KR101498705B1 - Primer and probe for diagnosing tuberculosis, a kit comprising the same and a tu-berculosis-diagnosing method using the kit - Google Patents

Abstract

The present invention relates to a primer, a probe, and a detection method using the primer, the probe, and a detection method using the same. More particularly, the present invention relates to a primer and a probe for detecting a gene of Mycobacterium tuberculosis present in a biological sample and an environmental sample, To a method for detecting a gene. According to the present invention, it is possible to rapidly and accurately detect in real time as compared with the conventional method of detecting Mycobacterium tuberculosis, and the dried product of the polymerase chain reaction mixture containing the primer and the probe has the same performance as the solution state, Lt; / RTI >

Description

TECHNICAL FIELD [0001] The present invention relates to a primer for diagnosis of tuberculosis, a probe, a kit containing the same, and a diagnostic method for diagnosing tuberculosis using the kit. BACKGROUND OF THE INVENTION 1. Field of the Invention [0002]

The present invention relates to a primer, a probe, a kit containing the same, and a method for diagnosing tuberculosis using the kit. More particularly, the present invention relates to a primer, a probe, and a probe for detecting a gene of a tubercle bacillus present in a biological sample And a method for detecting Mycobacterium tuberculosis by polymerase chain reaction.

Tuberculosis is a highly contagious disease in which about one-third of the world's population is already infected. Despite the introduction of anti-tuberculosis chemotherapy and global management, the absolute number of tuberculosis patients is increasing year by year. Occupy the top. Tuberculosis mainly affects the lungs. In most cases, infection with pathogens in the lungs is controlled by the immune system and does not cause symptoms, but when the immune system becomes weak, lung disease is activated. Symptoms of pulmonary tuberculosis include fever, fatigue, loss of appetite and weight, chills and persistent cough. Pleurisy causes water in the chest cavity, which eventually destroys part of the lungs.

There are many ways to diagnose tuberculosis, and the most useful method is the culture of tuberculosis. However, the culture can take 6 to 8 weeks to miss important treatment times. Therefore, a real-time polymerase chain reaction (PCR) method is widely used as a recent diagnostic method.

Thus, the present inventors have designed novel primers and probes specific to Mycobacterium tuberculosis. By performing real-time PCR using the primers, probes and kits containing them, By drying the polymerase chain reaction mixture necessary for the reaction, the storage period is improved while maintaining the performance of the mixed solution in the solution state, and the error is minimized by minimizing the mixing process, resulting in highly reproducible results The present invention has been completed.

The present invention has been made in view of the above needs, and an object of the present invention is to provide a tuberculosis diagnostic primer and a probe for use in real-time PCR.

It is another object of the present invention to provide a kit for detecting a Mycobacterium tuberculosis comprising the primer and the probe, wherein all the reagents required for the PCR reaction are mixed, dispensed and dried in accordance with the test volume once, The present invention provides a tuberculosis diagnostic kit.

Another object of the present invention is to provide a method for quantitative diagnosis of Mycobacterium tuberculosis quickly and accurately.

In order to accomplish the above object, the present invention provides a primer and a probe for detecting a Mycobacterium tuberculosis DNA through a real-time PCR reaction or a general polymerase chain reaction.

The real-time PCR reaction of the present invention monitors the reaction result in real time by using an oligonucleotide probe in which a primer and a fluorescent substance are chemically bonded. This probe binds to the complementary sequence in the nucleic acid of the sample like the two primers in the polymerase chain reaction, the position being a little away from the primer. The probe of the present invention may have a structure in which a fluorescent substance such as a reporter and a quencher is attached to both ends of the probe. In this case, if the reporter and the quencher are present in close proximity, fluorescence of the reporter is not detected , And as the amplification progresses, the reporter is detected if the reporter falls off the quencher. Therefore, the intensity of fluorescence gradually increases as the amplification cycle increases.

The primers and probes of the present invention include a portion of the Mycobacterium tuberculosis IS6110 gene, for example, a part of the Mycobacterium tuberculosis IS6110 gene (GenBank Accession No. AJ242908) or a part of the complementary base sequence thereof, preferably the 1300th to 2100th bases Preferably 5 to 40, preferably 19 to 25, nucleotides in SEQ ID NO: 3 or SEQ ID NO: 4, more preferably a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, Sequence reverse primer. In addition, the probe is preferably a nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6, and is a forward probe. The primers and probes of the present invention were prepared based on the IS6110 gene sequence (see FIG. 1), which is a specific gene of Mycobacterium tuberculosis using BLAST of National Center for Biotechnology Information (NCBI). Therefore, the DNA of Mycobacterium tuberculosis can be easily detected by using the primers and probes of the present invention.

