, conveniently, and continuously monitor of tuberculosis patients in Beijing and non-Beijing families.

Description

Composition for distinguishing between Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis and method for detecting Mycobacterium tuberculosis using the same}

The present invention relates to a composition for simultaneously detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family, specifically Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family While simultaneously detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family using a reagent composition necessary for real-time polymerase chain reaction (PCR), such as a primer and probe set that can be differentiated, It relates to a system or detection method capable of discriminating between the two.

Tuberculosis is a respiratory infectious disease in which Mycobacterium tuberculosis ( M. tuberculosis, MTB) is transmitted through the air and infects the body. 2015).Changes in tuberculosis control policy in Korea.KCDC 2015;Vol. 8, No. 28.). Accordingly, many countries are continuing their efforts to reduce the incidence of tuberculosis. Nevertheless, tuberculosis is still one of the top 10 causes of death worldwide, and as of 2018, about 10 million people (130 per 100,000 population) had tuberculosis, and about 1.5 million people (per 100,000 population) 20) are reported to have died. In particular, tuberculosis resistant to the most important drug, rifampicin (rifampin), has occurred in about 480,000 people, and about 80% of them are multidrug-resistant tuberculosis (MDR-resistant tuberculosis) resistant to isoniazid, isonicotinylhydrazine, INH, Laniazid, and Nydrazid. It was presumed to be a patient with multidrug-resistant tuberculosis (MDR-TB). Here, MDR-TB means a disease caused by Mycobacterium tuberculosis that is resistant to two or more antituberculosis drugs, including isoniazid and rifampin, which are the most important drugs for tuberculosis treatment.

Korea has been evaluated as a country with good tuberculosis control externally, but as of 2018, among the 36 OECD member countries, the tuberculosis incidence rate (66 per 100,000 population) was the highest, and the tuberculosis mortality rate (4.8 per 100,000 population) was Lithuania ( It ranked second after 5.6 per 100,000 people). This indicates that the tuberculosis incidence rate is about five times higher and the tuberculosis mortality rate is about 2.2 times higher than that of Japan, which is geographically close. In particular, since 2000, the rate of decrease in tuberculosis patients has slowed and the number of multidrug-resistant tuberculosis patients has increased (Centers for Disease Control and Prevention, Tuberculosis Guidelines (4th Edition), 2020.).

In particular, the proportion of foreign tuberculosis patients among domestic tuberculosis patients is increasing, and it is known that most foreign tuberculosis patients are from countries with high tuberculosis risk, such as China and Vietnam. tuberculosis isolated in Korea using IS6110 and MIRU-VNTR, 2015.). Mycobacterium tuberculosis, which occurs in most of East Asia including Korea, is known to occupy the genotype of the Beijing (East Asian) family, which is closely related to China. 82.4% of the genotypes isolated from Korean tuberculosis patients have been reported to be of Beijing family, and as the number of foreigners residing in Korea is increasing (Lee In-hyuk. ), it has become more important to distinguish the genotype of Mycobacterium tuberculosis introduced from outside and to understand the transmission route. Accordingly, although it is mandatory for foreigners from countries with a high incidence of tuberculosis to submit a tuberculosis diagnosis before entering Korea for a long-term stay in Korea, this method cannot prevent morbidity of patients with latent tuberculosis other than screening for active tuberculosis patients. There is a blind spot (Hajimin, et al. Mycobacterium tuberculosis genotyping characteristics analysis of foreign tuberculosis patients in Korea (2017-2018) Weekly Health and Disease, 2020, 13.1: 69-76.). Accordingly, continuous monitoring is required, and rapid and accurate techniques for this are required.

On the other hand, real-time polymerase chain reaction (Real-time PCR) is a method of confirming the increase amount of the amplification product by measuring the amount of fluorescence emitted from a probe bound to the amplification product of a nucleic acid in real time. Although there are many types of probes used in real-time PCR, the TaqMan™ probe method, which is very effective in detecting a specific nucleotide sequence of a target nucleic acid, is adopted in the present invention.

TaqMan™ probe is an oligomer having a fluorescent dye attached to the 5' end and a quencher attached to the 3' end to suppress the emission of the fluorescent dye attached to the 5' end. is composed During the annealing step of the PCR process, the TaqMan™ probe specifically bound to the template nucleic acid was obtained from Taq. It is hydrolyzed by the 5' → 3' exonuclease activity of the polymerase, and at this time, the fluorescence of the fluorescent dye away from the quencher that was suppressing the fluorescence is converted into real-time polymerase chain reaction ( Real-time PCR) is detected by a fluorescence meter attached to the device. Through this series of processes, the increase in nucleic acid amplification products can be converted to fluorescence and quantified. The real-time PCR method can interpret the results quickly and conveniently compared to the existing traditional polymerase chain reaction (PCR) method, and has very high diagnostic specificity and sensitivity. In addition, since it has the advantage of low risk of cross-contamination during the experimental process, it can be used as a technology applicable to an efficient tuberculosis control and monitoring system.

According to the above content, the present inventors have invented a primer and probe set capable of simultaneously and accurately detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing strain, but capable of distinguishing the two, and a composition comprising the same is a Peking strain closely related to multidrug resistance. Mycobacterium tuberculosis can be continuously monitored accurately and conveniently using real-time PCR.

It is an object of the present invention to provide a primer and probe set capable of simultaneously detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family.

