KR20000075180A - Method and Tool for detection of Mycobacteria - Google Patents

Abstract

PURPOSE: A method for diagnosing and identifying the species of Mycobacteria causing tuberculosis, and probes and primers used therefor are provided which improve diagnosis and identification for Mycobacteria in species level. Therefore, a number of species of Mycobacteria can be diagnosed and identified simultaneously by using the method. CONSTITUTION: The probes and primers for identifying Mycobacteria are designed based on the internal transcription (ITS) base sequence of Mycobacteria. The ITS base sequence is useful in distinguishing genotypes since it has more numerous polymorphism regions as well as conservative regions than 16S rRNA commonly used as a target DNA. The base sequences of M. fortuitum and M. chelonae are identified, and probes and primers are synthesized using their base sequences. The probes and primers for other Mycobacteria are designed by analyzing the base sequence obtained from public domain(Genbank) with multialignment and blast and by selecting the distinguishable regions by polymorphism. The method comprises the following steps of: a)culturing Mycobacteria in Ogawa medium and isolating genomic DNA therefrom by vortexing and centrifuging; b)constructing primers for amplifying ITS; c)performing PCR and confirming PCR products by agarose gel electrophoresis; d)cloning ITS of M. fortuitum and M. chelonae with pGEM-T vector and transforming E.coli JM 109 therewith by Heat-shock; e)DNA sequencing using a DNA auto sequencer and universal primer M13 by dye terminator; f)designing primers and probes from the ITS sequences.

Description

Diagnostic tools and methods of mycobacteria {Method and Tool for detection of Mycobacteria}

The present invention relates to a method for early diagnosis of tuberculosis by developing probes and primers for identifying tuberculosis bacteria, as well as a diagnostic method capable of accurately identifying the causative bacteria of tuberculosis bacteria by identifying the types of tuberculosis bacteria.

In the 1950s, it was reported that Mycobacteria other than tuberculosis (MOTT) can cause disease in humans.Since 1980, the M. avium complex has been known to cause systemic diseases in AIDS patients. Interest is growing. Non-tuberculous mycobacteria can be diagnosed with clinical findings and general pathology similar to tuberculosis, but non-tuberculosis mycobacteria are widely distributed in living environments, making it difficult to determine pathogenicity even when isolated from clinical specimens. In addition, since most of the anti-tuberculosis drugs have shown drug resistance, treatment is difficult and recurrence rate is known to be high. Strains resistant to various drugs are gradually increasing, and since the treatment of Mycobacterium tuberculosis bacteria and non-tuberculosis bacteria is different, there is a need for a rapid and accurate identification method for them.

With the increase in the incidence of tuberculosis and multidrug-resistant tuberculosis strains, there is a need for a faster and more accurate method for identifying TB bacteria as well as TB complexes for effective treatment and management of tuberculosis.

Early diagnosis of mycobacteria and the identification of Mycobacterium tuberculosis bacteria are of great importance for treatment, and various diagnostic methods have been studied and developed. Currently used identification and infection diagnosis methods and their advantages and disadvantages are as follows.

First, there is a microbiological method, smear and culture test. This method is not suitable for pathogenic microorganisms, such as mycobacteria, which require longer incubation times and higher risk of infection for the experimenter.

Second, the polymerase chain reaction (PCR) method has high sensitivity and specificity and is very useful for detecting pathogens that take a long time to separate and culture like mycobacteria. In particular, since a small amount of DNA is amplified and detected, only a small amount of pathogens present in the sample can be identified without going through the culture process of the strain. The detection by PCR has been introduced in various ways depending on the target DNA to be amplified. Among them, IS6110 and 16S rRNA, which exist in several copies, are mainly used.

Third, the physicochemical analysis of mycobacterial cell components detects fatty compounds in mycobacterial cells using gas chromatography and mass spectrometry. Although the specificity is high, expensive equipment is needed.

Fourth, the detection of cell components by serological methods includes the aggregation reaction using blood or latex particles which adsorb the antibody to the bacterial antigen, and the enzyme-linked immunoassay method in which the enzyme is bound to the antibody. However, this technique is very sophisticated and requires careful attention and is only possible in limited laboratories.