In addition, the present invention provides a kit for detecting Mycobacterium tuberculosis comprising the primer or the probe.

In addition to the primer or probe of the present invention, the kit may further include an amplification buffer solution, a dNTP, a control group, a detection reagent, and the like. The kit may be provided in a liquid or dry type, May include additional ingredients. The kit, which is provided in a dry state, has improved storage stability and can be used for a long period of time, and has a significant effect on the culture method, so that accurate results can be obtained in a shorter time.

The kit may further comprise an internal control primer and a probe. An internal positive control (hereinafter also referred to as 'IPC') template and primers suitable for the internal positive control (hereinafter also referred to as 'IPC') are prepared and put together when the PCR is performed, thereby easily confirming whether or not the PCR has been successfully performed. The primer includes, for example, a part of the Tobacco mosaic virus isolate Taigu movement protein (MP) gene (GenBank Accession No. FJ873800) or a part of the complementary base sequence thereof. Preferably, the primer is a 15 to 800 base The reverse primer is a forward primer comprising the nucleotide sequence of SEQ ID NO: 7 to SEQ ID NO: 9, and a reverse primer sequence of SEQ ID NO: 10 to SEQ ID NO: 12. In addition, the probe is preferably a base sequence represented by SEQ ID NO: 13 to SEQ ID NO: 15, and is a forward probe.

The primers and probes for the internal control were used as positive controls in the test, and when the negative judgment was made when the (real-time) PCR reaction was carried out using the present invention, that is, when the sample was found to be free of Mycobacterium tuberculosis, It is necessary to verify whether it is a real or non-existent M. tuberculosis, and should not interfere with tuberculosis detection when amplified together with the TB primer set of the present invention. If the internal control is positive, the polymerase chain reaction itself is not a problem.

The primers and probes for the tubercle bacilli detection or internal control may be any combination of two primers (one forward and one reverse), but preferably a forward primer described in SEQ ID NO: 1, 3 and the forward probe of SEQ ID NO: 5 can be used. The internal control primer and the probes may be any combination of two primers (one forward direction and one reverse direction) so long as they have one probe, preferably a forward primer described in SEQ ID NO: 7, And the forward probe described in SEQ ID NO: 13 can be used. The primers of the present invention can be used not only in real-time polymerase chain reaction but also in general polymerase chain reaction. Preferably, the reporter of the tuberculosis probe is FAM (6-carboxyfluorescein), the quencher is BHQ1 (2,5-di-tert-butylhydroquinone-1) and the reporter of the internal control probe is TAMRA (Carboxy-tetramethyl-hod -amine), and BHQ1 is preferably used as the quencher, but the present invention is not limited thereto.

In addition,

1) performing a polymerase chain reaction or a real-time PCR using the sample as a template using the above-described tuberculosis detecting primer and the above-mentioned tuberculosis detecting probe; And

2) detecting the presence or absence of amplification in step 1) or a fluorescence value for an amplification product.

According to the detection method of the present invention, it is possible to detect mycobacteria even when a very small amount of nucleic acid of Mycobacterium tuberculosis is present in the sample because of its high sensitivity. In particular, in real-time PCR, amplification can be observed immediately during amplification And the detection time can be reduced because no separate amplification product confirmation step is required. In the present polymerase chain reaction or real-time PCR, it is preferable to use IPC, but the present invention is not limited thereto. When the IPC template and the corresponding primer are prepared and put together, the PCR can be easily confirmed whether or not the PCR is performed well. In addition, the specimen can be obtained from clinical samples or environmental samples, but is not limited thereto.

By using the primers, probes and detection methods of the present invention, it is possible to detect the genes of Mycobacterium tuberculosis quickly and easily as compared with the conventional detection methods, and even the genes of very low concentrations of Mycobacterium tuberculosis in the sample can be detected can do. In addition, the development of the kit for the diagnosis of mycobacteria of the present invention is expected to accurately diagnose the early stage of the infection, and it is expected to contribute greatly to the confirmation of the drug resistance and the therapeutic effect of the mycobacteria through the monitoring of the drug therapy.