Another object of the present invention is to provide a composition capable of simultaneously detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family, including the primer and probe set, and a kit comprising the composition.

Another object of the present invention is to provide a method for simultaneously detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family using the primer and probe set.

In order to achieve the above object, the present invention is a first forward primer described in the nucleotide sequence of SEQ ID NO: 1; a second forward primer described in the nucleotide sequence of SEQ ID NO: 4; a first reverse primer described in the nucleotide sequence of SEQ ID NO: 2; a second reverse primer described in the nucleotide sequence of SEQ ID NO: 5; a first probe described in the nucleotide sequence of SEQ ID NO: 3; And it provides a set of primers and probes for simultaneous detection of Mycobacterium tuberculosis and the Beijing family Mycobacterium tuberculosis comprising a second probe described in the nucleotide sequence of SEQ ID NO: 6.

In addition, the present invention provides a composition capable of simultaneously detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family comprising the primer and probe set.

In addition, the present invention provides a kit for simultaneous detection of Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis comprising the composition.

In addition, the present invention includes the step of performing a real-time polymerase chain reaction (PCR) using a composition or kit comprising the primer and probe set using the nucleic acid isolated from the sample as a template To provide a method for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family.

The primer and probe set of the present invention is a single tube and a single step (one tube and one step) using a multiplex real-time polymerase chain reaction (PCR) method Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) And because it can simultaneously detect Mycobacterium tuberculosis of the Beijing family, it is possible to detect Mycobacterium tuberculosis quickly and conveniently with little cross-contamination. In addition, Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family can be detected separately from other pathogens, and the two can be detected separately. Therefore, through the present invention, it is possible to quickly and accurately detect dnaA-IS6110, a specific target gene for Mycobacterium tuberculosis of the Beijing family, which has a high incidence in Korea and is difficult to treat due to multi-drug resistance, with a sensitivity of 50 copies/uL, By continuously monitoring the pathogenesis and transmission of Mycobacterium tuberculosis, it can contribute to managing tuberculosis patients from neighboring countries and establishing an effective quarantine system.

1a is a multiplex real-time polymerase chain reaction (real-time PCR) using a primer and probe set for simultaneous detection of Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis of the present invention Beijing family (Beijing family) ) It is a graph showing the result of simultaneous detection of Mycobacterium tuberculosis in two channels (dnaA-IS6110: Mycobacterium tuberculosis target gene in Beijing, IS6110: Mycobacterium tuberculosis target gene).
1b is a multiplex real-time polymerase chain reaction (real-time PCR) using a primer and probe set for simultaneous detection of Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis of the present invention through non-Beijing (non -Beijing) It is a graph showing the result of simultaneous detection of Mycobacterium tuberculosis family in two channels (dnaA-IS6110: Beijing Mycobacterium tuberculosis target gene, IS6110: Mycobacterium tuberculosis target gene).
Figure 2 is a graph showing the results of a multiplex real-time polymerase chain reaction (real-time PCR) using the primer and probe set of the present invention in order to confirm the analytical sensitivity of Mycobacterium tuberculosis of the Beijing family.
Figure 3 is a non-Beijing (non-Beijing) line (family) to confirm the analytical sensitivity of Mycobacterium tuberculosis, multiplex real-time polymerase chain reaction (real-time PCR) results using the primer and probe set of the present invention This is the graph shown.
Figure 4a is a graph showing the detection result of Mycobacterium tuberculosis of the Beijing family in order to confirm the analytical specificity of the multiplex real-time polymerase chain reaction (real-time PCR) using the primer and probe set of the present invention ( dnaA-IS6110: Beijing strain Mycobacterium tuberculosis target gene, IS6110: Mycobacterium tuberculosis target gene).
Figure 4b is to confirm the analytical specificity of the multiplex real-time polymerase chain reaction (real-time PCR) using the primer and probe set of the present invention, non-Beijing (non-Beijing) family (family) Mycobacterium tuberculosis detection A graph showing the results (dnaA-IS6110: Beijing Mycobacterium tuberculosis target gene, IS6110: Mycobacterium tuberculosis target gene).
Figure 4c is a graph showing the detection results of pathogens other than Mycobacterium tuberculosis in order to confirm the analytical specificity of the multiplex real-time polymerase chain reaction (real-time PCR) using the primer and probe set of the present invention (dnaA-IS6110: Beijing strain Mycobacterium tuberculosis target gene, IS6110: Mycobacterium tuberculosis target gene).

Hereinafter, the present invention will be described in detail.

The present invention is a first forward primer described in the nucleotide sequence of SEQ ID NO: 1; a second forward primer described in the nucleotide sequence of SEQ ID NO: 4; a first reverse primer described in the nucleotide sequence of SEQ ID NO: 2; a second reverse primer described in the nucleotide sequence of SEQ ID NO: 5; a first probe described in the nucleotide sequence of SEQ ID NO: 3; And it provides a set of primers and probes for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family comprising a second probe described in the nucleotide sequence of SEQ ID NO: 6 (Table 1).

The primer set is used to specifically amplify the IS6110 gene region of Mycobacterium tuberculosis and the dnaA-IS6110 gene region of Mycobacterium tuberculosis of the Beijing family (Table 1). In an experimental example of the present invention, the primer set is a primer set described by the nucleotide sequences of SEQ ID NOs: 1, 2, 4 and 5, respectively.

