KR20080106616A - Method of detecting mycobacterium using denaturing high-performance liquid chromatography(dhplc) - Google Patents

Abstract

A method for analyzing the mutation of drug resistant gene of non-tuberculosis bacilli and tuberculosis bacilli, a primer pair for discriminating the mutation of drug resistant gene of tuberculosis bacilli, and a method for discriminating the mutation of drug resistant gene of tuberculosis bacilli by using the primer pair are provided to analyze the mutation of drug resistant gene of tuberculosis bacilli and non-tuberculosis bacilli by using base specific PCR. A method for analyzing the mutation of drug resistant gene of non-tuberculosis bacilli and tuberculosis bacilli comprises the steps of amplifying the gene part coding the RNA polymerasebeta-subunit-encoding gene (rpoB) capable of discriminating tuberculosis bacilli and non-tuberculosis bacilli by using a primer pair by base-specific PCR; and carrying out the denaturing high-performance liquid chromatography (DHPLC) with the obtained PCR product.

Description

Method of detecting Mycobacterium using denaturing high-performance liquid chromatography (DHPLC)

Figure 1 (a) is a tuberculosis bacteria (TB: Mycobacterium) tuberculosis ) and the location of each primer to amplify nontuberculous mycobacteria (NTM), (b) is a diagram showing the sequence of each primer (TB: M. tuberculosis H37Rv, AVI: M. avium , INT: M. intracellulare , KAN: M. kansasii ),

Figure 2 is an electrophoresis picture of a PCR product that can distinguish between TB and NTM depending on the presence or absence of amplification ((a) TB: PCR product using TB: Tbc1-TbcR5 primer set (235bp) (b) NTM: M5-RM3 primer set (136 bp) PCR product used),

Figure 3 is a photograph showing the Hae III restriction enzyme RFLP results of the product amplified with the MF-MR primer set (342bp) ((M ₁ ) 100bp marker (4) M. TB control, (8) M. TB 531 mutation TCG to TTG, (10) M. avium , (20) M. intracellulare , (M ₂ ) 25 bp marker, (14) M. TB 526 mutation CAC to GAC, (35) M. TB 526 mutation CAC to TGC, (54 ) M. parascrofulaceum , (60) M. gordonae , (64) M. terrae (86) M. fortuitum , (89) M. TB 526 mutation CAC to GAC, (M ₃ ) Sizing control ^* ),

Figure 4 is a photograph of the product amplified with MF-MR primer set (342bp) treated with Hae III restriction enzyme and analyzed by DHPLC (Patterns: (a) M. tuberculosis (b) M. avium (c) M. intracellulare ) (d) M. parascrofulaceum (e) M. gordonae (f ) M. terrae (g) M. fortuitum (h) Sizing control ^*

^{* Fragment Sizes: base pairs (bp} ) 80, 102, 174, 257, 267, 298, 434, 458, 587)

Figure 5 is a photograph of detection of drug-resistant bacteria using DHPLC ((DHPLC column temperature: 66 ℃). Patterns: (a) M. tuberculosis (b) 531 TCG to TTG; (c) 526 CAC to GAC; (d) 526 CAC to TGC; (e) 516 GAC to GTC),

Figure 6 is a pattern confirmed only by the sequence analysis of the results of the analysis by DHPLC ((a) M. tuberculosis (b) 531 TCG to TTG; (c) 526 CAC to GAC; (d) 526 CAC to TGC; (e) 516 GAC to GTC).

The present invention amplifies specific parts of Mycobacterium tuberculosis using molecular genetic techniques, and uses it to denature high-performance liquid chromatography (DHPLC) without the use of additional enzymes or electrophoresis. It also relates to analytical methods that enable the differentiation of.