FIG. 1 shows that IS6110 gene sequence of Mycobacterium tuberculosis was found using BLAST of National Center for Biotechnology Information (NCBI). Primers and probes of the present invention were prepared in the sequence.
2 and 3 show real-time PCR using an Exicycler ^TM Real-Time PCR System (manufactured by Bioneer, Korea) as all combinations of the tuberculosis primers and probes of the present invention shown in SEQ ID NOS: FIG. 2 is a graph showing the result of performing a real-time PCR reaction again by selecting one PCR-efficient set. FIG.
Figure 2: Graphs amplified with primers and probes of SEQ ID NOS: 1, 3 and 5
3: Graphs amplified with primers and probes of SEQ ID NOS: 2, 4 and 6
FIG. 4 to FIG. 6 are schematic diagrams showing the results of a real-time PCR using an Exicycler ^™ Real-Time PCR System (manufactured by Bioneer, Korea) as primers and probes of DNAs for internal control of the present invention shown in SEQ ID NOS: And a chain reaction.
Figure 4: Graphs amplified with primers and probes of SEQ ID NOS: 7, 10 and 13
5: Graphs amplified with primers and probes of SEQ ID NOs: 8, 11 and 14
6: Graphs amplified with primers and probes of SEQ ID NOS: 9, 12 and 15
FIG. 7 is a graph showing the relationship between the primer and the probe of the present invention, which are represented by SEQ ID NOS: 1, 3 and 5 and SEQ ID NOS: 7, 10 and 13, using a real-time Quantitative Thermal block of Exicycler ^TM 96 real- Of the present invention.
Green curves: Amplification curves of tuberculosis DNA at concentrations of 10 to 10 ⁷ copies each
Blue Curve: Amplification curve by self-made internal control DNA
FIG. 8 is a graph showing the relationship between the concentrations of the primers and the probes of the present invention as shown in SEQ ID NOS: 1, 3 and 5 and SEQ ID NOS: 7, 10 and 13, using the Exicycler ^TM 96 real- The standard curve of the reaction graph is shown (slope: -0.2961, R ² : 0.9995).
FIG. 9 shows the results of real-time PCR using a primer and a probe of the present invention as shown in SEQ ID NOS: 1, 3 and 5 and SEQ ID NOS: 7, 10 and 13 using a Real Time PCR System A graph of the polymerase chain reaction is shown.
Black curve: Amplification curve of tuberculosis template DNA by 10 to 10 ⁷ copies concentration
Purple curve: Amplification curves by internal DNA for internal control
FIG. 10 shows a standard curve of a real-time PCR reaction plot of the concentration-specific tuberculosis standard template using 7500 Fast Real-Time PCR System (slope: -3.266328, R ² : 0.999409).
FIG. 11 is a flow chart showing the steps of preparing a standard template for tuberculosis using the iQ ™ 5 Real-Time PCR Detection System using a combination of primers and probes of the present invention described in SEQ ID NOS: 1, 3 and 5 and SEQ ID NOS: 7, 10, Lt; RTI ID = 0.0 > PCR < / RTI >
Green curve: Amplification curve of tuberculosis DNA by 10 to 10 ⁷ copies concentration
Blue Curve: Amplification curve by self-made internal control DNA
12 shows the standard curve of the real-time PCR reaction plot of the concentration-specific tuberculosis standard template using the iQ ™ 5 Real-Time PCR Detection System (slope: -3.536, R ² : 0.996).
13 is a graph showing a real-time PCR reaction of a standard template of tuberculosis using a dry PCR product comprising the primers and probes of the present invention, which are represented by SEQ ID NOS: 1, 3 and 5 and SEQ ID NOS: 7, 10 and 13 A real-time polymerase chain reaction device, Exicycler ^TM 96 Real-Time Quantitative Thermal block.
Black curve: Amplification curve of tuberculosis template DNA by 10 to 10 ⁷ copies concentration
Blue Curve: Amplification curve by self-made internal control DNA
14 is Exicycler ^TM 96 Real-Time Quantitative thermal block shows the standard curve of the real-time PCR reaction curve (slope: -0.2932, R ² : 0.9999) when the concentration-specific tubercle standard template is applied to the dry PCR mixture.
Figure 15 illustrates a graph of the real-time polymerase chain reaction in tuberculosis standard template using a PCR mixture in dry state, real-time polymerase chain reaction device Exicycler ^TM 96 Real-Time Quantitative Thermal block.
Black curves: Amplification curves of ^{10 to} 10 ¹⁰ copies of TB standard template
NTC: Blank as a negative control group
FIG. 16 is a graph showing real-time PCR using a control mixture, which is a liquid PCR mixture, for the storage stability test of a PCR mixture in a dry state. Real-time polymerase chain reaction device Exicycler ^TM 96 Real-Time Quantitative Thermal Blocks were used, and the tests were conducted on standardized tuberculosis samples at a concentration of 10 ³ to 10 ⁶ copies. The formula at the top of the graph shows the standard curve of the real-time polymerase chain reaction graph using the concentration-specific tuberculosis standard template, which shows the slope of -0.3045 and the ratio of R ² : 0.9998.
Black curve: Amplification curve of tuberculosis DNA by concentration
Blue curve: Amplification curve of DNA for self-made internal control
NTC: Blank as a negative control group
FIGS. 17 to 22 are graphs showing the results of real-time PCR for a total storage period of 6 days after storing the dried PCR mixture at 40.degree. C. for storage stability test of the PCR mixture in a dry state. Real-time polymerase chain reaction device Exicycler ^TM 96 Real-Time Quantitative Thermal Blocks were used, and the tests were conducted on standardized tuberculosis samples at a concentration of 10 ³ to 10 ⁶ copies. The formula at the bottom of the graph shows the standard curve of the real-time polymerase chain reaction graph employing the concentration-specific tuberculosis standard template. The slope is in the range of -2.78 to-3.05 and the R ² value is in the range of 0.9989 to 0.9999. . 1 to 6 days indicates the total number of days stored at 40 ° C.
Black curve: Amplification curve of tuberculosis DNA by concentration
Blue curve: Amplification curve of DNA for self-made internal control
23 is a result of Mycobacterium tuberculosis (M.tuberculosis) extracting DNA from the positive specimens and is one using the dry PCR mixture perform real-time polymerase chain reaction graph obtained using ^{Exicycler TM 96 Real-Time Quantitative Thermal} block.
Green: Tuberculosis amplification curve
Blue: Amplification curve of DNA for internal control
FIG. 24 is a graph showing the real-time PCR using the dried PCR mixture after extracting the DNA from the tuberculosis negative sample and using the Exicycler ^TM 96 real-time quantitative thermal block. In the case of negative specimens, the amplification curve of tuberculosis does not appear similar to the result of NTC (blank trial).
Black: Tuberculosis amplification curve
Blue: Amplification curve of DNA for internal control
FIG. 25 is a graph showing the real-time PCR reaction using DNA polymerase extracted from atypical M. intracellulare (NTM) positive specimens and using the dried PCR mixture. As shown in FIG. 25, Exicycler ^TM 96 Real-Time Quantitative Thermal block As shown in Fig. In the case of NTM-positive specimens, the amplification curve of tuberculosis does not appear similar to the NTC results.
Black: Tuberculosis amplification curve
Blue: Amplification curve of DNA for internal control
Figure 26 is a graph of extracting DNA from a positive CT (Chlamydia trachomatis), and the sample, using a dry mixture PCR perform real-time polymerase chain reaction, Exicycler ^TM 96 Real-Time Quantitative Thermal block. In the case of CT-positive specimens, the amplification curve of tuberculosis does not appear similar to the NTC results.
Black: Tuberculosis amplification curve
Blue: Amplification curve of DNA for internal control
FIG. 27 is a table showing the results obtained by extracting DNA from 238 specimens, performing real-time RT-PCR using the dried PCR mixture, and comparing the results with those of the culture. Sensitivity is a sensitivity, Specificity is the specificity, PPV is the positive predictive value, NPV is the negative predictive value, and efficiency is the efficiency of the PCR mixture.

