KR100285253B1 - Diagnostic reagent for detecting Rifampin-resistant tuberculosis bacteria - Google Patents

Abstract

The present invention provides a primary PCR using a primary PCR primer that specifically amplifies only rpoB DNA of Mycobacterium tuberculosis without amplifying other bacteria's rpoB DNA, and 157 bp of mutants inducing mutations that cause rifampin resistance of Mycobacterium tuberculosis. The present invention relates to a nested PCR-SSCP for performing secondary PCR using a primer for secondary PCR amplifying rpoB DNA and a method for detecting rifampin-resistant Mycobacterium tuberculosis by using a single-step nested PCR-SSCP for shortening such a process. In the case of NPCR-SSCP using electric primers, due to the high specificity of the zero primer for the first PCR, only DNA of Mycobacterium tuberculosis can be specifically amplified to prevent false positives caused by non-tuberculosis acidophilic bacteria (MOTT), and SNPCR-SSCP In this case, a large number of samples can be processed, which can greatly save time and money.

Description

Rifampin-resistant Mycobacterium tuberculosis detection method using nested PCR-SCP and single-stage nested PCR-SCC targeting the R. gene gene

The present invention relates to nested polymerase chain reaction-single strand conformational polymorphism (PCS-SSCP) and single-step nested polymerase chain reaction-single strand targeting rpoB gene segments of Mycobacterium tuberculosis. The present invention relates to a method for detecting rifampin-resistant mycobacterium tuberculosis by conformational polymorphism. More specifically, the present invention provides a primary PCR using a primary PCR primer that specifically amplifies only rpoB DNA of Mycobacterium tuberculosis without amplifying rpoB DNA of another bacterium, and a mutation that causes rifampin resistance of Mycobacterium tuberculosis. Nested PCR-SSCP performing secondary PCR using primers for secondary PCR amplifying 157 bp rpoB DNA, and Rifampin-resistant Mycobacterium tuberculosis detection method using single-step Nested PCR-SSCP which shortened the procedure. It is about.

Tuberculosis has made great progress in the treatment and management of advanced countries since the mid-19th century due to improvements in living conditions, but remains a public health problem in developing countries. The six cases of tuberculosis carried out by the Korean Tuberculosis Association at five year intervals from 1965 to 1990 show that the prevalence of fungal pulmonary tuberculosis by smear or culture was 0.97% in 1965 and 0.19% in 1990. Significant decreases can be seen (see Ministry of Health and Social Affairs, Korean Tuberculosis Association, 6th National Tuberculosis Survey, 22-30, 1990). As such, the number of patients with tuberculosis has decreased significantly, but the emergence of multidrug-resistant tuberculosis bacteria (MDR Tb), which is resistant to anti-tuberculosis drugs, which causes many problems in developed countries, is gradually increasing in Korea.

In the case of tuberculosis treatment, short-term treatment is generally used. In order to suppress the emergence of Mycobacterium tuberculosis resistant to certain drugs, this method involves four drugs, namely isoniazid, rifampin, pyrazinamide, ethambutol or streptococcus, in the first two months. It is a treatment method that removes mycobacterium tuberculosis that is not suppressed in the initial treatment by administering mycin (streptomycin) together and only rifampin and isoniazid for 4 months thereafter. However, the current short-term treatment, which was expected to be effective, also shows no effect on the rapidly increasing multi-drug resistant tuberculosis.

An important problem of the multi-drug resistant tuberculosis bacteria is that the treatment for tuberculosis is limited, and in the case of drug-resistant tuberculosis is not easy to cure. In particular, rapid increases have been observed in immunocompromised patients, as well as in people not infected with HIV (Bloom, BR, Murray, CJ, Tuberculosis: Commentary on a Reemergent Killer, Science, 257 (5073)): 1055-1064 (1992). In foreign cases, 33% of the resistant patterns are at least one anti-tuberculosis agent, 26% isisoniazid (″ INH ″), 22% is rifampin (″ RIF ″), and 13% is streptomycin (streptomycin). , ″ SM ″) showed 1% resistance to pyrazinamide (″ PZA ″), and 19% reported simultaneous resistance to RIF and INH (Frieden, TR, Sterling, T., Pablos-Mendez A., Kilburn, JO, Cauthen, GM, Dooley, SW, The Emergence of Drug-resistant Tuberculosis in New York City., N. Engl. J. Med., 328 (8): 521-526 (1993).

According to the statistics of tuberculosis researchers, in the case of resistant tuberculosis bacteria isolated in Korea, the proportion of strains resistant to more than one drug is 27.4%. The highest proportion of INH resistant strains was 27.4%, and RIF resistance was reported at 7.1% in 1990 (see Ministry of Health and Social Affairs, Korean Tuberculosis Association, 6th National Tuberculosis Survey, 22-30, 1990). In the case of strains resistant to two or more anti-tuberculosis drugs, the ratio of strains resistant to both INH and RIF at the same time is 7.1%, it can be seen that the RIF-resistant tuberculosis bacteria have resistance to INH, which is the basic drug for tuberculosis treatment. That is, it means that rifampin resistance may be regarded as a marker representing whether or not multidrug resistance.

Due to the problems of drug resistance and high infectivity, a strict surveillance system and specific countermeasures against Mycobacterium tuberculosis have been urgently required. Therefore, it is emphasized that the efficiency of tuberculosis control is emphasized, and from the laboratory side, there is an absolute need for a method capable of quickly detecting MDR-TB. However, unlike general bacteria, tuberculosis bacteria grow very slowly, so it is disadvantageous that it takes about 12 weeks to confirm the results by a conventional culture test. For this reason, there has always been a demand for shorter inspection times.

Rifampin resistance, a marker of drug resistance, is due to its mechanism of resistance acquisition at the gene level (Telenti, A., Imboden, P., Marchesi, F., Lowrie, D., Cole, S., Colston, MJ, Matter, L., Schopfer, K., Bodmer, T., Detection of Rifampicin-resistance Mutations in Mycobacterium tuberculosis, Lancet, 1993, 341 (8846): 647-650), 3-4 by molecular biological methods You can check for rifampin resistance within days.