The primer set can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. This primer set can be modified using many means known in the art as long as it exhibits an effect of simultaneously detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family. Examples of such modifications include methylation, encapsulation, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg, methyl phosphonates, phosphotriesters, phosphoroamidates). , carbamates, etc.) or charged linkages (phosphorothioate, phosphorodithioate, etc.). Nucleic acids may contain one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (eg, acridine, proralene, etc.), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents.

In addition, if necessary, the primer set of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg, horseradish peroxidase, alkaline phosphatase), radioactive isotopes (eg, ³² P), fluorescent molecules and chemical groups (eg, biotin), and the like. There is this.

The probe set is used to specifically amplify the IS6110 gene region of Mycobacterium tuberculosis and the dnaA-IS6110 gene region of Mycobacterium tuberculosis of the Beijing family. In Experimental Example 1 of the present invention, the probe set is a probe described by the nucleotide sequences of SEQ ID NOs: 3 and 6, respectively.

As used herein, the term "probe" refers to a nucleic acid fragment corresponding to several to hundreds of bases capable of specifically binding to DNA or RNA, and an oligonucleotide probe, a single stranded DNA probe , a double-stranded DNA probe or an RNA probe may be manufactured.

The probe set may be further conjugated (attached) with a fluorescent dye to its 5' end. The fluorescent dye is 6-carboxyfluorescein (FAM), Texas red, fluorescein, fluorescein chlorotriazi nyl, HEX (2', 4', 5', 7'). -tetrachloro-6-carboxy-4,7-dichlorofluorescein), rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), oregon green , Alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dime thoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), Tetrachloro-Fluorescein (TET) , TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhoda mine), NED(N-(1-Naphthyl) ethylenediamine), VIC(2′-chloro-7′phenyl-1,4-dich loro-6-carboxy -fluorescein), cyanine-based dyes, and thiadicarbocyanine may be at least one selected from the group consisting of, but any known material that can be used as a fluorescent dye in the art may be used.

The probe set may be further conjugated (attached) with a quencher at its 3' end. The quencher is TAMRA, BHQ (black hole quencher) 1, BHQ2, BHQ3, NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (deep dark quencher), Blackberry Quencher (Blackberry Quencher), Iowa black (Iowa black) ) may be any one or more selected from the group consisting of, but any known material that can be used as a quencher in the art may be used.

In addition, the present invention provides a composition for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis comprising a primer and a probe set for simultaneous detection of Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis. to provide.

The composition may include reverse transcription polymerase, DNA polymerase, cofactors such as Mg ²⁺ , dATP, dCTP, dGTP, and dTTP in addition to the primer and probe sets. In order to amplify the reverse transcribed cDNA, various DNA polymerases may be used in the amplification step of the present invention, and examples of the DNA polymerase include Klenow fragment of E. coli DNA polymerase I, thermostable DNA polymerase or bacteriophage T7 DNA polymerization. There are enzymes. Polymerases can be isolated from the bacterium itself, purchased commercially, or obtained from cells expressing high levels of the cloned gene encoding the polymerase.

In a specific experimental example of the present invention, the inventors of the present invention have a first forward primer described in the nucleotide sequence of SEQ ID NO: 1, a second forward primer described in the nucleotide sequence of SEQ ID NO: 4, and a first forward primer described in the nucleotide sequence of SEQ ID NO: 2 A reverse primer, a second reverse primer represented by the nucleotide sequence of SEQ ID NO: 5, a first probe represented by SEQ ID NO: 3, and a second probe represented by SEQ ID NO: 6 were prepared (Table 1).

In addition, the present inventors performed multiplex real-time polymerase chain reaction (PCR) using the primer and probe set, Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis It was confirmed that only Mycobacterium tuberculosis was specifically detected, and at the same time, it was also confirmed to distinguish Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family (Table 6, Figs. 1a, 1b and 4a to 4c).

In addition, as a result of performing the multiplex real-time polymerase chain reaction (PCR), the Mycobacterium tuberculosis -specific target gene IS6110 and the Beijing family Mycobacterium tuberculosis-specific target gene It was confirmed that the minimum detection limit (sensitivity) for the detection of the phosphorus dnaA-IS6110 target gene was 50 copies/μL, respectively ( FIGS. 2 and 3 ).

Therefore, the primer and probe set can be usefully used for simultaneous detection and differentiation of Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family.

In addition, the present invention provides a kit for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis comprising a composition for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family of the present invention do.

The kit may further include one or more types of compositions, solutions, or devices suitable for the detection method. The kit includes a tube or other suitable container, a reaction buffer (with varying pH and magnesium concentrations), in addition to a primer set specific for the IS6110 gene region of Mycobacterium tuberculosis and the dnaA-IS6110 gene region of Mycobacterium tuberculosis of the Beijing family deoxynucleotides (dNTPs), enzymes such as Taq-polymerase, DNase inhibitors, DEPC-water and sterile water, and the like.

On the other hand, the components included in the kit may be prepared in a liquid form, or may be prepared in a dried form to increase the stability of the product by lowering the degree of freedom of the included components. In order to produce such a dried form, it is necessary to apply a drying step, and in this case, heating drying, natural drying, reduced pressure drying, freeze drying, or a combination process thereof may be used.

In addition, the present invention uses a nucleic acid isolated from a sample as a template, and uses a primer and a probe set for simultaneous detection of Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis of the present invention to perform multiple real-time polymerase chain reaction. It provides a method for simultaneous detection of Mycobacterium tuberculosis and the Beijing family Mycobacterium tuberculosis comprising the steps.