Hereinafter, the present invention will be described by way of examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1. Preparation of template DNA and internal control DNA

In order to carry out a real-time PCR reaction thereafter, a template DNA was first prepared. The IS6110 gene sequence of Mycobacterium tuberculosis was aligned to identify regions of high homology (Fig. 1). Then, 405 bp of the 1558th to 1962nd sequence including the primer and the probe sequence in the IS6110 gene (GenBank Accession No. AJ242908) was amplified by a gene synthesis method ( NBiochem. Biophys. Res. Commun. 1998, 248, 200-203) And cloned into pGEM-T-Easy Vector (Cat: A1360, Promega, USA).

Specifically, 5 μl of 2 × rapid ligation buffer (Promega, USA), 1 μl of T-Easy Vector (Promega, USA), 1 μl of T4 DNA ligase (Promega, USA) and 3 μl of gene synthesis material (8 μg) The mixture was placed in the same tube and incubated at 37 ° C for 1 hour. Thereafter, 5 μl of the above reaction solution was placed in 50 μl of E. coli competent cells. The reaction solution was placed on ice for 30 minutes, incubated at 42 ° C. for 90 seconds, and placed on ice again for 2 minutes. The reaction solution was inoculated on an LB plate containing ampicillin, IPTG, and X-Gal and cultured at 37 DEG C for 16 hours.