Rifampins are toxic to bacteria by binding to RNA polymerase and disrupting transcription. The mechanism of resistance of rifampin was first identified in E. coli (Jin, DJ, Gross, CA, Mapping and Sequencing of Mutations in the Escherichia coli rpoB Gene That Lead to Rifampicin Resistance, J. Mol. Biol., 1988, 202 (1) ): 45-58), among the 1342 amino acids encoding Escherichia coli RNA polymerase β subunit, resistance is shown by a mutation of four portions, that is, amino acid number 145, 507-533, 563-572 and 687 sites. In particular, there are genetic variations associated with most (70%) rifampin resistance at the 507-533 site. Even in the case of Mycobacterium tuberculosis, rifampin resistance occurs due to mutation of the rpoB gene encoding the β subunit of the RNA polymerase, a rifampin binding site. To date, almost all rifampin-resistant tuberculosis bacteria are known to have missense mutations between these amino acids 507-533 (see Telenti, A., Imboden, P., Marchesi, F). , Lowrie, D., Cole, S., Colston, MJ, Matter, L., Schopfer, K., Bodmer, T., Detection of Rifampicin-resistance Mutations in Mycobacterium tuberculosis., Lancet, 1993, 341 (8846) : 647-650), the present inventors have confirmed that the same in the case of rifampin-resistant tuberculosis bacteria confirmed in Korea (see: Kim, BJ, Kim, SY, Park, BH, Lyu, MA, Park, IK, Bai, GH) , Kim, SJ, Cha, CY, Kook, YH, Mutations in the rpoB gene of Mycobacterium tuberculosis that interfere with PCR-Single-Strand conformation polymorphism analysis for rifampin susceptibility testing., J. Clin.Microbiol., 1997, 35 (2 ): 492-494).

Under these backgrounds, the genetic variation is determined to determine the rifampin resistance of Mycobacterium tuberculosis. However, if the same result as the susceptibility test through culture was obtained, the resistance test time can be greatly saved. Many attempts have been made to save the time spent on screening for rifampin resistance of Mycobacterium tuberculosis using the PCR-SSCP method, which combines SSCP with PCR.

Briefly explaining the principle of SSCP, denatured single-stranded DNA forms a folded conformation by self complementarity and intramolecular interaction. Genetic variations in the structure of the single-stranded DNA changes. The presence or absence of such genetic mutations is confirmed by autoradiography to determine the difference in mobility between the original DNA and the mutated DNA, which occurs when the electro DNA is electrophoresed on a non-denaturing gel. By doing this, it is possible to determine whether Rifampin resistance of Mycobacterium tuberculosis.

Rifampin resistance is easier to find as PCR-SSCP compared to other drug resistance because the gene mutations related to rifampin resistance are not distributed in several genes like other drugs, but in the concentrated (amino acid number 507-533) site of rpoB. . However, as described above, many attempts have been made to detect Rifampin-resistant M. tuberculosis by PCR-SSCP by extracting DNA from purely cultured Mycobacterium tuberculosis, but at least six weeks are required for pure culture of M. tuberculosis and only 6 weeks are required for susceptibility testing. The result was a time saving.

As a result, the best method for determining the rifampin resistance of Mycobacterium tuberculosis is to minimize the time required by applying PCR-SSCP directly to a sample obtained from a patient without culture. However, the results of applying electro-PCR-SSCP directly to patient sputum are extremely rare, because there are some problems that are difficult to apply to patient samples.

First, unlike sputum samples, only a small amount of Mycobacterium tuberculosis bacteria was found in cultured samples, and rifampin resistance-related genes existed as a single copy (Donnabella, V., Martiniuk, F., Kinney, D., Bacerdo, M., Bonk, S., Hanna, B., Rom, WN, Isolation of the Gene for the Beta Subunit of RNA Polymerase from Rifampicin-resistant Mycobacterium tuberculosis and Identification of New Mutations, Am.J. Respir Cell.Mol Biol., 1994, 11 (6): 639-643) has a problem of sensitivity that is difficult to amplify.

Second, the presence of non-tuberculous acid-resistant bacteria (MOTT) is also a problem. Conventional primers TR9-TR8 can result in false positives by these MOTTs. This is because the corresponding site of MOTT (157bp) in the rpoB DNA that is the target of SSCP is very similar to Mycobacterium tuberculosis (see Korean Patent Application No. 97-35501). In conclusion, in order to increase the sensitivity and specificity, it can be seen that nested PCR-SSCP or single step nested PCR-SSCP using primers having high TB specificity should be used. There is already an example of a foreign investigator using nested PCR in the determination of the resistance of tuberculosis rifampin, but did not find satisfactory results because it did not find a specific sequence of the tuberculosis bacteria distinct from MOTT.

Accordingly, the present inventors have attempted to solve the electrical problems, nested PCR ("NPCR") and single step nested, which can be directly applied to samples such as sputum, blood, cerebrospinal fluid, etc. obtained from a patient. Tuberculosis specific primary primers for PCR (″ SNPCR ″) and secondary primers for rpoB mutation detection were developed. These primers were used to amplify Mycobacterium tuberculosis rpoB DNA of 205bp as a primary PCR product and 157bp as a secondary PCR product, and then subjected to SSCP analysis to determine the presence or absence of Mycobacterium tuberculosis in the specimen and whether or not the mycobacterium tuberculosis rpoB DNA was mutated. It was. This confirmed that the rifampin resistance of Mycobacterium tuberculosis was confirmed, and the present invention was completed.

After all, the main object of the present invention is to provide a tuberculosis specific primary primer pair for NPCR and SNPCR that can specifically amplify only rpoB DNA segment (205bp) of Mycobacterium tuberculosis.