The sample may be obtained from a clinical sample or an environmental sample. For example, the clinical sample may be a sample obtained from human sputum, bronchial lavage fluid, or cultured colonies.

Hereinafter, the present invention will be described in more detail by way of Examples and Experimental Examples. However, the present invention is not limited by the following Examples and Experimental Examples.

Experimental Example 1. Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) and Beijing family Mycobacterium tuberculosis detection primer and probe set production

After searching for gene sequences for Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family at the National Center for Biological Information (NCBI, https://www.ncbi.nlm.nih.gov/), the sequence was were sorted. 130 base sequence databases were secured for Mycobacterium tuberculosis , which includes both Beijing and non-Beijing families, and among them, the base sequence of Mycobacterium tuberculosis of the Beijing family The database includes 30.

The obtained nucleotide sequences were analyzed by a multi-alignment method in which several nucleotide sequences were bundled and aligned, and as a result of the multi-alignment, a conserved region in which the nucleotide sequence did not change was secured. Primer and probe sets were prepared by designing nucleotide sequences within the conserved region (Table 1). The prepared primer and probe sets (SEQ ID NOs: 1 to 6) were confirmed for nucleotide sequence homology with respect to the collected database. As a result, the primer and probe set of the Mycobacterium tuberculosis-specific IS6110 gene region of the present invention for the Beijing family Mycobacterium tuberculosis showed a theoretical 100% homology detection rate, and the Beijing family Mycobacterium tuberculosis-specific The primer and probe set of the dnaA-IS6110 gene region theoretically also showed a detection rate of 100% (1-130) homology (Table 2). The primer and probe set of the Mycobacterium tuberculosis-specific IS6110 gene region of the present invention for non-Beijing family Mycobacterium tuberculosis theoretically exhibited 100% homology detection rate, and Beijing family Mycobacterium tuberculosis-specific The primers and probe sets of the dnaA-IS6110 gene region are theoretically i) 100% forward primers (1-130), ii) 100% reverse primers (1-30), 22.7% (31-130), and iii) 100% probes (1-30), 33.3% (31-130) showed a detection rate of homology (Table 2).

pathogen primer and
probe sequence (5'-3') target gene SEQ ID NO: Beijing
strain Mycobacterium tuberculosis MTB Beijing-F GTG TTC TTC CGA CAA CGT TCT dnaA-IS6110 One MTB Beijing-R TCG ATG AAC CAC CTG ACA TGA C 2 MTB Beijing-P FAM CGG CAT GTC CGG AGA CTC CAG TTC BHQ1 3 Mycobacterium tuberculosis MTB-F GAA CCG TGA GGG CAT CGA IS6110 4 MTB-R AGG CCG AGT TTG GTC ATC AG 5 MTB-P VIC TGG CCA GAT GCA CCG MGBNFQ 6