Colonies of white color were taken and cultured in LB liquid medium for about 16 hours. After centrifuging, the supernatant was discarded and the plasmid DNA was extracted from the pellet using Accuprep Ðplasmid DNA prep kit (Bioneer, Korea). The plasmid DNA was measured for purity and purity by a UV spectrophotometer (Shimazu, Japan), and the purity was confirmed to be between 1.8 and 2.0. Based on the result of the concentration measurement, the copy number of the DNA was calculated by the following formula Respectively.

6.02 × 10 ²³ × concentration (concentration g / ml measured by UV spectrometer) / (3015 + 405) × 660 [3015 bp: size of T-easy vector, 405 bp: size of tubercle DNA]

The copy number of the template DNA was calculated and then 10-diluted with 1X TE + BSA buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.6% acetylated BSA) and stored at -70 ° C until use.

An internal control DNA was prepared in the same manner as the above-mentioned template DNA preparation. Internal control DNA is necessary to ensure that when negative results are obtained, the negative results are not due to amplification errors.

Tobacco mosaic virus isolate The 763 bp region of the 37th to 799th sequence including the primer and the probe sequence was used for gene amplification of the internal control DNA in the Taigu movement protein (MP) gene (GenBank Accession No. FJ873800) Based on the results of the concentration of extracted plasmid DNA, the number of DNA copies was calculated by the following formula.

[Size of the internal control DNA 3015 bp:: T-easy vector size, 763bp] 6.02 × 10 ²³ × concentration (the concentration g / ㎖ measured with a UV spectrometer) / (3015 + 763) × 660

The number of copies of DNA for internal control was calculated and then 10-diluted with 1X TE + BSA buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.6% acetylated BSA) and stored at -70 ° C until use.

Example 2. Primer and probe design

The nucleotide sequence of the tuberculosis IS6110 gene (GenBank Accession No. NC_002944) was selected between 1558 and 1962 to have a length of 19 to 25 bp and a Tm value of 55 to 65 ° C. Between 1558 and 1962, the sequence was arbitrarily selected between 20 and 30 bp and Tm value was between 67 and 77 ° C, and the Tm value was checked using the Primer 3 Plus program (Table 1) .

Tobacco mosaic virus isolate for internal control The nucleotide sequence of the Taigu movement protein (MP) gene (GenBank Accession No. FJ873800) was 17 to 23 bp in the nucleotide sequence of 37 to 799 and the Tm value was 55 to 62 ° C. Were selected as forward and reverse primers. Between 942 and 1708 of the nucleotide sequence, the base sequence was arbitrarily selected between 19 and 30 bp and the Tm value was between 67 and 72 ° C, and the Tm value was checked using the Primer3 Plus program (Table 2) .

First, after combining all of the TB germs primer and probe set to Exicycler ^TM Real-time quantitative system (Bioneer, USA) was used to perform real-time PCR (Table 3 and Table 4). Specifically, 5 .mu.l of 10X buffer, 2.5 .mu.l of wTfi polymerase, 20 .mu.M of dNTP, Thermostable Pyrophosphatase, PPi, stabilizer and the like were put into a tube, and the tuberculin DNA, the primer for detecting tuberculosis, probe and distilled water synthesized in Example 1 The mixture was mixed so as to have a volume of 50 μl, and the mixture was dispensed into a 96-well plate. At this time, the concentration of the forward primer, the reverse primer and the probe contained in the mixed solution having a total volume of 50 μl was 15 pmole, respectively. This was denatured at 95 DEG C for 10 minutes, and then subjected to 45 cycles of 95 DEG C for 20 seconds and 55 DEG C for 30 seconds. The amplified fluorescence values were continuously measured once at 55 ° C for 30 seconds after each PCR cycle. As a result of the reaction, test set 1 having a good PCR efficiency was selected (Table 1). That is, it was found that the primer and the probe having the highest PCR amplification efficiency were the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 3, and the forward probe of SEQ ID NO: 5 (FIGS.

In addition, the DNA for internal control and the primers and probes for internal control synthesized in Example 1 were combined in a set, and a real-time PCR was performed in the same manner as described above.

The reaction conditions were denaturation at 95 ° C for 10 minutes, followed by 45 cycles of 95 ° C for 20 seconds and 55 ° C for 30 seconds. As a result, primers and probes for internal control in which PCR amplification was efficiently confirmed were selected (Table 2). Among the primers and probes, the PCR primer having the highest PCR amplification efficiency was selected from the forward primer of SEQ ID NO: 7, the reverse primer of SEQ ID NO: , And a forward probe of SEQ ID NO: 13 (Figs. 4 to 6).