Another object of the present invention is to provide a second primer pair for NPCR and SNPCR, which can amplify r157B rpoB DNA fragments to find mutations using rpoB DNA fragments (205bp) amplified by electric primer pairs as templates. .

Still another object of the present invention is to provide a method for detecting rifampin-resistant Mycobacterium tuberculosis in a short time by connecting NPCR and SNPCR using an initial primary PCR primer and a secondary PCR primer to SSCP analysis in a patient sample. will be.

1 is a view showing a summary of the detection method of rifampin-resistant Mycobacterium tuberculosis using the nested PCR-SSCP or single-step nested PCR-SSCP of the present invention.

Figure 2 is a diagram showing a comparison of the time required for rifampin resistance test of Mycobacterium tuberculosis by nested PCR-SSCP or single-stage nested PCR-SSCP by conventional culture.

Figure 3 is a diagram showing the position on the rpoB DNA of the primers used in nested PCR-SSCP or single-stage nested PCR-SSCP.

Figure 4 is a diagram schematically showing the process of the rifampin resistance test and blind test (Blind test) by the culture in order to confirm the efficacy of nested PCR-SSCP and single-step nested PCR-SSCP using a tuberculosis specific primer pair to be.

Figures 5a, 5c and 5e are agarose gel electrophoresis pictures showing the first PCR results of the nested PCR performed on mycobacterial standard strains and tuberculosis bacteria including tuberculosis bacteria.

5B and 5D are photographs showing the results of Southern blots of FIGS. 5A and 5C.

6A and 6B are agarose gel electrophoresis photographs showing first and second PCR results of nested PCR performed using two pairs of primers, TB1-TB2 and TB3-TR8, on sputum specimens, respectively. .

Figure 7 is agarose gel electrophoresis picture showing the specificity of the primer TB1-TB2 for the primary PCR when performing a single-step nested PCR.

8 is agarose gel electrophoresis picture showing the sensitivity of the single-step nested PCR.

9A and 9B are photographs showing the results of performing nested PCR-SSCP and single-step nested PCR-SSCP on sputum specimens containing rifampin resistant bacteria, respectively.

Hereinafter, the present invention will be described in more detail.

The single strand conformational process of nested PCR ("NPCR") and single step nested PCR ("SNPCR") using the first primer for primary PCR and the primer for secondary PCR is SSCP (single strand conformational). In connection with polymorphism), a series of procedures for checking the resistance of M. tuberculosis to rifampin are shown in summary in FIG.

Figure 2 is a method for demonstrating the above-described process and its usefulness, the time required to find the missense mutations and determine whether rifampin resistance by DNA sequence analysis, and the rifampin resistance test through the conventional culture culture The figure shows the comparison of time. As can be seen in Figure 2, according to the method of the present invention can determine whether rifampin resistance within about 4 days, the conventional sensitivity test by the culture culture takes about 12 weeks. In addition, even if the PCR-SSCP is used, if the cultured bacteria are used, it is not performed directly on the patient specimens, it will require about 6 weeks for the culture.

First of all, the inventors of the present invention use a DNA obtained from a sample such as sputum, blood, cerebrospinal fluid, or culture bacteria of a patient as a template. primer) (TB1 (forward): 5'-ACGTGGAGGCGATCACACCGCAGACGT-3 '; TB2 (reverse): 5'-TGCACGTCGCGGACCTCCAGCCCGGCA-3') and 157 bp with a variation in rifampin resistance related to the product of the first-order PCR Inner primer (TB3 (forward): 5'-TCGCCGCGATCAAGGAGTTCTTC-3 '; TR8 (reverse): 5'- ^' TGCACGTCGCGGACCTCCA-3 ') to amplify rpoB DNA of It was designed based on the rpoB DNA fragment (360bp) nucleotide sequence of (MOTT) (see FIG. 3) (see Korean Patent Application No. 97-35501).

The genomic DNA of Mycobacterium tuberculosis, MOTT, and general bacteria was used as a template, and PCR products were analyzed using the primary PCR primer TB1-TB2 of the present invention. As a result, only 205 bp of the product was identified. Only rpoB DNA of Mycobacterium tuberculosis was specifically amplified. This could be confirmed by seeing that only the Mycobacterium tuberculosis DNA was hybridized even by Southern blot with a probe prepared by mixing the DNA of Mycobacterium tuberculosis and MOTT.

Based on these results, NPCR was performed in the same manner as above for the patient specimens, not the culture. As a result of the primary PCR, the majority of the samples had a small amount of the primary PCR product, but the secondary PCR was performed using the PCR product TB3-TR8 as the template and the 157bp in all samples. DNA was uniformly amplified in a large amount, and it was confirmed that NPCR using the primers of the present invention specifically amplified the tuberculosis rpoB DNA fragment of 157 bp, in which mutations were intensively associated with rifampin resistance of Mycobacterium tuberculosis.

Mutation of the generated 157bp DNA fragment was confirmed by SSCP analysis, which is commonly used to find genetic variations. That is, when rifampin-sensitive tuberculosis bacteria were used as a control group, if the SSCP pattern of the NPCR product performed on the sample was different from the control group, the rifampin-resistant strain could be determined as a rifampin-sensitive strain.

On the other hand, in the case of SNPCR performing each primer for the first PCR and the second PCR in one tube, the first PCR conditions, that is, denaturation (denaturation) is performed at 94 ℃ for 5 minutes, 94 ℃ 30 seconds → 82 ℃ By confirming that TB1-TB2 is selectively used under the conditions of 30 seconds to 82 ° C for 30 seconds, SNPCR-SSCP is a rifampin-resistant strain with similar accuracy to NPCR-SSCP, even though two pairs of primers are present at the same time. It was confirmed that can be detected. However, bands presumed to be amplification products of MOTT, which appear to be due to the non-specificity of the primer for secondary PCR, may be weakly generated.

