KR20130142220A - Improved method for diagnosing mycobacterium tuberculosis using one-tube nested real-time pcr - Google Patents

Abstract

The present invention relates to an improved method for diagnosing Mycobacterium tuberculosis using one-tube nested real-time PCR. The method of one-tube nested PCR of the present invention using two pairs of Mycobacterium tuberculosis-specific primers is quick and specific to Mycobacterium tuberculosis and has high sensitivity, thereby being very useful in diagnosis of tuberculosis.

Description

TECHNICAL FIELD The present invention relates to a method for diagnosing Mycobacterium tuberculosis using one-tube nested real-time PCR,

The present invention relates to a method for diagnosing improved Mycobacterium tuberculosis using one-tube nested real-time fish, wherein the one-tube nested PCR method of the present invention using two pairs of Mycobacterium tuberculosis specific primers is rapid, specific for Mycobacterium tuberculosis, It is useful for diagnosis of tuberculosis.

In general, tuberculosis is one of the infectious diseases caused mainly by respiratory infections caused by Mycobacterium tuberculosis . It is estimated that 1/3 of the world's population is infected with Mycobacterium tuberculosis, and WHO 2010 statistics show that 880 It is a major disease in which one million new TB patients develop and 1.1 million die from tuberculosis (Kumar V, Abbas AK, Fausto N, Mitchell RN, Robbins Basic Pathology, 2007. 8th Edition, p.152-160, Saunders Elsevier, Collingwood , Canada .; World Health Organization (WHO). Global tuberculosis control: WHO report 2011. 2011). In Korea, tuberculosis prevalence has been decreasing once, but tuberculosis patients such as women and children are increasing, and it is becoming a social problem. In order to reduce the incidence and mortality of these tuberculosis, treatment and management of tuberculosis are important, and rapid, specific and sensitive tests for tuberculosis are needed (World Health Organization (WHO).) Diagnostics for tuberculosis: global demand and market potential. ).

Bacteriological tests such as AFB smear test and cultured mycobacterial culture are widely used as diagnostic methods for tuberculosis. Although the conventional AFB smear test is inexpensive and quick to obtain test results, it is widely used around the world. However, it can be detected with more than 5 × 10 ^{3 to 4} mycobacteria, which is less sensitive than culture test. (Bates JH, 1979. 76: 757-763, Kent P, Kubica G. Public health mycobacteriology, A guide for the level III laboratory, 1985. p. .57-68 US Public Health service Washington DC: US Government Printing Office). In contrast, the culture test is a standard method for diagnosing tuberculosis. However, it can be detected even with 10 ^{1 or 2} mycobacteria, and it has high specificity. However, due to the characteristics of mycobacteria, it takes a long time (4 weeks ~ 8 weeks) (Tenover FC, Crawford JT, Huebner RE, Geiter LJ, Horsburgh CR, Jr., Good RC. The resurgence of tuberculosis: is your laboratory ready? J Clin Microbiol 1993. 31: 767-770; techniques in mycobacterial detection, Arch Pathol Lab Med 2001. 125: 122-126). In addition, in the case of sputum as a specimen, it is difficult to diagnose pulmonary tuberculosis. In case of patients with poor sputum, children and women, and patients with tuberculosis and HIV infection, And the diagnosis of tuberculosis is difficult.