Fifth, there is a method to insert the luciforase gene into mycobacteria L5 and infect it with mycobacteria to search for the luminescence with luciferin added in the medium. (WR JACOBS, RG BARLETTA, R. UDANI, J. CHAN , G. KALKUT, G. SOSNE, T. KIESER, GJ SARKIS, GF HATFUL, BR BLOOM. 1993, Science 260: 819-822)

Sixth, there is a method of identification by hybridization of oligonucleotide probes (A. TROESCH, H. NGUYEN, CGMIYADA, S. DESVARENNE, TR GINGERAS, PM KAPLAN, P. CROS and C. MABILAT. J. Clin.Microbiol. 1999, 37: 49-55.)

The present invention provides an oligonucleotide probe for use in diagnosing mycobacteria in response to specific nucleotide sequences of mycobacteria, and provides a method for diagnosing mycobacteria by enzymatic polymerase chain reaction using oligonucleotide primers. According to the present invention, it is possible to determine whether or not it is mycobacterium.

1 is a schematic diagram of the position of the primers used for the PCR amplification of ITS and ITS of mycobacteria, Figure 2 is a fragment of the mycobacteria using primer pairs of the nucleotide sequence of SEQ ID NO: 3, 4 containing 16S and 23S ITS electrophoresis after polymerase chain reaction (PCR), Figure 3 shows the ITS of Mycobacterium fortuitum and Mycobacterium chelonae obtained by the polymerase chain reaction Electrophoresis picture after cloning in a vector, FIG. 4 shows electrophoresis after polymerase chain reaction (PCR) with primer pairs of SEQ ID NOs: 3 and 6 and SEQ ID NOs: 4 and 5, which are specific sequences of the genus Mycobacteria. Photo, Figure 5 is a photograph of the electrophoresis after the nucleic acid sample and polymerase chain reaction of each species with a specific primer prepared with the species-specific base sequence of each species.

The present invention is a simple, high-accuracy and efficient method that can replace the existing bacterial culture and biochemical methods of bacterial identification by devising a common sequence region and genus specific sequence region of mycobacteria as probes or primers. Provide a method. To this end, identification probes and primers were designed from the ITS sequence of mycobacteria. ITS has more polymorphic regions and conservative regions than 16S rRNA, which is commonly used as target DNA, and therefore has high utility as target DNA for genotyping. The base sequence. In the present invention, the nucleotide sequence of M. fortuitum and M. chelonae, which have not yet been identified, are identified and the nucleotide sequence of the two strains and the disclosed nucleotide sequence information are analyzed by multialignment and blast to identify mycobacteria and probes and primers. To provide. In addition, theorem-specific probes and primers are designed by selecting sites that can be differentiated by polymorphism of the same sites.

The present invention is explained in more detail based on the following examples. However, the scope of the present invention is not limited to these examples.

Example 1 Culture of Mycobacterium Tuberculosis and Genomic DNA Isolation

The standard strains were obtained from Mycobacteria from Korean Collection for Type Culture (KCTC) and American Type Culture Collection (ATCC), and clinical strains were obtained through the Korean Tuberculosis Association and Tuberculosis Hospital. It was performed under the management of pathology and clinical microbiology professor. Mycobacterial DNA extraction was performed in the following manner. After incubating the mycobacteria in Ogawa medium, 100 μl of this was placed in a centrifuge tube, added with 200 μl of InstaGene matrix (Bio-Rad Co.), suspended for 30 minutes at 56 ° C., and then vortexed for 10 seconds. Heat-process for 8 minutes at 100 degreeC. After vortex again for 10 seconds, the mixture was centrifuged at 12,000 rpm for 3 minutes, and the supernatant was transferred to a new tube and stored at -20 ° C. 2 μl of the isolated DNA was used for the PCR reaction.