In the subsequent experiments, the results of the PCR primer and the probe having the highest PCR amplification efficiency (the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 3, the forward probe of SEQ ID NO: 5) (The forward primer of SEQ ID NO: 7, the reverse primer of SEQ ID NO: 10, and the forward probe of SEQ ID NO: 13).

Example 3. Standard template real-time polymerase chain reaction

Using the tuberculosis DNA prepared in Example 1 and the internal control DNA as templates, the tuberculosis primer and probe described in SEQ ID NOS: 1, 3 and 5 selected in Example 2, and the nucleotide sequences of SEQ ID NOS: 7, 10 and 13 applying a primer and a probe for an internal control that is to Exicycler ^TM Real-Time Quantitative Thermal Block (Bioneer Co., Korea), 7500 Fast Real-Time PCR System (applied Biosystems Co., USA) and iQ ™ 5 Real-Time PCR Detection System ( Real-time polymerase chain reaction was performed using BioRad (USA). A real-time PCR reaction of 45 cycles was performed under the same conditions and components as in Example 2, except that the negative control (blank without DNA microbial strain) was reacted together.

As a result, when the number of copies was calculated in accordance with the method of Example 1, the tubulin template DNA was detectable up to 10 copies (FIGS. 7, 9, 11). When a standard graph of the standard template real- The slope was -2.96 to-3.53 and the R ² value was 0.996 to 0.9995 (Figs. 8, 10 and 12). Here, R ² is a correlation coefficient indicating the linearity of the graph when the standard graph of the real-time PCR is drawn. It means that the closer to 1 (closer to the straight line) the PCR proceeds. In addition, it was confirmed that internal control amplification was independently performed without affecting the amplification of the tubercle DNA template even when 10 ⁵ copies of the DNA for internal control were reacted together.

Example 4. Standard template real-time polymerase chain reaction using dry type PCR composition

PCR Mixture (Premix) For the purpose of examining the thermal stability of the dried product, a PCR mixture having the same composition as in Example 2 was prepared and dried, and then a real-time polymerase chain reaction was performed using Exicycler ^™ Quantitative Thermal Block Respectively.

Specifically, 5 μl of 10 × Buffer, 10 μl of wTfi polymerase, 2.5 μl of dNTP, 2.5 μl of Thermostable Pyrophosphatase, PPi, and stabilizer were added to a tube and distilled water was added thereto to give a total volume of 25 μl. , SuperCentra 2 (Bionics Co., Korea) for 50 to 60 minutes. Then, the tuberculosis primers and probes described in SEQ ID NOS: 1, 3 and 5 selected in Example 2, and the primers and probes for internal control according to SEQ ID NOS: 7, 10 and 13 were mixed to a total volume of 5 μl The mixture was added to the primary dry matter and dried for 25 to 30 minutes.

To the dried PCR mixture, the tuberculosis DNA prepared in Example 1 and the DNA for internal control were added as a template and mixed with distilled water to give a total volume of 50 占 퐇, and the resulting mixture was thoroughly mixed so that the dried material was well pulverized. Real-time polymerase chain reaction (PCR) was performed under the same conditions and components as in Example 2, except that Exicycler ^TM Real-Time Quantitative Thermal Block (manufactured by BIONIA Co., Ltd., Korea) was used and a negative control (blank sample without tubercle DNA template) Reaction was carried out.

As a result, the number of copies was calculated according to the method of Example 1, and the tubulin template DNA was detectable up to 10 copies (FIG. 13). When a standard graph of the standard template real-time PCR was prepared, the slope was -2.932 , And the R ² value was 0.9999 (Fig. 14). Here, R ² is a correlation coefficient indicating the linearity of the graph when the standard graph of the real-time PCR is drawn. It means that the closer to 1 (closer to the straight line) the PCR proceeds. These results show that the two methods of real-time polymerase chain reaction using the mixture of the solution and the dry mixture maintain the same performance.

A Quantitative Thermal Block of Exicycler ^TM (Bionics Co., Ltd., Korea) was used to confirm the dynamic range (concentration range of the tuberculosis DNA detectable in one reaction) using the dried PCR mixture in the same manner as described above. , 45 cycles of real-time PCR were carried out under the same conditions as in Example 2. [ Tuberculous template DNA was diluted 10 times at a concentration ranging from 10 ¹⁰ copies to a minimum of 10 copies at a concentration of 10 times in accordance with the method of Example 1, and tuberculous DNA was detected normally in a range of 10 log (FIG. 15) .