When the frequency of occurrence of the last 10 years occurred in the United States Mycobacterium tuberculosis (Mycobacterium tuberculosis, TB) accounts for 50% of total non-tuberculosis hangsanseonggyun (nontuberculous mycobacteria, NTM) have accounted for the remaining 50%. However, more than 93% of tuberculosis infections in Korea are caused by non-tuberculosis acidophilic bacteria, which occur 3 to 10% of the time. Tuberculosis has continued to decline until the late 1980s due to the effective use of anti-tuberculosis drugs, but it has been gradually increasing in the 1990s due to the increase in resistant tuberculosis bacteria and the increase in patients with acquired immunodeficiency. Especially in Korea, the death of tuberculosis is the highest among infectious diseases due to the increase of homelessness caused by IMF, and more than 3000 people per year are reported to die from tuberculosis.

Non-tuberculosis acidophilic bacteria are clinically most immunocompromised, but the elderly are ill and clinical findings are similar to tuberculosis. Although the incidence rate is relatively low compared to tuberculosis in Korea, it is widely distributed in living environment, and it is difficult to determine whether it is pathogenic even if separated from clinical specimens, and diagnosis is not easy. It is difficult and is open to high recurrence rate. In addition, these non-tuberculosis acidophilic bacteria have been reported to cause disease in patients without a decrease in immune function. Since the spread of human immunodeficiency virus (HIV) infection since 1980, it is known that non-tuberculosis acidophilic bacteria cause systemic disseminated infections in immunocompromised patients.

In Korea, the prevalence of tuberculosis is gradually decreasing, and patients with acquired immunodeficiency syndrome and the use of immunosuppressive agents are gradually increasing. Therefore, the incidence of infections caused by NTB is relatively high.

Mycobacterium spp. Are treated differently because the anti-tuberculosis resistance pattern is different for each species. Therefore, differentiation of species of Mycobacterium spp. Into species level is essential for the healing of patients. The biochemical identification method has the disadvantage that it takes a long time and requires a skilled person because of the slow development of Mycobacterium spp. There are methods for identification by lipid analysis of cell walls, such as high performance liquid chromatography (HPLC) and thin layer liquid chromatography (TLC), but these methods are also difficult to perform and costly. do. Traditional Mycobacterium The identification of genus species is time consuming to identify and diagnose because of the biological nature of the slow growth rate of these organisms (slow growth → 2-3 months).

In order to overcome the problems of the existing identification methods, Mycobacterium after the 1990s Diagnostic methods using molecular biological methods that target chronometer molecules, such as 16S rDNA and RNA polymerase genes, which show the systematic relationship of the genus species, are developed. However, the identification method using 16S rDNA has been reported a number of problems, such as the heterogeneity of genes in an individual. Mycobacterium There is an urgent need to develop alternative chronometer molecules that can complement the problems of conventional chronometers in diagnostics. The use of alternative chronometer molecules enables the development of new fungal diagnostic methods, as well as the replacement of imported tuberculosis and non-tuberculosis mycobacterium diagnostic kits, which have a large share in the infectious disease diagnosis market, as well as the export of original technologies to developed countries. It is thought to be. Therefore, the Mycobacterium gene using the hsp65 gene, which is an alternative chrinometer molecule There is a need to develop new diagnostic methods that can be used to identify genus species.

Therefore, in the present invention, a new molecule targeting a gene that enables not only the diagnosis of Mycobacterium tuberculosis but also the discrimination of the non-tuberculosis acidophilic species to the species level by replacing the existing identification and diagnosis methods that take a lot of time and miss the medicinal healing time Developed genetic diagnostics.

The present invention is to solve the problems of the method of classifying the species of TB, NTM and the method of classifying mutations of drug-resistant genes of TB, which has been used in the prior art, and an object of the present invention is to amplify specific gene regions of TB and NTM and By using denaturing high performance liquid chromatography, there is provided an analysis method for identifying mutations in drug-resistant genes of TB, NTM and TB without RFLP method or electrophoresis.