For more rapid and accurate testing, nucleic acid amplification tests (PCR) to confirm the DNA of Mycobacterium tuberculosis by polymerase chain reaction (PCR) and real-time PCR (molecular biology) amplification test, NAAT) (Beavis KG, Lichty MB, Jungkind DL, Giger O. Evaluation of Amplicor PCR for direct detection of Mycobacterium tuberculosis from sputum specimens. J Clin Microbiol. 1995. 33: 2582-2586; Woods GL. Molecular techniques in mycobacterial detection. Arch Pathol Lab Med. 2001. 125: 122-126). PCR, one of the representative NAAT, amplified more than 2 ³⁵ specific regions of the sample DNA and confirmed by electrophoresis (Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. DNA polymerase is a thermostable DNA polymerase that is capable of providing rapid and accurate results within the same day as the smear test and culture, (Levin et al., 1983), and the results of the present study are as follows: (1) the presence of a nucleic acid E, Gesouli E, Pappa C, Stefanou D. Four-year experience of using the Cobas Amplicor system for rapid detection of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens in Greece. Eur J Clin Microbiol Infect Dis. 2003. 22: 349-356; Tang YW, Meng S, Li H, Stratton CW, Koyamatsu T, Zheng X. PCR Enhances acid-fast bacillus stain-based rapid detection of Mycobacterium tuberculosis J Clin Microbiol. 2004. 42: 1849-1850). In this regard, real-time PCR, which can detect amplification products in real time using a specific oligonucleotide probe labeled with a fluorescent dye, has been widely used for the diagnosis of tuberculosis (Kraus G, Cleary T, Miller N, Seivright R, Young AK, Spruill G, Hnatyszyn HJ, Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes and real-time PCR. Mol Cell Probes. 2001 15: 375-383). Most commercialized studies or kits based on real-time PCR are used for identification and diagnosis by performing a single real-time PCR.

However, AFB staining is used to confirm the presence of a small number of Mycobacterium tuberculosis in the specimens. In order to identify the tuberculosis bacterium positive, at least 10 ⁵ bacterial cells per 1 ml of sputum are available. Thus, a sensitive diagnostic method is needed to diagnose a small amount of Mycobacterium tuberculosis in a specimen. In addition, among the patients with tuberculosis, detection of tuberculosis in urine is easy in patients with renal tuberculosis. In other cases, it is difficult to detect tuberculosis with urine. However, even in the case of pulmonary tuberculosis, the tuberculosis bacillus is decomposed in the lungs and cell-free nucleic acid is passed through the blood circulation system into the kidney and is discharged into the urine, so that it is known that the tuberculosis DNA (transrenal DNA) can be detected in the urine. However, since most of the tuberculosis bacterium that is discharged into the urine is filtered through the glomerulus network of the kidney, a small amount of tubercle bacillus that can not be detected in the renal glomerulus network is detected in order to be discharged into the urine and a nucleic acid of about 70 kDa or 100 bp It is known that tuberculosis can be detected in the urine (Green C, Huggett JF, Talbot E, Mwaba P, Reither K and Zumla AI. Rapid diagnosis of tuberculosis

through the detection of mycobacterial DNA in urine by nucleic acid amplification methods.Lancet Infect Dis. 2009 Aug; 9 (8): 505-11).

As a target of amplification, IS6110 has 1 to 20 copies of copy number, and in many studies, IS6110-targeted PCR is known to have the highest sensitivity. However, there is a disadvantage in that MTB complex sequences are similar to those of various NTMs, and recent strains that do not have this gene in the MTB strain have also been reported. Therefore, we tried to construct a sensitive and specific real-time PCR by solving the false positives and false negatives by simultaneously amplifying the rpoB gene that acts as a target RNA polymerase.

Nested PCR is a technique to increase the specificity and sensitivity of PCR amplification. It is usually used to amplify low purity DNA or RNA. In this method, 1 ^st PCR is performed using the primer of the target gene as successive PCR with two kinds of primers, 2 ^nd PCR is performed using the second primer located inside the first PCR product,

In the present invention, when real-time PCR is performed, one-tube nested PCR, in which two genes are used and two pairs of primers specific to each gene are amplified in the same tube, And to provide a method for diagnosing tuberculosis in various specimens by improving sensitivity and sensitivity.

As a related patent, Korean Patent Publication No. 1020030063935, a primer for amplifying the hsp65 gene of Mycobacterium sp., And a method for identifying the mycobacterium genus using the primer pair, comprises a pair of primers specific to mycobacterium sp. The present invention relates to a method for identifying a mycobacterial species, and more particularly, to a method for identifying a hsp65 gene of Mycobacterium spp. By amplifying a hsp65 gene of Mycobacterium sp. Discloses a method for identifying an Mycobacterium sp. Strain by RFLP analysis using a specific restriction enzyme site in a nucleic acid (Restriction Fragment Length Polymorphism Analysis).

The present invention solves the above problems and aims to provide a method for diagnosing tuberculosis in various specimens by improving specificity and sensitivity.

Another object of the present invention is to provide a composition capable of diagnosing tuberculosis in various specimens by improving specificity and sensitivity.