As such, SNPCR amplifies desired DNA sequentially according to PCR conditions when two pairs of primers are present at the same time, thereby saving time and cost when screening a large number of samples, and contaminating that may occur when performing NPCR ( There is an advantage to prevent carry over contamination. The minimum amount of Mycobacterium tuberculosis DNA that could be detected by electrical SNPCR was up to 10 fg.

The detection results of Rifampin-resistant M. tuberculosis bacteria obtained by NPCR-SSCP and SNPCR-SSCP using two types of primer pairs of the present invention are prospective by blind test comparing the results by performing the test separately from the conventional bacterial culture assay. A prospective study was conducted to confirm that the two results were in agreement. Thus, it can be seen that the electric NPCR-SSCP and SNPCR-SSCP can be applied directly to the patient sample to detect rifampin resistance of Mycobacterium tuberculosis simpler and more sensitively than any conventional method.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Comparative Example 1 Identification of Mycobacterium Tuberculosis and Rifampin Susceptibility Testing

M. tuberculosis was isolated, identified, and tested for rifampin sensitivity. The WHO cooperative tuberculosis tuberculosis researcher (WHO / IUATLD designated Supranational Reference Laboratory for Global Drug Resistance Surveillance) was performed.

Comparative Example 1-1: Mycobacterium tuberculosis culture and identification

It was performed according to the conventional tuberculosis culture and identification method. Using sputum from a newly diagnosed or treated patient with tuberculosis, the solution was dissolved in an equivalent amount of 4% NaOH for 15 minutes, dissolved, and then centrifuged at 4,000xg for 15 minutes to collect bacteria and Ziehl-Nelsen. The acidophilic staining method was observed under a microscope. Electrolyzed samples were washed twice more with phosphate buffered saline (PBS) at pH 7.0 and then inoculated in Leuwenstein-Jensen medium and incubated at 37 ° C. for 8 weeks. The cultured bacteria were niacin, catalase heat resistance test (68 ° C., 20 minutes), 500 μg / ml of p-nitrobenzoic acid (PNB) and 10 μg / ml of thiophene-2. It was confirmed that the tuberculosis bacteria through the development test in the medium containing carboxylic acid hydrazide (thiophene-2-carboxylic acid hydrazide, TCH).

Comparative Example 1-2: Rifampin Sensitivity Test of Mycobacterium Tuberculosis

Rifampin susceptibility testing of cultured Mycobacterium tuberculosis was performed using the resistance ratio method (see Canneti, G., Fox, W., Khomenko, A., Mahler, HT, Menon, NK, Mitchison, DA, Rist, N., and Smelev). , NA, Advances in Techniques of Testing Mycobacterial Drug Sensitivity and the Use of Sensitivity Tests in Tuberculosis Control Programs, Bull., WHO 1969, 41: 21-431969). After culturing the cultured tuberculosis bacteria in a medium containing 40 μg / ml of rifampin for 4 weeks, the number of tuberculosis bacteria grown on the rifampin-containing medium was compared to the number of tuberculosis on the rifampin-free medium as compared to the growth of tuberculosis on the rifampin-free medium. If it is 1% or more, it was determined to be resistant.

Example 1: Genomic DNA Isolation of Cultured Mycobacterium Tuberculosis and Mycobacterium Tuberculosis

In order to extract genomic DNA, bead-beater / phenol (BB / P) method using vitreous spheres was used (Hunt JM, Roberts GD, Stockman L, Felmlee TA, Persing DH, Detection of a genetic). locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens, Diagn Microbiol.Infect.Dis., 1994, 18: 219-27). Smear positive sputum specimens positive for microbial observation were used in the same manner as in Comparative Example 1-1, and cultured tuberculosis bacteria were suspended in 200 µl of TEN buffer solution (Tris-HCl 10 mM, EDTA 1 mM, NaCl 100 mM), 100 μl of vitreous spheres (diameter 0.1mm, glass beads 11079-110; Biospec Products, Bartlesville, Okla., USA), PCI solution (phenol: chloroform: isopropylalcohol = 50: 49: 1 (v / v / v) ) 250 μl was added and shaken for 1 minute with a Mini beater (74005-0722, Biospec Products, Bartlesville, Okla., USA). After cell disruption, the supernatant was obtained by centrifugation at 12,000 rpm for 5 minutes, and 10 µl of 3M sodium acetate and 250 µl of ethanol were added to the supernatant, followed by 10 minutes at -20 ⁰ C to obtain a DNA precipitate. . The DNA thus obtained was dissolved in 60 μl of TE buffer, and then used as template DNA for PCR in Examples described later.

Example 2: Identification of Mycobacterium Tuberculosis by PCR

Apart from the identification of Mycobacterium tuberculosis by mycobacterium tuberculosis incubation, the presence of Mycobacterium tuberculosis in sputum specimens prior to NPCR-SSCP and SNPCR-SSCP was confirmed by detecting Mycobacterium tuberculosis DNA in the extracted DNA. After performing a PCR reaction using an IS6110 amplification kit (TB-CR, TB detection kit, Bionia, Korea) and a thermocycler (Thermocycler Model 9600, Cetus, USA), which are known as Mycobacterium tuberculosis specific insertion elements, Electrophoresis on 1% agarose gel confirmed the product of 532 bp.

Example 3 Preparation of Primers for Nested PCR (″ NPCR ″) and Single Step Nested PCR (″ SNPCR ″)

Example 3-1 Preparation of Primer Pairs for Primary PCR

Primary primer pair TB1-TB2 for NPCR and SNPCR that can specifically amplify only r205B rpoB fragments of Mycobacterium tuberculosis without amplifying the rpoB gene segments of non-tuberculosis acid bacterium (MOTT) or normal bacteria Was prepared based on the rpoB fragment (306bp) nucleotide sequence of Mycobacterium tuberculosis and MOTT (see Korean Patent Application No. 97-35501) (see FIG. 3).