An object of the present invention is a base-specific polymerase chain reaction step of amplifying a portion of the gene encoding the RNA polymeraseβ-subunit-encoding gene ( rpoB ) to distinguish between tuberculosis and non-tuberculosis using a primer pair; And a step of denaturing high-performance liquid chromatography (DHPLC) of the PCR product amplified by the PCR reaction. The present invention provides an analysis method for identifying mutations in drug resistance genes.

Still another object of the present invention is to provide primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 for determining mutations in drug resistance genes of Mycobacterium tuberculosis.

Still another object of the present invention is to provide a method for discriminating a mutant group of drug-resistant genes of Mycobacterium tuberculosis using the above primer pairs.

The present invention relates to a method for discriminating Mycobacterium tuberculosis using denatured high performance liquid chromatography.

The present invention is a base-specific polymerase chain reaction step of amplifying a portion of the gene encoding the RNA polymerase β-subunit-encoding gene ( rpoB ) to distinguish between tuberculosis and non-tuberculosis using a primer pair; And a step of denaturing high-performance liquid chromatography (DHPLC) of the PCR product amplified by the PCR reaction. Assays for determining mutations in drug resistance genes.

The present invention includes primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 for determining mutations in drug resistance genes of Mycobacterium tuberculosis.

The present invention includes a method for discriminating a mutant group of drug-resistant genes of Mycobacterium tuberculosis using the above primer pairs.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

<Example 1; Mycobacterium DNA Extraction>

After receiving sputum from patients with tuberculosis, incubated in Ogawa medium, and then treated with 1ml of 1M NaOH, and boiled for 15 minutes with boiling water. Equivalent amount of PCI-9 was added to this pretreated solution, centrifuged at 12,000 rpm, and the supernatant was transferred to a new tube. 2M sodium acetate and 100% ethanol were added thereto, followed by washing to extract DNA. The extracted DNA was confirmed by electrophoresis for 30 minutes at 100V in 0.8% agarose gel.

<Example 2; Distinguishing between Mycobacterium tuberculosis and non-tuberculosis acidophilic bacteria>

The RNA polymeraseβ-subunit-encoding gene ( rpoB ) contains specific nucleotides of Mycobacterium tuberculosis and nonmycobacterium tuberculosis. Use this difference to set Tbc1 (5'-CGT ACG GTC GGC GAG CTG ATC CAA-3 ') TbcR5 (5'-C CAC CAG TCG GCG CTT GTG GGT CAA-3') primer set and M5 (5'-G GAG CGG ATG ACC ACC CAG GAC GTC-3 ')-RM3 (5'CAG CGG GTT GTT CTG GTC CAT GAA-3') primer set was prepared. The first primer set is amplified 235bp DNA sequence from Mycobacterium tuberculosis, and the second primer set is amplified 136bp DNA sequence from non-tuberculosis acidophilic bacteria (Figure 1). PCR premix (2.5 U Taq polymerase, 250 uM dNTP, 1.5 mM MgCl ₂ , Each primer (10 pmol) and extracted DNA (2 ul) were added to the final buffer to 20 ul. The mixture was reacted at 94 ° C. for 10 minutes in an initial denaturation step using a thermal cycler (Applied Biosystems, USA, GeneAmpPCR System 2700), followed by denaturation step 94 ° C. 30 sec, annealing step 60 ° C. 30 sec, and extension step 72 ° C. 40 The reaction was carried out for 33 cycles under the condition of seconds. Thereafter, the reaction was carried out at 72 ° C. for 5 minutes in the last extension step. The amplification product was confirmed by electrophoresis for 30 minutes at 100 V on a 1.5% agarose gel. As shown in FIG. 2, (a) is a tuberculosis bacterium with a DNA sequence of 235 bp, and (b) is a non-tuberculous bacterium with a sequence of 136 bp. It could be confirmed (Fig. 2).