It is still another object of the present invention to provide a primer and / or a probe capable of diagnosing tuberculosis in various specimens.

In order to achieve the above object, the present invention provides a primer set consisting of the primers of SEQ ID NOS: 1 to 4 and SEQ ID NOS: 6 to 9.

The present invention also provides a kit for detecting Mycobacterium tuberculosis comprising the primer set of the present invention.

In a preferred embodiment of the present invention, the kit preferably further comprises a probe, and more preferably, the probe is selected from the group consisting of SEQ ID NO: 5 and SEQ ID NOs: 10 to 11, but is not limited thereto.

In another embodiment of the present invention, the 5 'terminal and the 3' terminal of the probe are preferably labeled with a fluorescent substance, and the fluorescent substance labeled at the 5 'terminal is 6-carboxyfluorescein, FAM ), Hexachloro-6-carboxyfluorescein (HEX), tetrachloro-6-carboxyfluorescein, and Cyanine-5 (Cy5) And the fluorescent substance labeled at the 3 'end is 6-carboxytetramethyl-rhodamine (TAMRA) or black hole quencher-1,2,3 (BHQ-1,2 , 3), but is not limited thereto.

In another embodiment of the invention, the Mycobacterium tuberculosis is Mycobacterium tuberculosis, M. bovis, M. africanum, M. canetii, and M. microti.

The present invention also relates to a method for obtaining DNA from a sample and real-time PCR using the DNA as a template, wherein the primer of SEQ ID NOs: 1 to 2 and SEQ ID NOs: 6 to 7 is used as a first primer, And amplifying the primers of SEQ ID NOS: 3 to 4 and SEQ ID NOS: 8 to 9 in the same tube as the DNA.

In one embodiment of the present invention, the tube further comprises a probe, and the probe is preferably a probe selected from the group consisting of SEQ ID NO: 5 and SEQ ID NOs: 10 to 11, The terminal is more preferably labeled with a fluorescent substance, but is not limited thereto.

The kit of the present invention includes a polymerase for performing PCR, buffer distilled water, dNTP, and the like, and may include instructions for use.

In another embodiment of the present invention, the concentration of the first primer and the second primer is preferably 10 picogram (pg), but is not limited thereto.

In another embodiment of the present invention, the sample is preferably but not limited to sputum, urine blood or tumor tissue.

The present invention also relates to a method for preparing a gene sample, comprising the steps of: a) preparing a gene analysis sample by mixing the DNA of a bacterium extracted from a sample with the primer composition of SEQ ID NOS: 1 to 4 and SEQ ID NOS: 6 to 9 present in one tube; b) real-time nested PCR is performed on the gene analysis sample of step (a) to obtain a PCR product; c) quantification of the PCR product of step (b) to obtain a quantitative curve; And d) quantifying the rpoB and IS6110 gene-specific genes present in the DNA of the bacterium extracted from the sample using the above quantitative curve.

Also, the present invention provides a method for detecting Mycobacterium tuberculosis using Nested Real-time PCR, wherein the first primer is selected from the group consisting of SEQ ID NOs: 1 to 2 and SEQ ID NOs: 6 to 7, 4 and SEQ ID NOS: 8 to 9 in the same tube to amplify Mycobacterium tuberculosis, M. bovis, M. africanum, M. canetii, and M. microti in order to improve the sensitivity and specificity for M. tuberculosis.

Hereinafter, the present invention will be described.

Mycobacterium tuberculosis strains were used to compare the sensitivity of one-tube nested real-time PCR with that of single-real-time PCR.

Among the nested PCR methods, two-tube nested PCR is usually used when the amount of DNA in the sample is very limited and can not be detected by one PCR. The first PCR reaction is performed and the second amplification , A new reagent is added so that the reaction can occur smoothly and a pair of primers responds to a relatively small number of times so that the sensitivity and specificity can be increased to about 100 times that of the conventional PCR .

However, in order to solve this problem, two-pair primers were added to one tube to try one-tube nested PCR to reduce the problem of contamination. At the same time, it was confirmed that the sensitivity was better than the conventional PCR.