Example 2. Preparation of Primer to Amplify ITS of Mycobacteria

In the mycobacteria, the nucleotide sequence near the ITS is shown in FIG. Therefore, in order to amplify ITS, some nucleotide sequences of conserved regions of 16S rRNA and 23S rRNA of mycobacteria were selected and prepared as primers. The primer design was selected by computer analysis of the nucleotide sequences of 16S and 23S rRNAs of mycobacteria, which have already been published. The selected primers had the nucleotide sequences of SEQ ID NOs: 3 and 4, and were used to selectively amplify the ITS region to about 500 bp with 16S rRNA and a part of 23S rRNA. Amplification with the above primer is shown in FIG. All primers used in the experiment, including the ITS amplification primers, were made at a concentration of 50 nmol by Perkin-Elmer DNA synthesizer by BioBasic (Canada).

Example 3 PCR Reactions and Identification of Products

The composition of the PCR reaction is as follows. 500 mM KCl, 100 mM Tris HCl (pH 9.0), 1% Triton X-100, 0.2 mM deoxynucleoside triphosphates (dATP, dGTP, dTTP and dCTP) 1.5 mM MgCl ₂ , 1 pmol primer, 1U Taq DNA polymerase (Bio Basic Inc .) The mixed solution was reacted for 5 minutes at 94 ° C., sufficiently denatured, and then reacted 30 times for 1 minute at 94 ° C., 1 minute at 60 ° C., and 1 minute at 72 ° C., and finally extended at 72 ° C. for 10 minutes. After the reaction, the PCR product was confirmed by electrophoresis on 1.5% agarose gel. As predicted from Genbank's data, the size of ITS amplified by conservative primers of 16S and 23S rRNA was about 500 bp, but the mycobacterium smegmatis and mycobacterium M. agri, Mycobacterium moricaens, and Mycobacterium gallinarum (M. gallinarum) were 50 bp larger than other strains. Therefore, it can be seen that the size of the ITS according to the type of strain is about 270 ~ 350bp.

Example 4 Cloning of M. fortuitum and M. chelonae ITS

After confirming the PCR reaction, the reactions of ITS of M. fortuitum and M. chelonae, whose ITS sequences were not disclosed, were purified by PCR purification kit (QIAGEN) and used for cloning. PCR products of purified ITS were cloned into pGEM-T vector (Promega Co. Madison, Wis.). The genotyping method using heat-shock was induced into E. coli JM 109, a host, and plated on X-gal / IPTG / Amp + / LB agar medium prepared with transformants and incubated for 16-18 hours in a 37 ° C. incubator. After incubation, only white colonies were picked out and grown in LB (Amp +) liquid medium to separate and purify the plasmid. Plasmid DNA was isolated with a QIAprep kit and cut and inserted into DNA fragments with restriction enzymes (FIG. 3).

Example 5. DNA Sequencing

DNA was quantified at a concentration of 200 μmol and used for the reaction. DNA sequences were determined by dye terminator method using universal primer M13 as a DNA auto sequencer (Perkin Elmer, ABI prim 377 sequencer).

Example 6 Selection and Synthesis of Oligonucleotide Primers

The nucleotide sequence of the ITS region of M. fortuitum and M. chelonae obtained through Example 5 and other mycobacterial ITS sequences obtained from Genbank were analyzed by multialignment and blast, corresponding to the conserved sequences of ITS of mycobacteria. It is designed to identify mycobacterial primers, and to designate strain-specific primers that correspond to nucleotide sequences with high polymorphism.

The positions and their nucleotide sequences within the ITS of the designed primers are shown in Table 1.