Example 4 Identification Using Restriction Section Length Formation

In the DNA (2 ul) obtained in Example 1, Mycobacterium- specific primers MF (5'-CGA CCA CTT CGG CAA CCG-3 ') and MR (5'-TCG ATC GGG CAC ATC CGG-3'), respectively, were prepared. Pmol was added and PCR was performed with a final volume of 20 μl of the PCR premix (2.5 U Taq polymerase, 250 uM dNTP, 1.5 mM MgCl ₂ , buffer). The mixture was reacted at 94 ° C. for 10 minutes in an initial denaturation step using a thermal cycler (Applied Biosystems, USA, GeneAmpPCR System 2700), followed by denaturation step 94 ° C. 30 sec, annealing step 60 ° C. 30 sec, and extension step 72 ° C. 40 The reaction was carried out for 33 cycles under the condition of seconds. The resulting amplified product was identified in detail using restriction fragment length polymorphism (RFLP). Restriction enzyme was reacted for 1 hour at 37 ℃ using Hae III (Promega Corporation, USA). After that, electrophoresis was performed for 35 minutes at 100V in 3% agarose gel. As a result, TB was identified in lane 3 and TB mutations were observed in lane 7, lane 8 and lane 13. Non-tuberculosis NTM was detected in lane 4 ( M. avium ), lane 5 ( M. intracellulare ) and lane. 9 ( M. parascrofulaceum ), lane 10 ( M. gordonae ), lane 11 ( M. terrae ), lane 12 ( M. fortuitum ). (Fig. 3 lane _{1: (M 1) 100bp marker} , Lane 2: (4) M. TB control , lane 3: (8) M. TB 531 mutation TCG to TTG, Lane 4: (10) M. avium, Lane 5: (20) M. intracellulare , lane 6: (M ₂ ) 25 bp marker, lane 7: (14) M. TB 526 mutation CAC to GAC, lane 8: (35) M. TB 526 mutation CAC to TGC, Lane 9: (54) M. parascrofulaceum , lane 10: (60) M. gordonae , lane 11: (64) M. terrae , lane 12: (86) M. fortuitum , lane 13: (89) M. TB 526 mutation CAC to GAC, lane 14: (M ₃ ) Sizing control ^*

^{* Fragment Sizes: base pairs (bp} ) 80, 102, 174, 257, 267, 298, 434, 458, 587).

In addition, the product amplified with MF-MR primer set (342bp) was treated with Hae III restriction enzyme, and the PCR amplification product treated with restriction enzyme was analyzed by length using non-denaturing method, one of DHPLC analysis methods. This analysis method uses the principle that the longer the DNA fragment is, the longer the residence time in the column, and the pattern of DHPLC varies according to the length of DNA fragments cut by restriction enzyme. As shown in Figure 4, the tuberculosis bacteria (a) M. tuberculosis , non-tuberculosis bacteria (b) M. avium (c) M. intracellulare (d) M. parascrofulaceum (e) M. gordonae (f ) M . terrae (g) M. fortuitum It was confirmed to be. (h) Sizing control ^*

( ^* Fragment Sizes: base pairs (bp) 80, 102, 174, 257, 267, 298, 434, 458, 587,)