As can be seen from the present invention, the present invention can be used to diagnose the presence or absence of tubercle bacilli directly without culture, or to use one-tube nested tubercle bacilli using two pairs of Mycobacterium tuberculosis-specific primers in a small number of pathogens such as sputum specimens, The PCR method is rapid and specific for M. tuberculosis and has high sensitivity and is useful in the diagnosis of tuberculosis.

Figure 1 shows the sensitivity of single real-time PCR of the rpoB gene using the standard strain of Mycobacterium tuberculosis H37Rv DNA;
Figure 2 shows the sensitivity of the one-tube nested real-time PCR of the rpoB gene using the standard strain of Mycobacterium tuberculosis H37Rv DNA;
Figure 3 shows the sensitivity of single real-time PCR with primer concentration control;
Figure 4 shows the sensitivity of single real-time PCR with amplification cycle and primer concentration control;
a) Sensitivity of single real-time PCR performed at 10 and 40 cycles using 10p primer concentration
b) Sensitivity of single real-time PCR performed at 10 and 40 cycles using 20p primer concentration
Figure 5 compares the detection sensitivity of one-tube nested real-time PCR with primer concentration for detection of Mycobacterium tuberculosis;
Figure 6 shows the sensitivity of single real-time PCR of IS6110 gene using the standard strain of Mycobacterium tuberculosis H37Rv DNA;
FIG. 7 shows the sensitivity of one-tube nested real-time PCR of the IS6110 gene using the standard strain of Mycobacterium tuberculosis H37Rv DNA;
FIG. 8 shows the sensitivity of the rpoB gene and the IS6110 gene using dual-one tube nested real-time PCR using the standard strain of Mycobacterium tuberculosis H37Rv DNA;
FIG. 9 shows the sensitivity of the rpoB gene and the IS6110 gene using dual-one tube nested real-time PCR using the standard strain of Mycobacterium tuberculosis H37Rv DNA.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: Standard strain DNA  detach

The strain Mycobacterium tuberculosis H37Rv (ATCC 25618), which was used as a standard strain, was purchased from the Department of Microbiology at Yonsei University and DNA was isolated by the following method (Lemarkchand et al., 2005). 50 μl of 10 mg / ml lysozyme (Sigma Chem. Co., USA) was added, mixed well and reacted overnight at 37 ° C. 5 ul of 10 mg / ml proteinase K (Sigma Chem. Co., USA) and 70 ul of 10% (w / v) SDS (Sodium Dodexyl Sulfate, Sigma Chemical Co., USA) React for 10 minutes. Add 100 ul of 5 M NaCl (Sodium Chloride, Ducksan, Korea) and mix well. Add 100 μl of CTAB (Cetyl Trimethyl Ammonium Bromide, Sigma Chem. Co., USA) preheated to 65 ° C, mix well and react at 65 ° C for 10 minutes. Chloroform: isoamylalcohol (= 24: 1 (v / v)) 700 μl is added, mixed well and centrifuged. Transfer only the supernatant containing DNA into a new tube, add 450 ul of isopropanol (Ducksan, Korea), mix, and precipitate DNA at -20 ° C for 30 minutes. After centrifugation at 4 ° C and 12,000 rpm for 30 minutes, the supernatant is removed and 1 ml of 70% ethanol is added and the tube is inverted about 5 times. After centrifugation at 4 ° C for 10 minutes, discard the supernatant, dry it for 30 minutes, and dissolve the DNA in 50 μl of TE. After the concentration of the dissolved DNA is confirmed, it is diluted to 1 ng / ul and used for PCR. In case of failure of culture, a portion of the cells was well solubilized in 400 μl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and DNA was isolated by the same procedure as above. The separated DNA was used for real-time PCR by confirming the concentration and diluting 10 times.

Example  2: Clinical specimen separation

Among the clinical samples, the nucleic acid was isolated from sputum specimens by the following procedure. As a pretreatment, 4% NaOH (3 ml) of the same amount as the sputum was treated at room temperature for 5 minutes and centrifuged at 200 x g for 10 minutes at 4 ° C. The separated supernatant was removed, washed with 1 ml of sterile PBS (pH 7.4), and centrifuged. After removal of the separated supernatant, 1 ml of TRIZOL reagent was added and 0.2 mm glass beads were bead-beaded for 60 seconds in Precellys 24 (Bertin Technologies, bis Avenue Ampeere, Montigny-le-Bretonneux, France) , Centrifuged at 12,000 × g for 15 minutes, and the supernatant layer was transferred to a new 1.5 ml microcentrifuge tube to separate DNA and RNA.