TB1 (Forward): 5'- ACGTGGAGGCGATCACACCGCAGACGT-3 '

TB2 (Reverse): 5′-TGCACGTCGCGGACCTCCAGCCCGGCA-3 ′

Example 3-2 Preparation of Primer Pairs for Secondary PCR

A 205 bp rpoB fragment of Mycobacterium tuberculosis amplified by the primary PCR was used as a template to prepare a pair of inner primers for secondary PCR for NPCR and SNPCR to specifically amplify the 157 bp fragment in which mutations are concentrated. Forward primer TB3 was prepared based on the rpoB fragment (306bp) nucleotide sequence of MOTT (see Korean Patent Application No. 97-35501) (refer to FIG. 3), and reverse primer TR8 was prepared from a conventional primer (Telenti, A). ., Imboden, P., Marchesi, F., Lowrie, D., Cole, S., Colston, MJ, Matter, L., Schopfer, K., Bodmer, T., Detection of Rifampicin-resistance Mutations in Mycobacterium tuberculosis , Lancet, 1993, Mar. 13; 341 (8846): 647-650).

TB3 (Forward): 5-′TCGCCGCGATCAAGGAGTTCTTC-3 ′

TR8 (Reverse): 5′- ^' TGCACGTCGCGGACCTCCA-3 ′

Example 4 First PCR Reaction and Southern Blot of NPCR with DNA of Standard Strains and General Bacteria

The DNA of 15 kinds of general bacteria, such as Mycobacterium tuberculosis and 18 non-tuberculosis acidophilic bacteria (MOTT), Rhodococcus genus 3, Nocardia genus 2 and Corynebacterium genus 2, shown in Table 1 below, prepared in Example 3-1 PCR primers were prepared by mixing 20 pmol of each primer primer TB1-TB2 and PreMix-Top (Bionia, Korea), and adding distilled water to a final volume of 20 μl. At this time, PCR is subjected to a 30-minute process of denaturation at 94 ° C for 5 minutes, 94 ° C 30 seconds → 82 ° C 30 seconds → 82 ° C 30 seconds using a thermocycler (Thermocycler, Model 9600, Cetus, USA). The final extension process was performed at 82 ° C. for 5 minutes.

The amplified product was electrophoresed on 1.5% agarose gel (see FIGS. 5A and 5C).

Mycobacteria and General Bacteria Used to Determine Mycobacterium Tuberculosis Specificity of Primary Primers for NPCR and SNPCR Mycobacteria M. avium (ATCC 25291) M. chelonae (ATCC 35749) fortuitum (ATCC 6841) M. gastri (ATCC 15754) gordonae (ATCC 14470) M. intracellulare (ATCC 13950) M. kansasii (ATCC 12478) M. malmoense (ATCC 29571) M. nonchromogenicum (ATCC 19530) M. phlei (ATCC 11758) scrofulaceum (ATCC 19981) M. simiae (ATCC 25275) smegmatis (ATCC 19420) M. szulgai (ATCC 35799) M. terrae (ATCC 15755) M. tuberculosis H37Rv (ATCC 27294) M. triviale (ATCC 23292) M. ulcerans (ATCC 19423) vaccae (ATCC 15483) 2. General bacteria Rhodococcus equi Rhodococcus erythropolis Rhodococcus rhodniiNocardia otitidiscaviarum Nocardia nova Corynebacterium diphtheriae Corynebacterium glutamicum Staphylococcus aureus Micrococcus luteus

Figures 5a, 5c and 5e shows the results of PCR performed by the above method using the DNA of mycobacterial standard strain (ATCC strain) and general bacteria including tuberculosis bacteria.

In FIG. 5A, M is a DNA size marker as øⅩ174 digested with Hae-III; Lanes 1 to 15 include M. tuberculosis H37Rv, M. tuberculosis (patient isolate), M. avium, M. fortuitum, M. gastri, M. gordonae, M. intracellulare, M. kansasii, M. malmoense, M. nonchromogenicum, M. phlei, M. sucrofulaceum, M. simiae, M. smegmatis, M. terrae, respectively; M in FIG. 5C is a DNA size marker as øⅩ174 digested with Hae-III; Lanes 1 to 16 were M. tuberculosis H37Rv, M. tuberculosis (Korean patient isolate strain), M. triviale, M. vaccae, M. chelonae, M. szulgai, M. ulcerans, S. aureus, S. epidermidis , M. luteus, S. pneumoniae, S. faecalis, S. pyogenes, C. diphtheriae, N. meningitidis, H. influenzae, respectively; In FIG. 5E, M is øⅩ174 DNA size marker cleaved with Hae-III; Lanes 1 to 9 include M. tuberculosis H37Rv, M. tuberculosis (patient isolate), R. equi, R. erythropolis, R. rhodnii, N. nova, N. otitidiscaviarum, C. diphtheriae, C. glutamicum Represent each.

5A and 5C, amplification of Mycobacterium tuberculosis and MOTT DNA was performed by using a DNA of the standard strain (ATCC strain) as a template and performing a primary PCR using an outer primer TB1-TB2. As a result, it was confirmed that 205 bp of DNA was amplified only in the first and second lanes representing Mycobacterium tuberculosis (see arrows of FIGS. 5A and 5C).

5B and 5D show the results of Sudden blot for the primary PCR product obtained above. Each lane of FIGS. 5B and 5D represents the same strain as each lane of FIGS. 5A and 5B, respectively.

Southern blot was performed through the following process:

PCR products electrophoresed with agarose gels were transferred to a nylon membrane using transfer buffer (10X SSC) and membranes were dried at 90 ° C. for 2 hours in a vacuum dry oven. fixed to the membrane.

As the probe, 157 bp rpoB DNA was used as the amplification product using the second primer (inner primer). That is, the DNA of Mycobacterium tuberculosis and 18 non-tuberculosis acidophilic bacteria (MOTT) was used as a template, followed by electrophoresis by amplification with TB3-TR8 primers, which amplify all 157bp rpoB DNA. QIAGEN, Hilden, Germany) and added 2.5 ng / strains so that the total amount was 50 ng. This mixed DNA was labeled with [P ³² ] -dCTP using the Ready prime kit (Amersham, UK), purified on a Sephadex G-50 column and used as a probe.