<Example 5; Detection of Drug-Resistant Mycobacterium Tuberculosis Using DHPLC>

TB, which is resistant to antibiotics, is resistant to treatment because the drug is resistant to treatment. Therefore, in order to provide a suitable medicine for patients, the drug-resistant Mycobacterium tuberculosis bacteria should be examined for mutations. However, the conventional method requires an antibiotic test, so it may take a long time for the patient to get worse. Therefore, the rapid detection of drug-resistant Mycobacterium tuberculosis is required so that TR8 (5'-TCG CCG CGA TCA AGG AGT-3 '(SEQ ID NO: 1)) and TR9 (5'-TGC ACG TCG CGG ACC TCA-) The polymerase chain reaction was performed using 3 '(SEQ ID NO: 2) primer set. The primer (10 pmol) and the DNA (2 ul) extracted in Example 1 were added to the PCR premix as described above to make the final 20 ul. The mixture was reacted for 10 minutes at 94 ° C. in an initial denaturation step using a thermal cycler (Applied Biosystems, USA, GeneAmpPCR System 2700), followed by a denaturation step 94 ° C. 30 sec, annealing step 61 ° C. 30 sec, and extension step 72 ° C. 40 PCR reactions were performed in 33 cycles under the conditions of seconds. The amplified PCR product was denatured at 95 ° C. for 10 minutes using a thermal cycler, and then slowly reanealed at room temperature for 45 minutes to form a heteroduplex. For example, when the above experiment is explained with TB and NTB of FIG. 5, the PCR product of TB and the PCR product of NTB are mixed and denatured according to the above conditions, and then cooled at room temperature to form a heterozygote and the PCR products of TB of TB. In the case of mixing, homozygotes are formed. In the DHPLC analysis method, the heterozygote has a non-complementary base sequence, so that a bubble phenomenon occurs on the base sequence and the homozygote does not occur. Therefore, since the homozygotes are more tightly bonded than the heterozygotes in the coupling with the column, the retension time is different, so that different patterns of chromatography are drawn in DHPLC. Can be distinguished. This product was analyzed using Transgenomic WAVE system (DHPLC) (Transgenomic, Inc., Omaha, NE, USA). Analyzes were performed from 65 ° C to 67 ° C and detection of resistant tuberculosis bacteria was best at 66 ° C. As mobile phase, 0.1 M triehylammonium acetate (TEAA), pH7 (buffer A) and 0.1 M TEAA (buffer B) containing 25% acetonitrile were used. As a result, TB ( Mycobacterium tuberculosis ) 5 (b) shows that 531 TCG is TTG, (c) 526 CAC is GAC, (d) 526 CAC is TGC, and (e) 516 GAC is mutated to GTC. As a result, the sequencing of the microorganisms was performed by Macrogen Co., Ltd., and it was confirmed that the results were consistent with those of Fig. 5 (Fig. 5- (DHPLC column temperature: 66 ° C). Patterns: (a) M . tuberculosis (b) 531 TCG to TTG; (c) 526 CAC to GAC; (d) 526 CAC to TGC; (e) 516 GAC to GTC). Samples with different patten chromatograms were identified using a saline sequencer (Fig. 6-Patterns: (a) M. tuberculosis (b) 531 TCG to TTG; (c) 526 CAC to GAC; (d) 526 CAC to TGC; (e) 516 GAC to GTC).

The tuberculosis detection kit of the present invention replaces the existing identification and diagnosis methods that miss the time of medicinal healing due to the long time-consuming process, and therefore, tuberculosis and non-tuberculosis can be diagnosed at the species level of non-tuberculosis acidophilic bacteria. It is a new diagnostic method that has the effect of exporting the original technology to developed countries as well as the import substitution effect of the acidophilic bacterium diagnostic kit.

<110> Industry-Academic Cooperation Foundation, Dankook University          PARK, YONG HYEUN <120> Method of detecting Mycobacterium using denaturing          high-performance liquid chromatography (DHPLC) <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tcgccgcgat caaggagt 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgcacgtcgc ggacctca 18  

Claims (3)

A base-specific polymerase chain reaction step of amplifying a portion of a gene encoding an RNA polymeraseβ-subunit-encoding gene ( rpoB ) capable of distinguishing tuberculosis from non-tuberculosis with primer pairs; And a step of denaturing high-performance liquid chromatography (DHPLC) of the PCR product amplified by the PCR reaction. Assay method for determining mutation of drug resistance gene. Primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 for determining mutations in drug resistance genes of Mycobacterium tuberculosis. A method for discriminating a mutant group of drug-resistant genes of Mycobacterium tuberculosis using the primer pair of claim 2.

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