Among the clinical samples, urine sample DNA was isolated by the following procedure. 1 ml of the urine sample was dispensed into 1.5 ml microcentrifuge tubes and centrifuged at 15,000 × g for 3 minutes. After removing the supernatant, 1 ml of sterile PBS (pH 7.4) was added and vortexed vigorously. After centrifugation, the supernatant was removed. The same procedure was repeated twice. After washing, 50 μl of DNA extraction buffer containing resin was added, boiled at 100 ° C for 20 minutes, centrifuged at 15,000xg for 3 minutes, and the supernatant was used as DNA.

Example  3: Single Real - time PCR  Perform

Target specific to Mycobacterium tuberculosis The rpo B gene and IS6110 gene were used, and single real-time PCR was performed for each gene in the following manner. For the real-time PCR, 10 pmole of the forward primer and reverse primer were added, and 10 pmole of the probe was added. 5 μl of DNA was added to the final volume Lt; / RTI > to 20ul. The nucleotide sequences of each primer and probe are shown in Table 1.

The PCR reaction was performed once for 10 minutes at a denaturation temperature of 95 ° C using an ABI 7500 Fast (Applied Biosystem), and repeated for 40 cycles at a denaturation temperature of 95 ° C for 15 seconds and an annealing temperature of 60 ° C for 30 seconds. After each annealing process, fluorescence was measured and fluorescence values were measured for each cycle.

One-tube nested real-time PCR was performed using 20 pmole each of Forward primer and Reverse primer for comparison under the same conditions. PCR was performed once for 10 minutes at denaturation temperature of 95 ° C using ABI 7500 Fast (Applied Biosystem) , A denaturation temperature of 95 캜 for 15 seconds, and an annealing temperature of 60 캜 for 30 seconds was repeated 50 times. After each annealing process, fluorescence was measured and fluorescence values were measured for each cycle.

Example  4: One tube nested real - time PCR  Perform

One-tube nested real-time PCR was performed on rpo B and IS6110 genes, and one-tube nested real-time PCR was performed using both rpoB and IS6110 genes to improve specificity and sensitivity. For the real-time PCR reaction, 10 pmole each of 2.5, 5, and 10 pmole, 2 ^nd forward primer and reverse primer were added to the real-time PCR master mix (TOYOBO, Osaka, Japan), 1 ^st forward primer and reverse primer Add 10 μl of probe, add 5 μl of DNA, and proceed to a final volume of 25 μl. The nucleotide sequences of each primer and probe are shown in Table 1.

The PCR reaction was performed once for 10 minutes at a denaturation temperature of 95 ° C using ABI 7500 Fast (Applied Biosystem), 10 cycles of denaturation at 95 ° C for 15 seconds, and annealing at 65 ° C for 30 seconds, A cycle of denaturation at 95 DEG C for 15 seconds and annealing at 60 DEG C for 30 seconds was repeated 40 times. After each annealing process, fluorescence was measured and fluorescence values were measured for each cycle.

Target Objective Name 5 '
modification Sequence 3 'modification rpo B gene e 1 ^st primer Sense- rpo B GCG TCG GTC GCT ATA AGG TCA AC (SEQ ID NO: 1) antisense- rpo B ACC CTC GTG CAA GCG GAC CAG ATA T (SEQ ID NO: 2) 2 ^nd primer Sense- rpo B GGT CGC TAT AAG GTC AAC AAG AAG (SEQ ID NO: 3) antisense- rpo B ACC CTG GTG CAA GCG GAC CAG (SEQ ID NO: 4) rpo B probe FAM CTG CAT GTC GGC GAG CCC ATC ACG (SEQ ID NO: 5) BHQ1 IS6110 1 ^st primer Sense-IS6110 AAC GGC TGA TGA CCA AAC TC (SEQ ID NO: 6) antisense-IS6110 AAG CGG CGC TGG ACG AGA TC (SEQ ID NO: 7) 2 ^nd primer Sense-IS6110 GTC GAA CGG CTG ATG ACC AAA CT (SEQ ID NO: 8) antisense-IS6110 TCC GAA GCG GCG CTG GAC GA (SEQ ID NO: 9) IS6110 probe-1 FAM ACC CGC GGC AAA GCC CGC AGG (SEQ ID NO: 10) BHQ1 IS6110 probe-2 FAM ACC ACG ATC GCT GAT CCG GCC ACA (SEQ ID NO: 11) BHQ1