Membranes were prehybridized with 6X SSC, 5X Denhardt solution, 0.5% SDS and 100 mg of denatured salmon sperm DNA for 1 hour and hybridized with labeled probes. (hybridization). 5 minutes at room temperature with a solution containing 2X SSC, 0.5% SDS, 15 minutes with a solution containing 2X SSC, 0.1% SDS, and 1 hour membrane washing at 68 ° C with a solution containing 0.1X SSC, 0.5% SDS, It was dried and autoradiography.

As shown in FIGS. 5B and 5D, it was observed that only the first and second lanes of Mycobacterium tuberculosis DNA were detected by the prepared probes capable of detecting rpoB DNA of Mycobacterium tuberculosis and MOTT (see FIGS. 5B and 5D). arrow).

On the other hand, Rhodococcus, Nocardia, Corynebacterium known as genus close to the genus Mycobacterium was confirmed that there is no amplification product (see Fig. 5c).

Therefore, it was confirmed that only rpoB DNA of Mycobacterium tuberculosis was specifically amplified by primary PCR using the primary primer TB1-TB2 for NPCR and SNPCR.

Example 5: Secondary PCR Reaction of Nested PCR (NPCR)

10^-3In concentration Dilute the template DNA, the amplification product of the primary PCR reaction using TB1-TB2, and the primer TB3-TR8 for the secondary PCR into 20 pmol and PreMix-Top (Bionia, Korea), respectively. Distilled water was added to a volume of 20 μl to prepare a reaction mixture, and the second PCR was performed. PCR was performed for 5 minutes at 94 ° C for denaturation using a thermocycler (Thermocycler, Model 9600, Cetus, USA), followed by 30 times at 94 ° C 30 seconds → 72 ° C 30 seconds → 72 ° C 30 seconds. After the final extension was performed for 5 minutes at 72 ° C. Amplification products were electrophoresed on 1.5% agarose gel.

Example 6: Nested PCR (NPCR) on Sputum Specimen

The primary PCR primer TB1-TB2, which confirmed the specificity of Mycobacterium tuberculosis in the standard strain, and the primer primer TB3-TR8, which amplify the rpoB DNA fragment, were targeted for sputum specimens of tuberculosis patients. Was performed (FIGS. 6A, 6B).

M in FIG. 6A is DNA size marker as øⅩ174 digested with Hae-III; Lanes 1-14 show the results of primary PCR using DNA obtained from patient sputum as a template and primers TB1-TB2. In FIG. 6B, M is DNA as øⅩ174 digested with Hae-III. Size markers; Lanes 1 to 14 are obtained by diluting the primary PCR amplification products obtained above to 10 ^-3 concentration as template DNA, and performing secondary PCR using primers TB3-TR8.

As shown in Figure 6a, the amount of the primary PCR product using the tuberculosis-specific primer TB1-TB2 in the majority of samples was very small, it can be seen that a small amount of tuberculosis bacteria in the sputum, lanes 8, 9 and It was observed that a large amount of 205 bp DNA was amplified only in lane 12. However, in the second PCR using the inner primer TB3-TR8 which purifies and dilutes the amplification products into template DNA and amplifies the rpoB DNA fragment, the 157bp DNA is uniformly large in all samples as expected. Amplified was observed (see FIG. 6B). As a result, it was confirmed that a small amount of Mycobacterium tuberculosis in sputum could be specifically detected by NPCR using two pairs of primers, that is, a primer for primary PCR and a primer for secondary PCR.

Example 7: Single Stage Nested PCR (SNPCR)

Example 7-1: Confirming Annealing Specificity of Primary Primer Pairs when Performing SNPCR

Prior to performing the SNPCR, the following experiment was performed to confirm that only the primary PCR product is produced even if the primary PCR primer and the secondary PCR primer exist simultaneously under the primary PCR conditions:

Three PCR mixtures were prepared by simultaneously adding 20 pmol of primer pair TB1-TB2 for primary PCR, 20 pmol of primer TB3-TR8 for secondary PCR, and 20 pmol of two primers TB1-TB2 and TB3-TR8 respectively. That is, after proceeding 30 times at 95 ℃ 5 minutes, 94 ℃ 30 seconds → 82 ℃ 30 seconds → 82 ℃ 30 seconds, PCR was performed at 82 ℃ 5 minutes to perform 1.5% agarose gel electrophoresis (see Fig. 7). In Figure 7, M is ø 174 DNA size marker cleaved with Hae-III; The first and second lanes (designated as ″ 1 ″) are primer pairs TB1-TB2 for primary PCR only, and the third and fourth lanes (designated as ″ 2 ″) are designed for primer pairs TB3-TR8 for secondary PCR However, the 5th lane and the 6th lane (indicated by "1 + 2") show the results of SNPCR under primary PCR conditions using two types of primer pairs TB1-TB2 and TB3-TR8 simultaneously.

As shown in FIG. 7, when the primer for primary PCR was used (″ 1 ″) and when the primary and secondary PCR primers were added at the same time (″ 1 + 2 ″), the primer for primary PCR of 205 bp was added. Amplification products were observed, but when only the primer TB3-TR8 for secondary PCR was used (″ 2 ″), no amplification products were observed. Therefore, even when the primary PCR primers and the secondary PCR primers are used simultaneously when performing SNPCR, only the primary PCR primers TB1-TB2 are selectively used among the two types of primer pairs under the conditions of the primary PCR performed initially. Could confirm.