Example 5. Stability test according to storage period of dry type PCR composition

Using the same composition and method as in Example 4, the tuberculosis primers and probes described in SEQ ID NOS: 1, 3 and 5 and the primers and probes for internal control according to SEQ ID NOS: 7, 10 and 13, The mixture was incubated at 40 ° C for 6 days at an interval of 1 day, and the dry type PCR composition for each storage period was analyzed using Exicycler ^TM Real-Time Quantitative Thermal Block (Bionea, Korea) Real - time PCR was performed under the same conditions. This is presumed to be stored at -20 ° C for about 64 days when stored at 40 ° C for one day in a short-term predictable manner by accelerating the performance maintenance period of the dried PCR composition at the actual storage recommended temperature of -20 ° C (Table 5).

Specifically, the dry type PCR composition was prepared in a batch of 7 days in the same batch, and then a portion of the mixture immediately after drying was used in a real-time PCR reaction of 45 cycles under the same conditions as in Example 2, . All other dry type PCR compositions were placed in a 40 ° C thermostat at the same time and taken out at intervals of one day as needed for the reaction, and real-time PCR was performed. At this time, the number of copies was calculated according to the method of Example 1, and the reaction was carried out using four concentrations ranging from a maximum of 10 ⁶ to a minimum of 10 ³ copies. The slope value and the R ² value of the results of the real-time PCR of the dry mixture for each storage period obtained using Exicycler ^TM Real-Time Quantitative Thermal Block (Bionics Co., Korea) were compared with the control results, And 40 ℃ storage days.

The slurry was -3.04 and the R ² value was 0.9998 (FIG. 16) in the liquid mixture of the PCR mixture, and the dry type PCR composition stored at 40 ° C. Was found to have a slope of -2.94 to-3.13 and an R ² value of 0.9999 to 1.0 (Figs. 17 to 22). This means that the same results as those obtained immediately after drying even after 6 days of 40 ° C. stationary setting are obtained, and it is possible to obtain a result similar to that immediately after drying for about 384 days in terms of storage days at -20 ° C.

N.Temp: Normal temperature means normal temperature.

Example 6. Detection test of tuberculin specimen using dry type PCR composition

Using the dry type PCR composition comprising the tuberculosis primers and probes described in SEQ ID NOS: 1, 3 and 5 and the primers and probes for internal control described in SEQ ID NOs: 7, 10 and 13 prepared in Example 4, Were subjected to detection tests. DNA extracted from a specimen determined to be positive or negative for tuberculosis by culturing using sputum and a specimen determined to be positive for CT ( Chlamydia trachomatis ) by culturing using patient urine was analyzed using Exicycler ^TM Real-Time Quantitative Thermal Block Real-time polymerase chain reaction was performed under the same conditions as in Example 2.

Specifically, Bead beating method (Tuberculosis Research Institute) was used to extract DNA from 1 ml of Sputum-positive specimen. The extraction procedure was carried out according to the recommended method and stored at -70 ° C until use.

In addition, the DNA extracted from specimens determined to be positive for tuberculosis, negative specimens similar to tuberculosis (NTM: M. intracellulre ) and those positive for CT ( Chlamydia trachomatis ) were analyzed by Exicycler ^TM Real-Time Quantitative Real-time polymerase chain reaction was performed under the same conditions as in Example 2 using a thermal block. this is In order to confirm whether the primer and the probe of the present invention have the same result when applied to the standard template of Example 3 as well as the actual tuberculosis positive specimen, In order to confirm the presence or absence of a cross-reaction.

As a result, all of the TB genes were amplified for the above-mentioned tuberculosis-positive specimens. In order to determine the cross-reactivity, DNA was extracted from the tuberculosis negative specimens and CT positive specimens, Respectively. As a result, it was confirmed that the primers and probes of the present invention were able to simultaneously detect four kinds of tuberculosis genes and did not cross-react with irrelevant pathogens (FIGS. 23 to 26).

Example 7. Detection of tuberculin specimen using dry type PCR composition Specificity (sensitivity) test

A detection test was performed on tuberculin specimens using the dry type PCR mixture prepared in Example 4 above. The specimen is 238 specimens determined to be positive or negative by culturing using patient sputum, and includes 43 tuberculosis positive specimens and 195 DNA extracted from tuberculosis negative specimens. The tuberculosis sample was extracted from 1 ml of sputum using bead beating method (Tuberculosis Research Institute) and used for the test after freezing at -20 ℃.