Table 1 shows primers and probes for real-time PCR for diagnosis of Mycobacterium tuberculosis

The results of the above embodiment are as follows.

rpoB  Gene Single real - time PCR and one tube nested Real - time PCR Of Mycobacterium tuberculosis detection sensitivity

To compare the sensitivity of single-real-time PCR and one-tube nested real-time PCR for detection of Mycobacterium tuberculosis, H37Rv DNA, a standard strain of Mycobacterium tuberculosis, was diluted 10-fold to 10 fg Respectively. In the single real-time PCR of the rpoB gene, ct values ranging from 10 ng to 10 fg were 17.36 to 37.18 when using a 10p primer (Figure 1). One-tube nested real-time PCR showed ct values of 10ng to 10fg Was 10.33 to 27.92, respectively (Fig. 2). Therefore, it was confirmed that the method using one-tube nested real-time PCR is about 100 times more sensitive than the method using single real-time PCR.

rpoB  Gene Single real - time PCR Of Mycobacterium tuberculosis detection sensitivity

To investigate the effect of primer concentration and cycle number on the one-tube nested real-time PCR method as a cause of improved sensitivity compared to single real-time PCR. The sensitivity of single-real-time PCR and single-tube nested real-time PCR was evaluated by performing 40 cycles and 50 cycles (Figs. 4A and 4B) using 2 ^nd primer concentrations of 10p and 20p, respectively The effect was confirmed.

As a result, sensitivity was not increased even when the concentration of primer was changed from 10p to 20p in 40 cycles. When 50 cycles, which is the same cycle number as one tube nested real-time PCR, were used, the primer concentration was changed from 10p and 20p to 40 the sensitivity was lower than that when the cycle was performed. It was confirmed that the sensitivity of one-tube nested real-time PCR was higher than that of single real-time PCR even when primers and cycles of the same concentration were not enough to perform 50 cycles as one pair of primers.

rpoB  Gene one tube nested Real - time PCR Of Mycobacterium tuberculosis detection sensitivity

One-tube nested real-time PCR was used to determine the sensitivity of the primers. (Fig. 5A), 5p (Fig. 5B) and 10p (Fig. 5C) were used for the 1 ^st primer concentration and 10p for the 2 ^nd primer.

As a result, it was confirmed that the sensitivity of Ct was 30.48 at the concentration of 2.5p, the Ct value was 29.42 at the concentration of 5p, and the value of Ct was 27.42 at the concentration of 10p. The sensitivity was 10 times higher than that of primer.

These results suggest that the sensitivity of the one-tube primed real-time PCR is 100 times higher than that of the 1- ^st primer using 10p and the 2 ^nd primer at 10p 2).

Table 2 compares the detection sensitivity of single-real-time PCR and one-tube nested real-time PCR for the detection of Mycobacterium tuberculosis in the rpoB gene.

IS6110  Using a gene single real - time PCR and one tube nested Real - time  PCR detection sensitivity of Mycobacterium tuberculosis

To compare the sensitivity of single-real-time PCR and one-tube nested real-time PCR for detection of Mycobacterium tuberculosis, H37Rv DNA, a standard strain of Mycobacterium tuberculosis, was diluted 10-fold to 10 fg Respectively. As a result, ct values of 10 ng to 10 fg were 14.95 to 35.71 when using a 10p primer in single real-time PCR of IS6110 gene (Fig. 6). In one-tube nested real-time PCR, the ct values from 10 ng to 10 fg were 5.83 to 30.62 (Fig. 7). Therefore, it was confirmed that the method using one-tube nested real-time PCR is about 100 times more sensitive than the method using single real-time PCR.