No. Category Reference % Identity of the Beijing strain Mycobacterium tuberculosis
Primer and Probe Sets Mycobacterium tuberculosis
Primer and Probe Sets MTB
Beijing-F MTB
Beijing-R MTB
Beijing-P MTB-F MTB-R MTB-P One Beijing line
Mycobacterium tuberculosis CP007803 100 100 100 100 100 100 2 CP017920 100 100 100 100 100 100 3 AP018036 100 100 100 100 100 100 4 CP007803 100 100 100 100 100 100 5 CP011510 100 100 100 100 100 100 6 CP012090 100 100 100 100 100 100 7 CP017596 100 100 100 100 100 100 8 CP022704 100 100 100 100 100 100 9 HG813240 100 100 100 100 100 100 10 AP018034 100 100 100 100 100 100 11 AP018035 100 100 100 100 100 100 12 AP018036 100 100 100 100 100 100 13 CP002871 100 100 100 100 100 100 14 CP002882 100 100 100 100 100 100 15 CP002883 100 100 100 100 100 100 16 CP002885 100 100 100 100 100 100 17 CP007809 100 100 100 100 100 100 18 CP009426 100 100 100 100 100 100 19 CP012506 100 100 100 100 100 100 20 CP013475 100 100 100 100 100 100 21 CP016794 100 100 100 100 100 100 22 CP016888 100 100 100 100 100 100 23 CP017593 100 100 100 100 100 100 24 CP017594 100 100 100 100 100 100 25 CP017595 100 100 100 100 100 100 26 CP017597 100 100 100 100 100 100 27 CP017598 100 100 100 100 100 100 28 CP022014 100 100 100 100 100 100 29 AP018034 100 100 100 100 100 100 30 JXXH01000016 100 100 100 100 100 100 31 Non-Beijing system
Mycobacterium tuberculosis CP025599 100 22.7 33.3 100 100 100 32 CP025594 100 22.7 33.3 100 100 100 33 CP046728 100 22.7 33.3 100 100 100 34 LC005472 100 22.7 33.3 100 100 100 35 LC005475 100 22.7 33.3 100 100 100 36 CP025607 100 22.7 33.3 100 100 100 37 CP025598 100 22.7 33.3 100 100 100 38 CP008961 100 22.7 33.3 100 100 100 39 CP022704 100 22.7 33.3 100 100 100 40 CP025607 100 22.7 33.3 100 100 100 41 CP009172 100 22.7 33.3 100 100 100 42 LR699570 100 22.7 33.3 100 100 100 43 CP046728 100 22.7 33.3 100 100 100 44 CP025595 100 22.7 33.3 100 100 100 45 CP025597 100 22.7 33.3 100 100 100 46 CP019612 100 22.7 33.3 100 100 100 47 CP022577 100 22.7 33.3 100 100 100 48 CP054013 100 22.7 33.3 100 100 100 49 CP019610 100 22.7 33.3 100 100 100 50 CP025606 100 22.7 33.3 100 100 100 51 CP047163 100 22.7 33.3 100 100 100 52 CP025605 100 22.7 33.3 100 100 100 53 CP009191 100 22.7 33.3 100 100 100 54 CP044345 100 22.7 33.3 100 100 100 55 CP046529 100 22.7 33.3 100 100 100 56 CP025602 100 22.7 33.3 100 100 100 57 CP025597 100 22.7 33.3 100 100 100 58 CP023640 100 22.7 33.3 100 100 100 59 CP053092 100 22.7 33.3 100 100 100 60 CP048071 100 22.7 33.3 100 100 100 61 CP025603 100 22.7 33.3 100 100 100 62 CP025599 100 22.7 33.3 100 100 100 63 CP023603 100 22.7 33.3 100 100 100 64 CP025593 100 22.7 33.3 100 100 100 65 CP025604 100 22.7 33.3 100 100 100 66 CP025605 100 22.7 33.3 100 100 100 67 CP054014 100 22.7 33.3 100 100 100 68 LR027516 100 22.7 33.3 100 100 100 69 CP025595 100 22.7 33.3 100 100 100 70 CP023624 100 22.7 33.3 100 100 100 71 CP025596 100 22.7 33.3 100 100 100 72 CP025596 100 22.7 33.3 100 100 100 73 CP046308 100 22.7 33.3 100 100 100 74 CP022578 100 22.7 33.3 100 100 100 75 CP025593 100 22.7 33.3 100 100 100 76 CP017598 100 22.7 33.3 100 100 100 77 CP023589 100 22.7 33.3 100 100 100 78 CP025593 100 22.7 33.3 100 100 100 79 CP008980 100 22.7 33.3 100 100 100 80 CP023614 100 22.7 33.3 100 100 100 81 CP023630 100 22.7 33.3 100 100 100 82 CP048071 100 22.7 33.3 100 100 100 83 LR027516 100 22.7 33.3 100 100 100 84 CP047163 100 22.7 33.3 100 100 100 85 CP025599 100 22.7 33.3 100 100 100 86 CP046529 100 22.7 33.3 100 100 100 87 MK862423 100 22.7 33.3 100 100 100 88 CP000611 100 22.7 33.3 100 100 100 89 CP025603 100 22.7 33.3 100 100 100 90 CP007027 100 22.7 33.3 100 100 100 91 CP047163 100 22.7 33.3 100 100 100 92 CP046728 100 22.7 33.3 100 100 100 93 CP054013 100 22.7 33.3 100 100 100 94 CP054014 100 22.7 33.3 100 100 100 95 CP025600 100 22.7 33.3 100 100 100 96 CP049108 100 22.7 33.3 100 100 100 97 CP019610 100 22.7 33.3 100 100 100 98 KP844720 100 22.7 33.3 100 100 100 99 CP022578 100 22.7 33.3 100 100 100 100 CP022704 100 22.7 33.3 100 100 100 101 CP025603 100 22.7 33.3 100 100 100 102 CP025603 100 22.7 33.3 100 100 100 103 CP023635 100 22.7 33.3 100 100 100 104 LR027516 100 22.7 33.3 100 100 100 105 CP023601 100 22.7 33.3 100 100 100 106 CP023634 100 22.7 33.3 100 100 100 107 CP025606 100 22.7 33.3 100 100 100 108 CP022577 100 22.7 33.3 100 100 100 109 CP023592 100 22.7 33.3 100 100 100 110 CP002882 100 22.7 33.3 100 100 100 111 CP023638 100 22.7 33.3 100 100 100 112 CP019610 100 22.7 33.3 100 100 100 113 CP048071 100 22.7 33.3 100 100 100 114 CP023639 100 22.7 33.3 100 100 100 115 CP048071 100 22.7 33.3 100 100 100 116 CP053092 100 22.7 33.3 100 100 100 117 CP025598 100 22.7 33.3 100 100 100 118 CP025605 100 22.7 33.3 100 100 100 119 CP022577 100 22.7 33.3 100 100 100 120 CP025599 100 22.7 33.3 100 100 100 121 CP044345 100 22.7 33.3 100 100 100 122 CP018304 100 22.7 33.3 100 100 100 123 CP046728 100 22.7 33.3 100 100 100 124 CP023638 100 22.7 33.3 100 100 100 125 CP025597 100 22.7 33.3 100 100 100 126 CP047164 100 22.7 33.3 100 100 100 127 CP023640 100 22.7 33.3 100 100 100 128 CP025605 100 22.7 33.3 100 100 100 129 CP023638 100 22.7 33.3 100 100 100 130 CP016972 100 22.7 33.3 100 100 100

Experimental Example 2. Mycobacterium tuberculosis using the primer and probe set of the present invention ( Mycobacterium tuberculosis ) and Beijing family Mycobacterium tuberculosis detection

By performing multiplex real-time PCR using the primer and probe set prepared in Experimental Example 1, Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis were distinguished and detected. .