Identification strain Probe name Location in ITS Sequence number Mycobacteria MYC1MYC2MYC3MYC4 Depends on species 5678 TB COMPLEX MTB1MTB2MTB3MTB4MTB5MTB6MTB7MTB8MTB9 166-18565-8420-3941-6060-7981-100125-144139-158203-222 91011121314151617 M. Avium-M. intracellularae (MAC) MAC1MAC2MAC3MAC4 241-260142-16192-111117-136 18192021 M. fortuitum FOR1FOR2FOR3FOR4FOR5FOR6FOR7FOR8FOR9FOR10 41-6045-6465-8479-9890-109110-129115-134136-155158-177248-267 22232425262728293031 M. chelonae CHE1CHE2CHE3CHE4CHE5CHE6CHE7CHE8 41-6045-6459-7879-98110-129133-152172-191247-266 3233343536373839

Sympathetic strain Probe name Location in ITS SEQ ID NO: M. gordonae GOR1GOR2GOR3GOR4GOR5 84-103249-268216-235201-220223-242 4041424344 M. terrae TER1TER2TER3TER4TER5 178-197237-25624-4370-8989-108 4546474849 M. scrofluceum SCO1SCO2SCO3 118-137131-150210-229 505152 M. kansasii KAN1KAN2 35-50238-257 5354 M. szulgai SZU1SZU2 124-143209-228 5556 M.marinum and M. ulcerans MAR-ULC1MAR-ULC2 85-104128-147 5758 M. gastri GAS1GAS2 85-104145-164 5960 M. xenopi XEN1 190-209 61 M. genavense GEN2 85-104 62 M. malmoense MAL1MAL2 203-22229-48 6364 M. shimoidei SHI1 89-108 65 M. habana HAB1 86-105 66 M. farcinogen FAR1FAR2 122-141111-130 6768 M. simiae SIM1SIM2SIM3 83-102129-148208-227 697071 M. sp SP1 225-244 72

Example 7 Results of Polymerase Reaction by Mycobacterial Diagnostic Primer

As a result of PCR reaction of the primer pairs having the nucleotide sequences of SEQ ID NOs: 3 and 6 and the nucleotide sequences of SEQ ID NOs: 4 and 5, which are specific to the mycobacteria, 350 bp and 200 were identified for the mycobacterial strains. Although amplified to bp, Staphylococcus aureus, Enterococcus faecium, and Serratia marcescens, other intestinal microorganisms, did not show amplified results, indicating that mycobacteria could be diagnosed with the above probes and primers. 4 is a photograph showing electrophoresis of the PCR reaction results according to Example 7.

Example 8. Results of Polymerase Reaction by Diagnostic Primers of TB Complex and MOTT Strains

The oligonucleotide probe according to the present invention was prepared as a primer to determine whether each strain can be specifically identified by PCR amplification. For identification of the TB complex, sequences of SEQ ID NO: 3 and 10 were selected, Among the MOTT strains, the nucleotide sequences of SEQ ID NOs: 21 and 18 were selected for the identification of MAC, the nucleotide sequences of SEQ ID NOs: 31 and 23 were selected for the identification of M. fortuitum, and the sequences of SEQ ID NOs. 34 and 37 for the identification of M. chelonae. Select base sequence, select nucleotide sequence of SEQ ID NO: 40 and 42 to identify M. gordonae, select base sequence of SEQ ID NO: 47 and 46 to identify M. terra, and sequence number to identify M. scrofulaceum Primers with the sense strand of the first nucleotide sequence and the primer with the antisense strand of the second nucleotide sequence by selecting the nucleotide sequences of 51 and 52 As a result of the polymerization and electrophoresis of the mycobacterial strains, the polymerase chain reaction of FIG. 7 was observed. As shown in FIG. 7, each of the mycobacterial strains could be diagnosed by PCR with each primer pair. .

As described above, in the present invention, the hybridization reaction and the enzymatic polymerase chain reaction are performed using the oligonucleotide probe and the primer of the present invention for the detection and identification of mycobacteria, thereby quickly obtaining the identification result, and whether the tuberculosis infection is present. It provides a means to judge easily and accurately.

Claims (2)

An oligonucleotide probe having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5-8, hybridizing to a specific base sequence of an internal transcription region of mycobacteria and used as a means for diagnosing mycobacteria. Select one pair of the nucleotide groups of SEQ ID NOs: 5-8 to obtain a primer having a sense strand of one of the base sequences, and another primer having an antisense strand of the remaining sequences Mycobacteria diagnostic method, characterized in that to perform the enzyme polymerization reaction (PCR) and to determine whether the amplification to a predetermined size mycobacteria.