rpoB  Gene and IS6110  Using a gene one tube nested Real - time PCR Of Mycobacterium tuberculosis detection sensitivity

To improve the sensitivity of rpoB one-tube nested real-time PCR, IS6110 gene was added to perform dual-one tube nested real-time PCR. To compare the sensitivity of one-tube nested real-time PCR for detection of Mycobacterium tuberculosis, H37Rv DNA, a standard strain of Mycobacterium tuberculosis, was used as template DNA for real-time PCR from 10 ng to 10 fg by 10-fold dilution. As a result, ct values from 10 ng to 10 fg were 4.99 to 24.9 when using a 10p primer (Fig. 8). In order to increase the sensitivity, 15, 25 cycles were performed at 10, 40 cycled, and the sensitivity from 10 fg to 1 fg was confirmed (Fig. 9).

Therefore, it was confirmed that the real-time PCR using dual-one tubes was more than 100 times more sensitive than the one-tube nested real-time PCR method.

In conclusion, we performed a dual-one tube nested real-time PCR with 15 cycles of 1 ^st primer concentration and 10p of 2 ^nd primer, followed by 25 cycles And the sensitivity was at least 100 times higher (Table 3).

Table 3 compares the detection sensitivity of single-real-time PCR and one-tube nested real-time PCR and dual one-tube nested real-time PCR for detection of Mycobacterium tuberculosis in rpoB gene and IS6110 gene.

Single-real-time PCR with standard strains and dual one-tube nested real-time PCR Detection specificity  Confirm

The specificity of single real-time PCR and dual one-tube nested real-time PCR using standard strain DNA was confirmed. As a result, it was confirmed that only the MTB complex reacts specifically (Table 4).

Table 4 shows specificity using the standard strain.

Evaluation of the sensitivity of single PCR and dual one-tube nested real-time PCR in clinical specimens

Clinical specimens were used to determine the sensitivity and specificity of the newly developed dual one-tube nested real-time PCR. Specificity was checked by sputum and urine of normal subjects, and the sensitivity of the patient was confirmed using specimens of tuberculosis patients.

7-1) Evaluation of the sensitivity of single-PCR and dual one-tube nested real-time PCR in clinical specimens

We compared the sensitivities of the newly developed single-tube nested real-time PCR and single PCR using Real-MTB-ID (Korea), which has been commercialized to confirm the detection rate of M. tuberculosis in clinical specimens. Specificity was confirmed using normal sputum. As a result of the experiment using patient sputum, the detection rate of Mycobacterium tuberculosis was 12/26 (46.1%) in single PCR, whereas it was 26/26 (100%) in dual one-tube nested real-time PCR ).

Table 5 shows the sensitivity and specificity of sputum specimens.

7-2) Usefulness of sensitivity in single PCR and dual one-tube nested real-time PCR in clinical urine specimens

To confirm the detection rate of Mycobacterium tuberculosis in clinical urine specimens, single PCR using AdvanSure ^™ TB / NTM real-time PCR (LG Life Sciences, Daejeon) and newly developed dual one-tube nested real-time PCR Were compared.

Specificity was checked using normal urine. The sensitivity of PCR in clinical urine specimens was 3/56 (5.4%) in single PCR and 29/56 (51.8%) in dual one-tube nested real-time PCR Table 6).

Table 6 shows the sensitivity and specificity of urine specimens.

7-3) Evaluation of the sensitivity of single-PCR and dual one-tube nested real-time PCR in clinical tissue specimens

In order to confirm the detection rate of M. tuberculosis in tissue specimens of clinical granuloma (granuloma, tumor-like masses or nodules of granulation tissue containing actively growing fibroblasts and capillary tuberculosis), Real MTB-ID (M & D, Korea) The sensitivity and specificity of the newly developed single-tube nested real-time PCR was 25/76 (32.9%) in Single PCR, whereas the sensitivity of single-tube nested real- / 76 (57.9%), respectively (Table 7).

Single PCR nested qPCR MTB 25 (32.9%) 44 (57.9%) - 51 (67.1%) 32 (42.1%) total 76 76

Table 7 shows the usefulness evaluation of sensitivity in single PCR and one-tube nested PCR in clinical tissue (Granuloma).