Example 7-2: Single Stage Nested PCR (SNPCR)

SNPCR was performed by mixing DNA from the sputum sample, 0.5 pmol each of the primary primer pair TB1-TB2, 20 pmol each of the secondary primer pair TB3-TR8, and PreMix-Top (Bionia, Korea). At this time, the SNPCR is subjected to a denaturation process at 94 ° C. for 5 minutes using a thermocycler (Thermocycler Model 9600, Cetus, USA), and 15 times at 94 ° C. 30 seconds → 82 ° C. 30 seconds → 82 ° C. 30 seconds. After proceeding, the process of denaturation at 94 ° C. for 5 minutes, the process at 94 ° C. 30 seconds → 72 ° C. 30 seconds → 72 ° C. 30 seconds was performed 30 times, and final extension was performed at 72 ° C. for 5 minutes.

Example 7-3: Sensitivity Measurement of Single Step Nested PCR (SNPCR)

In order to determine the minimum Mycobacterium tuberculosis DNA concentration that can be detected by SNPCR, DNA of M. tuberculosis H37Rv was gradually diluted and subjected to SNPCR as a template. Amplified products were electrophoresed on 1.5% agarose gel (see FIG. 8). In FIG. 8, M is a øⅩ174 DNA size marker cleaved with Hae-III; Lanes 1 to 8 were obtained by SNPCR using DNA of M. tuberculosis H37Rv at concentrations of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg; Lane 9 represents the negative control, respectively.

As shown in FIG. 8, it was observed that SNPCR can detect up to 10 fg of Mycobacterium tuberculosis DNA (arrow). As a result, it was found that the minimum detectable Mycobacterium tuberculosis DNA concentration of SNPCR, which is useful for performing a large number of sputum samples, was 10 fg.

Example 8: Single strand conformational polymorphism (SSCP) reaction

NPCR and SNPCR were performed by adding [P ³² ] -dCTP (NEN, NEG 513H, USA) to the PCR mixture. 2 μl of the reaction product generated above and 4 μl of the stop solution were mixed and denatured at 95 ° C. for 5 minutes, and 4 μl of the MDE gel (AT Biochem, a nondenaturation gel) was used. Electrophoresis using Malvern, PA; AT Biotech, 1-500-01). MDE gel was mixed with 25 ml of MDE, 6 ml of 10X TBE, and 69 ml of distilled water to make 100 ml. Then, 800 µl of 10% ammonium persulfate and 40 µl of TEMED were added and poured into a gel plate to harden for 3 hours. The denatured PCR product was placed in each well by 5 μl / well, alternating with the control, electrophoresed for 6 hours at 6 W with 0.6 X TBE, followed by drying and autoradiography. The control DNA was extracted and used by the method described in Example 1 from M. tuberculosis H37Rv which is rifampin-sensitive.

On the other hand, in order to demonstrate the practical efficacy of the detection and discrimination of Mycobacterium tuberculosis rifampin resistance of NPCR-SSCP and SNPCR-SSCP using the primer pair of the present invention, the same test for sputum specimens at the same time as the tuberculosis researchers in blind test (blind test) Prospective study was performed (see FIG. 4). In addition, the conventional TR9-TR8 primer (see Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, Matter L, Schopfer K, Bodmer T. PCR-SSCP using rifampicin-resistance mutations in Mycobacterium tuberculosis.Lancet 1993 Mar 13; 341 (8846): 647-650) was performed simultaneously and compared. PCR was denatured at 95 ° C. for 5 minutes; 95 ° C. 30 sec → 65 ° C. 30 sec → 72 ° C. 45 sec, proceeded 30 times; Extension was carried out at 72 ° C. for 5 minutes and SSCP was carried out in the manner described above.

Mycobacterium tuberculosis culture test As a result of the rifampin resistance test, it was confirmed that, among the 56 samples identified as smear and culture positive specimens, 21 specimens contained rifampin-resistant tuberculosis bacteria. In the NPCR-SSCP and SNPCR-SSCP, 21 specimens with different band shift patterns were found, which were consistent with those identified as resistant strains in the sensitivity test by the culture culture (see Table 2). The remaining specimens were susceptible to susceptible tuberculosis bacteria that showed the same pattern as the control strain.

Comparison of results of conventional rifampin susceptibility test with tuberculosis bacterium cultured by blind test of tuberculosis patients' sputum and results of NPCR-SSCP / SNPCR-SSCP * Method Sample Susceptibility Results SSCP Genetic variation Method Sample Susceptibility Results SSCP Genetic variation One S － No variation 29 R + 526 _His → 526 _Tyr (CAC → TAC) 2 S － No variation 30 S － No variation 3 R + 526 _His → 526 _Tyr (CAC → TAC) 31 S － No variation 4 R + 526 _His → 526 _Arg (CAC → CGC) 32 R + 531 _Ser → 531 _Leu (TCG → TTG) 5 R + 531 _Ser → 531 _Leu (TCG → TTG) 33 R + 531 _Ser → 531 _Leu (TCG → TTG) 6 R + 526 _His → 526 _Tyr (CAC → TAC) 34 S － No variation 7 R + 531 _Ser → 531 _Leu (TCG → TTG) 35 R + 513 _Gln → 513 _Pro (CAA → CCA) 8 S － No variation 36 S － No variation 9 R + 516 _Asp → 516 _Val (GAC → GTC) 37 S － No variation 10 R + 531 _Ser → 531 _Leu (TCG → TTG) 38 S － No variation 11 S － No variation 39 S － No variation 12 S － No variation 40 S － No variation 13 S － No variation 41 S － No variation 14 S － No variation 42 R + 526 _His → 526 _Tyr (CAC → TAC) 15 S － No variation 43 R + 531 _Ser → 531 _Leu (TCG → TTG) 16 S － No variation 44 S － No variation 17 R + 531 _Ser → 531 _Leu (TCG → TTG) 45 S － No variation 18 R + 531 _Ser → 531 _Leu (TCG → TTG) 46 S － No variation 19 S － No variation 47 R + 531 _Ser → 531 _Leu (TCG → TTG) 20 S － No variation 48 R + 531 _Ser → 531 _Leu (TCG → TTG) 21 R + 531 _Ser → 531 _Leu (TCG → TTG) 49 S － No variation 22 S － No variation 50 S － No variation 23 S － No variation 51 S － No variation 24 R + 531 _Ser → 531 _Leu (TCG → TTG) 52 R + 531 _Ser → 531 _Leu (TCG → TTG) 25 S － No variation 53 S － No variation 26 S － No variation 54 S － No variation 27 S － No variation 55 S － No variation 28 R + 531 _Ser → 531 _Leu (TCG → TTG) 56 S － No variation

* R / S: rifampin resistance / sensitivity, +/-; SSCP pattern is different / same as control

9A and 9B show the results of NPCR-SSCP and SNPCR-SSCP on 21 resistant strains of 21 specimens identified as rifampin-resistant Mycobacterium tuberculosis, respectively, and C represents Rifampin-sensitive tuberculosis (M. tuberculosis H37Rv). Lanes 1 to 7 represent each sputum sample.