Specifically, for this detection experiment, each target DNA sample of Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family was obtained from Masan National Hospital. The final concentrations of the primer and probe sets of the present invention were adjusted to the final concentrations shown in Table 3 to prepare a reaction composition, and the final volume of the reaction composition was prepared to be 20 μL (Table 4). Using the prepared reaction composition, multiple real-time polymerase chain reactions (ABI7500; Applied Biosystems 7500 Real-time PCR Instrument System, Applied Biosystems, USA) were performed under the experimental conditions shown in Table 5, and the results were analyzed with detection software (ABI Version 2.3, USA) was used for analysis.

As a result, when performing multiple real-time polymerase chain reaction using the primer and probe set of the present invention, Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis are simultaneously detected, but they can be detected separately was confirmed (FIGS. 1A and 1B).

pathogen Primers and Probes final concentration (nM) Mycobacterium tuberculosis of the Beijing strain MTB Beijing-F 250 MTB Beijing-R 250 MTB Beijing-P 100 Mycobacterium tuberculosis MTB-F 250 MTB-R 250 MTB-P 100

reaction composition Volume (μL) ^* Master Mix 10 Primer and Probe Mix 5 template DNA sample 5 Sum 20

^* Master Mix: PowerAmp ^TM Real-time PCR Master Mix Ⅲ (Cat No. M0108), Kogen Biotech (Korea)

Temperature (℃) time number of cycles 50 2 minutes One 95 10 minutes One 95 15 seconds 35 ^* 60 1 min

^* 60: fluorescence detection step

Experimental Example 3. Confirmation of specificity of multiple real-time polymerase chain reaction using the primer and probe set of the present invention

In order to confirm the specificity of the multiple real-time polymerase chain reaction using the primer and probe set of the present invention, DNA samples of 10 types of Mycobacterium tuberculosis of Beijing and non-Beijing family (Table 6), non-tuberculous mycobacterium 5 Species DNA samples (Table 7), RNA/DNA samples of 9 types of respiratory disease viruses (Table 8), and 16 types of RNA/DNA samples (Table 9) including other pathogenic bacteria were subjected to detection experiments.

Specifically, multiple real-time polymerase chain reactions were performed under the same conditions and methods as those described in Experimental Example 2, except that the nucleic acid samples shown in Tables 6 to 9 below were used as templates.

As a result, in the Beijing system, even with the same Mycobacterium tuberculosis, only 5 types of Mycobacterium tuberculosis of the Beijing strain were detected, and in the Mycobacterium tuberculosis system, all 10 strains of Mycobacterium tuberculosis of the Beijing and non-Beijing strains were detected (Table 6). On the other hand, 5 non-tuberculous mycobacteriaceae (Table 7), 9 respiratory disease viruses (Table 8), and 16 other pathogenic bacteria (Table 9) were not detected in both Beijing and Mycobacterium tuberculosis systems. This indicates that the primer and probe set of the present invention can specifically detect Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family, and at the same time can detect them separately ( FIGS. 4A to 4C ).

division number pathogen source Beijing system Mycobacterium tuberculosis system Beijing strain Mycobacterium tuberculosis One M. tuberculosis Beijing family (1) National Masan Hospital Tuberculosis Specimen Bank + + 2 M. tuberculosis Beijing family (2) National Masan Hospital Tuberculosis Specimen Bank + + 3 M. tuberculosis Beijing family (3) National Masan Hospital Tuberculosis Specimen Bank + + 4 M. tuberculosis Beijing family (4) National Masan Hospital Tuberculosis Specimen Bank + + 5 M. tuberculosis Beijing family (5) National Masan Hospital Tuberculosis Specimen Bank + + Non-Beijing strain Mycobacterium tuberculosis 6 M. tuberculosis non-Beijing family (1) National Masan Hospital Tuberculosis Specimen Bank - + 7 M. tuberculosis non-Beijing family (2) National Masan Hospital Tuberculosis Specimen Bank - + 8 M. tuberculosis non-Beijing family (3) National Masan Hospital Tuberculosis Specimen Bank - + 9 M. tuberculosis non-Beijing family (4) National Masan Hospital Tuberculosis Specimen Bank - + 10 M. tuberculosis non-Beijing family (5) National Masan Hospital Tuberculosis Specimen Bank - +

division number pathogen source Beijing system Mycobacterium tuberculosis system Non-tuberculous mycobacteria One Mycobacterium smegmatis ^a ATCC 700084 ^{- -} 2 Mycobacterium phlei ATCC 11758 - - 3 Mycobacterium massiliense ^b KCTC 19086 ^{- -} 4 Mycobacterium marinum ATCC BAA-535 - - 5 Mycobacterium kansasii KCTC 9515 - -

^a ATCC: American Type Culture Collection

^b KCTC: Korean Collection for Type Cultures

division number pathogen source Beijing system Mycobacterium tuberculosis system respiratory disease virus One SARS-CoV 2 ^c NCCP 43326 - - 2 Influenza A virus H1N1 ^d KBPV-VR-76 - - 3 Influenza A virus H3N2 KBPV-VR-85 - - 4 Influenza B virus (Yamagata lineage) NCCP 43234 - - 5 Influenza B virus (Victoria lineage) KBPV-VR-72 - - 6 Human Parainfluenzavirus 1 MBC037 (Vircell) - - 7 Respiratory syncytial virus A KBPV-VR-73 - - 8 MERS-Cov2 MBC132 (Vircell) - - 9 adenovirus KBPV-VR-1 - -