Specifically, the dry type PCR composition comprising the tuberculosis primers and probes described in SEQ ID NOS: 1, 3 and 5 and the primers and probes for internal control described in SEQ ID NOS: 7, 10 and 13, The tuberculosis DNA prepared in Example 1 and the DNA for internal control were added as a template and mixed with distilled water to give a total volume of 50 μl, and the mixture was thoroughly mixed so that the dry composition was well pulverized. At this time, except for the template DNA, the DNA extracted from each sample was substituted for the same components as above for the sample test. 45 cycles of real-time PCR were performed under the same conditions as in Example 2 using Exicycler ^TM Real-Time Quantitative Thermal Block (Bioneer, Korea).

As a result, all 43 tuberculosis specimens were found to be positive in 100 specimens and 10 specimens were positive in microscopic examination. Patients were followed-up for an additional positive specimen, and the patient was already diagnosed with tuberculosis and was being treated or cured (Table 6). Based on the culture results, the sensitivity and specificity of the dry type PCR composition provided in the present invention were 95.5% and 95.3%, respectively, and the positive predictive value was 79.2% (FIG. 27).

<110> BIONEER Corporation <120> PRIMER AND PROBE FOR DETECTION OF TUBERCULOSIS, KIT COMPRISING          THE SAME, AND DETECTION METHOD OF TUBERCULOSIS USING THE SAME <130> 2010-opa-4518 <160> 15 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Tuberculosis <400> 1 cagggttagc cacactttgc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Tuberculosis <400> 2 agtttggtca tcagccgttc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Tuberculosis <400> 3 gcgaactcaa ggagcacatc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Tuberculosis <400> 4 gccaactacg gtgtttacgg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward probe for Tuberculosis <400> 5 tagttggcgg cgtggacgcg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward probe for Tuberculosis <400> 6 cagggttagc cacactttgc 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for IPC <400> 7 agatttcagt tcaaggtcgt tc 22 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for IPC <400> 8 tcagttcaag gtcgttcc 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for IPC <400> 9 acaattgcag aggaggtg 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for IPC <400> 10 gaaacccgct gacatctt 18 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for IPC <400> 11 gagaaagcgg acagaaacc 19 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for IPC <400> 12 tgggaacgac cttgaact 18 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward probe for IPC <400> 13 acgcgatgaa aaacgtctgg caagt 25 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward probe for IPC <400> 14 acgcgatgaa aaacgtctgg caagt 25 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward probe for IPC <400> 15 ctggtggaca aaaggatgga aagagc 26

Claims (18)

A reverse primer of SEQ ID NO: 3, and a probe of SEQ ID NO: 5. The method according to claim 1,
A forward primer of SEQ ID NO: 2, a reverse primer of SEQ ID NO: 4, and a probe of SEQ ID NO: 6. delete delete The method according to claim 1,
Wherein the probe is a structure having a reporter and a quencher attached thereto. The method of claim 5,
Wherein the reporter is FAM. The method of claim 5,
Wherein the quencher is BHQ1. A kit for detecting Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 3, and a probe of SEQ ID NO: 5. The method of claim 8,
The kit for detecting Mycobacterium tuberculosis further comprises an internal control gene, a primer, and a probe. The method of claim 8,
The kit for detecting Mycobacterium tuberculosis is a dry type. The method of claim 9,
Wherein the internal control gene, primer and probe are derived from a nucleotide sequence of Tobacco mosaic virus isolate Taigu movement protein (MP) gene (GenBank Accession No. FJ873800). The method of claim 11,
The primer is selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 7 to SEQ ID NO: 12, and the probe is selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 13 to SEQ ID NO: 1) performing a polymerase chain reaction or a real-time PCR using a specimen as a template using a composition for detecting mycobacteria comprising the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 3, and the probe of SEQ ID NO: 5; And
2) detecting the presence or absence of amplification in step 1) or the fluorescence value for the amplification product. 14. The method of claim 13,
Wherein the step of performing the chain reaction is performed by further using an internal control gene, a primer, and a probe. 15. The method of claim 14,
Wherein the internal control gene, the primer and the probe are derived from a nucleotide sequence of Tobacco mosaic virus isolate Taigu movement protein (MP) gene (GenBank Accession No. FJ873800). 16. The method of claim 15,
Wherein the primer is selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 7 to SEQ ID NO: 12, and the probe is selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 13 to SEQ ID NO: The method of claim 8,
A forward primer of SEQ ID NO: 2, an inverse primer of SEQ ID NO: 4, and a probe of SEQ ID NO: 6. 14. The method of claim 13,
Wherein the composition for detecting Mycobacterium tuberculosis further comprises a forward primer of SEQ ID NO: 2, a reverse primer of SEQ ID NO: 4, and a probe of SEQ ID NO: 6.

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