9a and 9b, the band pattern of each sample was different from the control group was found to be rifampin resistant bacteria. In FIG. 9A, the new band, which is not visible in the first lane of FIG. 9B, was shown to be amplified by the rpoB DNA of MOTT due to the nonspecificity of the primer for the secondary PCR, which was confirmed to be MOTT by sequencing analysis.

On the other hand, the results of PCR-SSCP using the conventional TR9-TR8 primers carried out with the culture were also the same as the results of NPCR-SSCP and SNPCR-SSCP performed directly on the sputum sample.

Example 9: Gene Variation Pattern of Rifampin Resistant Mycobacterium Tuberculosis

The nucleotide sequence of a sample judged to be resistant by NPCR-SSCP and SNPCR-SSCP was amplified by PCR using a primer pair (Rpo665-Rpo998) capable of amplifying rpoB DNA (342 bp) of mycobacteria (see Korean patent) Application No. 97-35501) The nucleotide sequence was analyzed by PCR using the purified DNA.

As compared with the rpoB DNA sequence of the control strain M. tuberculosis H37Rv, all 21 PCR products identified as rifampin resistance showed gene mutations related to rifampin resistance (see Table 2). The most frequent variation was 531 _Ser- > 531 _Leu (TCG-> TTG)-14 specimens (66.7%). In addition, five (23.8%) samples were 526 _His , and the other two (9.5%) were 516 _Asp and 513 _Gln , respectively. Genetic variation corresponding to 90.5% was found in 531 _Ser and 526 _His sites.

As a result, the NPCR-SSCP or SNPCR-SSCP method using the Mycobacterium tuberculosis-specific primer pair of the present invention can obtain the same results as the susceptibility test by culture culture, and can quickly detect rifampin-resistant Mycobacterium tuberculosis from the sputum sample. It was confirmed that it is an alternative method with advantages.

As described and demonstrated in detail above, in the present invention, by detecting the tuberculosis bacteria directly from the patient sample using NPCR-SSCP and SNPCR-SSCP targeting the rpoB gene segment of Mycobacterium tuberculosis, to determine whether the tuberculosis rifampin resistance A primer for primary PCR and a primer for secondary PCR were developed. In the case of NPCR-SSCP using the above primers, due to the high specificity of the primers for primary PCR, only DNA of Mycobacterium tuberculosis can be specifically amplified to prevent false positives caused by non-tuberculosis acidophilic bacteria (MOTT). In this case, a large number of samples can be processed, which can greatly save time and money.

Claims (6)

A pair of primers having the following sequence to specifically amplify the rpoB gene segment (205 bp) of M. tuberculosis: TB1 (Forward): 5'-ACGTGGAGGCGATCACACCGCAGACGT-3 ' TB2 (Reverse): 5'-TGCACGTCGCGGACCTCCAGCCCGGCA-3 ' Primer pairs having the following sequences to amplify rpoB gene segments (157 bp) of M. tuberculosis and non-tuberculosis acidophilic bacteria (MOTT): TB3 (Forward): 5'-TCGCCGCGATCAAGGAGTTCTTC-3 ' TR8 (Reverse): 5'-TGCACGTCGCGGACCTCCA-3 ' Each primer pair below is included as an active ingredient, and genomic DNA extracted from Mycobacterium tuberculosis contained in a patient's sample is used as a template, and a primary PCR reaction is performed using the following primer 1 as the primary primer, followed by the first generation A diagnostic reagent for detecting rifampin-resistant Mycobacterium tuberculosis by analyzing the SSCP profile of a nested PCR amplification product which performs a secondary PCR reaction using a PCR product as a template and a primer 2 as a secondary primer; ① Primer 1 TB1 (Forward): 5'-ACGTGGAGGCGATCACACCGCAGACGT-3 ' TB2 (Reverse): 5'-TGCACGTCGCGGACCTCCAGCCCGGCA-3 ' ② Primer 2 TB3 (Forward): 5'-TCGCCGCGATCAAGGAGTTCTTC-3 ' TR8 (Reverse): 5'-TGCACGTCGCGGACCTCCA-3 ' The method of claim 3, wherein The patient's sample is sputum, blood or cerebrospinal fluid Diagnostic reagent. Each primer pair is included as an active ingredient, and the first PCR reaction using genomic DNA extracted from Mycobacterium tuberculosis contained in a patient sample as the template, and the following primer 1 as the primary primer, and the first generated PCR product A diagnostic reagent for detecting rifampin-resistant tuberculosis bacteria by analyzing the SSCP pattern of a single step nested PCR amplification product which simultaneously performs a secondary PCR reaction using the following primer 2 as a secondary primer: ① Primer 1 TB1 (Forward): 5'-ACGTGGAGGCGATCACACCGCAGACGT-3 ' TB2 (Reverse): 5'-TGCACGTCGCGGACCTCCAGCCCGGCA-3 ' ② Primer 2 TB3 (Forward): 5'-TCGCCGCGATCAAGGAGTTCTTC-3 ' TR8 (Reverse): 5'-TGCACGTCGCGGACCTCCA-3 ' The method of claim 5, The patient's sample is sputum, blood or cerebrospinal fluid Diagnostic reagent.