^c NCCP: National Culture Collection for Pathogens

^d KBPV: Korea Bank for Pathogenic Viruses

division number pathogen source Beijing system Mycobacterium tuberculosis system other pathogenic bacteria One Bordetella pertussis ATCC 10380 - - 2 Haemophilus Influenzae MBC020 (Vircell) - - 3 Streptococcus pneumoniae MBC070 (Vircell) - - 4 Legionella pneumophila ATCC 33152 - - 5 Mycoplasma gallisepticum ATCC qCRM-19610D - - 6 Bacillus cereus ATCC 14579 - - 7 Campylobacter coli ATCC 33250 - - 8 Clostridium perfringens ATCC 12916 - - 9 Enterococcus hirae ^e KVCC-BA0700653 - - 10 Escherichia coli ATCC 43888 - - 11 Listeria monocytogenes ATCC 19113 - - 12 Pseudomonas aeruginosa ATCC 10145 - - 13 Salmonella Enteritidis NCCP 12236 - - 14 Staphylococcus aureus ATCC 23235 - - 15 Vibrio vulnificus ATCC 27562 - - etc 16 Human genomic DNA G304A (Promega) - -

^e KVCC: Korea Veterinary Culture Collection

Experimental Example 4. Sensitivity confirmation of multiple real-time polymerase chain reaction using the primer and probe set of the present invention

The sensitivity of the multiple real-time polymerase chain reaction using the primer and probe set of the present invention is a Mycobacterium tuberculosis -specific target gene IS6110 and a Beijing family Mycobacterium tuberculosis-specific target gene dnaA-IS6110 Complementary nucleotide sequence Each of the included DNA samples was confirmed by serial dilution by concentration. The sensitivity was determined as the lowest concentration when the detection rate was 95% or more.

Experimental Example 4-1. Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) Preparation of DNA samples for specific IS6110 and Beijing family Mycobacterium tuberculosis specific dnaA-IS6110 target genes

The nucleotide sequence information of Mycobacterium tuberculosis of each region including the sequence of the primer and probe set of the present invention described in Table 1 was collected from NCBI, and oligomers were synthesized by requesting an oligomer synthesis company (Bionics, Korea). Using the synthesized oligomer as a template, a singleplex real-time polymerase chain reaction was performed for each Mycobacterium tuberculosis under the same conditions and methods as those described in Experimental Example 2. The amplification products of each polymerase chain reaction were purified using a purification kit (QIAquick PCR Purification Kit, Qiagen, Germany). A plasmid was prepared by ligating the purified polymerase chain reaction amplification product of each Mycobacterium tuberculosis to pGEM T vector (pGEM T Vector System I, Promega, USA), and then, soluble cells (MAX Efficiency DH5a Competent Cell, Invitrogen , USA) was transformed.

Thereafter, each plasmid DNA was isolated using a kit (QIAprep Spin Mini prep Kit, Qiagen) to obtain synthesized DNA. The obtained DNA sample was quantified using a spectrophotometer, and the number of DNA copies was calculated using Equations 1 to 3 below.

[Equation 1]

Number of moles of dsDNA (mol) = Weight of dsDNA (g) / {(Length of ssRNA (nt) × 617.96 g/mol) + 36.04 g/mol}

[Equation 2]

Number of moles of dsDNA ends (mol) = Number of moles of dsDNA (mol) × 2

[Equation 3]

DNA copy number = number of moles of dsDNA × 6.022 × 10 ²³ copies/mol.

Experimental Example 4-2. Quantified Mycobacterium tuberculosis -specific IS6110 and Beijing family Mycobacterium tuberculosis-specific dnaA-IS6110 Serial dilution and sensitivity determination of DNA of each target gene

Mycobacterium tuberculosis -specific IS6110 and Beijing family Mycobacterium tuberculosis-specific dnaA-IS6110 DNA of each target gene quantified in Experimental Example 4-1 200 copies/μL, 100 copies/μL, 50 copies/μL , was prepared by serial dilution with Nuclease-free water (Applied Biosystems™, USA) to 25 copies/μL, 12.5 copies/μL. Multiple real-time polymerase chain reactions were performed under the same conditions and methods as those described in Experimental Example 2, except for each DNA template prepared in five concentration sections (Tables 10 and 11).

As a result, the primer and probe set of the present invention can effectively detect each of Mycobacterium tuberculosis-specific IS6110 and Beijing family Mycobacterium tuberculosis-specific dnaA-IS6110 target gene up to 50 copies/μL (Fig. 2). , Figure 3 and Tables 10 to 11).

Target Concentration Ct mean detection rate Mycobacterium tuberculosis Beijing strain (M. tuberculosis)
Beijing family) 2.0 × 10 ² copies/μL 30.6 100% 1.0 × 10 ² copies/μL 31.8 100% 5.0 × 10 ¹ copies/μL 33.1 100% 2.5 × 10 ¹ copies/μL 34.7 66.7% 1.25 × 10 ¹ copies/μL ND 0%

Target Concentration Ct mean detection rate M. tuberculosis 2.0 × 10 ² copies/μL 30.9 100% 1.0 × 10 ² copies/μL 31.9 100% 5.0 × 10 ¹ copies/μL 33.1 100% 2.5 × 10 ¹ copies/μL 34.8 66.7% 1.25 × 10 ¹ copies/μL ND 0%