<110> Yonsei University Wonju Industry-Academic Cooperation Foundation <120> Improved method for diagnosing Mycobacterium tuberculosis using          one-tube nested real-time PCR <160> 11 <170> Kopatentin 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gcgtcggtcg ctataaggtc aac 23 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 accctcgtgc aagcggacca gatat 25 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggtcgctata aggtcaacaa gaag 24 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 accctcgtgc aagcggacca g 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 5 ctgcatgtcg gcgagcccat cacg 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aacggctgat gaccaaactc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aagcggcgct ggacgagatc 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gtcgaacggc tgatgaccaa act 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tccgaagcgg cgctggacga 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 10 acccgcggca aagcccgcag g 21 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 11 accacgatcg ctgatccggc caca 24

Claims (18)

A primer set consisting of the primers of SEQ ID NOs: 1-4 and SEQ ID NOs: 6-9. Mycobacterium tuberculosis detection kit comprising a primer set of claim 1. The kit for detecting Mycobacterium tuberculosis according to claim 2, wherein the kit further comprises a probe. The kit for detecting Mycobacterium tuberculosis according to claim 3, wherein the probe is a probe selected from the group consisting of SEQ ID NO: 5 and SEQ ID NOs: 10 to 11. The kit for detecting Mycobacterium tuberculosis according to claim 3, wherein the 5 'end and the 3' end of the probe are labeled with a fluorescent material. 6. The method of claim 5, wherein the 5'-end labeled fluorescent material is selected from the group consisting of 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein (HEX) Tetrachloro-6-carboxyfluorescein, and Cyanine-5 (Cy5).   6. The method according to claim 5, wherein the fluorescent substance labeled at the 3 'end is 6-carboxytetramethyl-rhodamine (TAMRA) or black hole quencher-1,2,3 (BHQ -1,2,3). &Lt; / RTI &gt;  The method of claim 2, wherein the Mycobacterium tuberculosis is Mycobacterium tuberculosis, M. bovis, M. africanum, M. canetii, and M. microti. When real-time PCR is performed using the DNA as a template, the primers of SEQ ID NOs: 1 to 2 and SEQ ID NOs: 6 to 7 are used as a first primer and the primers of SEQ ID NO: 3 To 4 and SEQ ID NOS: 8 to 9 in a DNA-like tube. 10. The method of claim 9, wherein the tube further comprises a probe. 11. The method for detecting Mycobacterium tuberculosis according to claim 10, wherein the probe is a probe selected from the group consisting of SEQ ID NO: 5 and SEQ ID NOs: 10 to 11. [Claim 11] The method according to claim 10, wherein the 5 'and 3' ends of the probe are labeled with a fluorescent material.  10. The method of claim 9, wherein the Mycobacterium tuberculosis is Mycobacterium tuberculosis, M. bovis, M. africanum, M. canetii, and M. microti. 10. The method of claim 9, wherein the concentration of the first primer and the second primer is 10 picograms (pg). 10. The method of claim 9, wherein the sample is sputum, urine, blood or tumor tissue. a) preparing a gene analysis sample by mixing the DNA of the bacteria extracted from the sample with the primer composition of SEQ ID NOS: 1 to 4 and SEQ ID NOS: 6 to 9 present in one tube;
b) real-time nested PCR is performed on the gene analysis sample of step (a) to obtain a PCR product;
c) quantifying the PCR product of step (b) to obtain a quantitative curve; And
d) A method of quantifying rpoB and IS6110 gene specific genes present in the DNA of the bacteria extracted from the sample using the above quantitative curve. 17. The method of claim 16, wherein the method further comprises a probe selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 10 to 11 in the tube. 1 to 2 and SEQ ID NOs: 6 to 7 as the first primer, SEQ ID NOS: 3 to 4 as the second primer, and SEQ ID NOs: 3 to 4 as the second primer, in a method for detecting Mycobacterium tuberculosis using Nested real- The primers 8 to 9 were amplified in the same tube to obtain Mycobacterium tuberculosis, M. A method for improving the sensitivity and specificity for Mycobacterium tuberculosis selected from the group consisting of bovis, M. africanum, M. canetii, and M. microti.

Images