<110> KoGene BioTech Co., LTD. <120> Composition for distinguishing between Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis and method for detecting Mycobacterium tuberculosis using the same <130> 2021P-05-010 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MTB Beijing-F <400> 1 gtgttcttcc gacaacgttc t 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MTB Beijing-R <400> 2 tcgatgaacc acctgacatg ac 22 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MTB Beijing-P <400> 3 cggcatgtcc ggagactcca gttc 24 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MTB-F <400> 4 gaaccgtgag ggcatcga 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MTB-R <400> 5 aggccgagtt tggtcatcag 20 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> MTB-P <400> 6 tggccagatg caccg 15

Claims (18)

The following nucleotide sequences (a) to (c) selected from the dnaA-IS6110 gene region present only in the Beijing family Mycobacterium tuberculosis and the following nucleotide sequence (d) selected from the IS6110 gene region present in Mycobacterium tuberculosis A composition for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family, comprising (f), comprising Mycobacterium tuberculosis, Mycobacterium tuberculosis of the Beijing family and other pathogens:
(a) the first forward primer base sequence represented by SEQ ID NO: 1 and specifically amplifying the dnaA-IS6110 gene region of Mycobacterium tuberculosis of Beijing lineage
(b) a first reverse primer base sequence represented by SEQ ID NO: 2 and specifically amplifying the dnaA-IS6110 gene region of Mycobacterium tuberculosis of Beijing lineage
(c) the first probe base sequence represented by SEQ ID NO: 3 and specifically amplifying the dnaA-IS6110 gene region of Mycobacterium tuberculosis of Beijing lineage
(d) a second forward primer base sequence represented by SEQ ID NO: 4 and specifically amplifying the IS6110 gene region of Mycobacterium tuberculosis
(e) a second reverse primer base sequence represented by SEQ ID NO: 5 and specifically amplifying the IS6110 gene region of Mycobacterium tuberculosis; and
(f) a second probe base sequence represented by SEQ ID NO: 6 and specifically amplifying the IS6110 gene region of Mycobacterium tuberculosis. delete According to claim 1,
The base sequences (c) and (f) are TaqMan probe technology applied, and Mycobacterium tuberculosis characterized in that a fluorescent dye and a quencher are further attached to its 3' end at the 5' end and Beijing strains ( Beijing family) composition for simultaneous detection of Mycobacterium tuberculosis. 4. The method of claim 3,
The fluorescent dye is 6-carboxyfluorescein (FAM), Texas red, fluorescein, fluorescein chlorotriazi nyl, HEX (2', 4', 5', 7'). -tetrachloro-6-carbpxu-4,7-dichlorofluorescein, rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), oregon green , Alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dime thoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), Tetrachloro-Fluorescein (TET) , TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhoda mine), NED(N-(1-Naphthyl) ethylenediamine), VIC(2′-chloro-7′phenyl-1,4-dich loro-6-carboxy -fluorescein), mycobacterium tuberculosis, characterized in that at least one selected from the group consisting of cyanine-based dyes and thiadicarbocyanine; composition for simultaneous detection of Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis . 4. The method of claim 3,
The quencher is TAMRA, BHQ (black hole quencher) 1, BHQ2, BHQ3, NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (deep dark quencher), Blackberry Quencher (Blackberry Quencher), Iowa black (Iowa black) ) Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) and Beijing family (Beijing family) composition for simultaneous detection of Mycobacterium tuberculosis, characterized in that at least one selected from the group consisting of. delete According to claim 1,
The other pathogen is Mycobacterium tuberculosis, characterized in that it comprises any one or more selected from non-tuberculous mycobacterium, respiratory disease virus, and other pathogenic bacteria, and a composition for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family. delete delete delete 8. The method of any one of claims 1, 3 to 5 and 7, wherein
The composition is a multiplex real-time polymerase chain reaction (Polymerase Chain Reaction, PCR), characterized in that used for Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) and Beijing family (Beijing family) composition for simultaneous detection of Mycobacterium tuberculosis. 8. The method of any one of claims 1, 3 to 5 and 7, wherein
The composition is a composition for simultaneous detection of Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis, characterized in that the minimum detection limit is 50 copies/μL, respectively. delete According to claim 1,
The composition is Mycobacterium tuberculosis, characterized in that it further comprises any one or more selected from a reverse transcription polymerase, a DNA polymerase, a cofactor such as Mg2+, dATP, dCTP, dGTP and dTTP ( Mycobacterium tuberculosis ) and Beijing family Mycobacterium tuberculosis composition for simultaneous detection of The kit for simultaneous detection of Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis, characterized in that it comprises the composition of claim 1. 1) performing multiple real-time polymerase chain reactions by adding the composition of claim 1 to obtain an amplification product; and
2) Simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis of the Beijing family, comprising the step of detecting and analyzing the amplification product. 17. The method of claim 16,
The composition of step 1) is a single tube and a single step (one tube and one step), characterized in that the cross-contamination is small Mycobacterium tuberculosis (Mycobacterium tuberculosis) and Beijing family (Beijing family) tuberculosis simultaneous detection method. 18. The method of claim 16 or 17,
The simultaneous detection method is Mycobacterium tuberculosis and Beijing family Mycobacterium tuberculosis, characterized in that i) Mycobacterium tuberculosis and other pathogens, ii) Beijing strain Mycobacterium tuberculosis and non-Beijing strain Mycobacterium tuberculosis, or i) and ii) are distinguished. Simultaneous detection method of

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