KR100343623B1 - Oligonucleotide for detection and identification of mycobacteria - Google Patents

Abstract

The present invention relates to oligonucleotide primers or probes that can be used for the identification and differential diagnosis of mycobacterial strains, Mycobacterium fortuitum ( M. fortuitum ), mycobacteria Leeum four cellos (M. chelonae), Mycobacterium epseo Seuss (M. abscessus), Mycobacterium beche (M. vaccae), Mycobacterium Plata bay sense (M. flavescens), Mycobacterium Asia The interior of M. asiaticum , Mycobacterium forcinum , Mycobacterium acapulcensis , and Mycobacterium Diernhoferi As a primer for PCR or hybridization reaction, the nucleotide sequence of the internal transcribed spacer region (ITS) can be identified, and the nucleotide sequence can be identified and identified for each mycobacteria species. An oligonucleotide having the nucleotide sequence of SEQ ID NO: 10 to 241 provides an oligonucleotide.

Description

Oligonucleotide for identification of mycobacteria species {OLIGONUCLEOTIDE FOR DETECTION AND IDENTIFICATION OF MYCOBACTERIA}

The present invention relates to oligonucleotides that can be used for the identification of mycobacterial species, and more specifically, Mycobacterium fortuitum , which is a type of non-tuberculosis mycobacteria, Mycobacterium chelonae ), Mycobacterium abscessus , Mycobacterium vaccae , Mycobacterium flavescens , Mycobacterium asiaticum , Mycobacterium asiaticum the nucleotide sequence of the formate when num (Mycobacterium porcinum), Mycobacterium ah ride sensor system (Mycobacterium acapulcensis), and Mycobacterium diethoxy other inside the transfer area of the hopper Li (Mycobacterium diernhoferi) (ITS, internal Transcribed Spacer region) Using these ITS sequences, identification of the genus Mycobacteria and the identification of individual Mycobacterium species Possible oligonucleotides intended to provide an oligonucleotide primer or probe.

Tuberculosis is steadily decreasing compared to the past, but there are still around eight million new cases and three million deaths worldwide each year. Moreover, in developing countries, chronic carriers with drug-resistant bacteria are increasing due to inadequate treatment due to the lack of anti-tuberculosis drugs, and in developed countries, tuberculosis reduction is stagnating and increasing again in the 1980s due to the increase of AIDS patients. The aspect is showing. Thus, this trend is expected to generate 12 million new patients around 2000 (JP Natain, MC Raviglione, and A. Kochi, Tubercle and Lung Disease, 73 : 311-321, 1992; Murray CJL. And Lopez). AD.The global burden of disease.Global burden of disease and injury series.Vol. 1.Cambridge, Mass: Harvard University Press, 1996, p349-350; Global Tuberculosis Program: Anti-tuberculosis drug resistance, WHO Report 1997: World Health Organization, 1997).

In the 1950s, it was reported that non-tuberculous mycobacteria (NTM) could also cause disease in humans. Since 1980, the M. avium complex (MAC) has been reported in AIDS patients. Since being known to cause systemic diseases, interest in non-tuberculosis mycobacteria has increased. In non-tuberculosis mycobacterial diseases, clinical and general pathological findings are similar to those of tuberculosis. However, non-tuberculosis mycobacteria are widely distributed in the surrounding living environment and difficult to determine whether they are pathogenic even if they are separated from clinical specimens, and they are difficult to diagnose, and they are difficult to treat and have high recurrence rates. It is known. As non-tuberculosis mycobacterial strains that are resistant to various drugs are gradually increasing, a rapid and accurate identification method for these diseases is required because different methods must be used for diseases caused by Mycobacterium tuberculosis. . In addition, due to the increased incidence of tuberculosis and multidrug-resistant tuberculosis strains, more rapid and accurate identification of NTM strains as well as TB complexes is required for effective treatment and management of tuberculosis.

Since early diagnosis of mycobacterial infection and the identification of mycobacterial strains play an important role in the treatment, various diagnostic methods have been researched and developed. Among them, currently used identification and diagnosis methods of infection are as follows.

First is the microbiological method, smear and culture test. However, this method is not suitable for application to pathogenic microorganisms, such as mycobacteria, which require longer incubation periods and involve the risk of infection to the experimenter.

Secondly, there is a polymerase chain reaction (PCR) method, which is very useful for the detection of pathogens that take a long time to isolate and culture such as mycobacteria due to their high sensitivity and specificity. In particular, since a small amount of DNA is amplified and detected, there is an advantage that 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 is 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 (Bauer J, Andersen AB, Kremer K, and Miorner). H, Usefulness of spoligotyping to discriminate IS6110 low-copy-number Mycobacterium tuberculosis complex strains cultured in Denmark, 1999, J. Clin.Microbiol . 37 : 2602-2606; Troesch, A., H. Nguyen, CG Miyada, S. Desvarenne , TR Gingeras, PM Kaplan, P. Cros and C. Mabilat. 1999, Microbacterium species identification and rifampin resistance testing with high-density DNA probe arrays, J. Clin. Microbiol. 37 : 49-55).

The third method is the physicochemical analysis of mycobacterial cell components, and a method for detecting fatty compounds in the mycobacterial cell components using HPLC or GC and mass spectrometry. This method has a high specificity but has the disadvantage of requiring expensive equipment.

Fourth, as a method for detecting cell components by serological methods, agglutination reaction using blood cells or latex particles which adsorb the antibody to the fungal antigen, and enzyme-linked immunoassay method in which the enzyme is bound to the antibody are used. However, this technique is very sophisticated and requires careful attention and is only possible in limited laboratories, and it is difficult to distinguish the difference between current and previous infections.

Fifth, there is a method to search for mycobacteria by inserting luciferase gene into mycobacterial phage L5 and infecting with mycobacteria, and then 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, and BR Bloom. 1993, Science 260: 819-822).

Sixth, there is a method of identification by hybridization of oligonucleotide probes (A. Troesch, H. Nguyen, CG Miyada, S. Desvarenne, TR Gingeras, PM Kaplan, P. Cros and C. Mabilat. 1999. J. Clin.Microbiol . 37 : 49-55).

On the other hand, non-tuberculosis mycobacteria causing disease in the human body, in addition to the above-mentioned mycobacterium avium complex (MAC) mycobacteria, M. fortuitum ( M. fortuitum ), mycobacterium cellone complex ( M chelonae complex), Mycobacterium tere ( M. terrae ) and Mycobacterium becé ( M. vaccae ) are known. Among these, the Mycobacterium Cellone Complex is classified as Mycobacterium Cellone ( M. chelonae ) and Mycobacterium Epsomus ( M. abscessus ). Not known

1 is a schematic diagram of the location of the primers used for amplification of the Mycobacteria internal transcription region (ITS) and polymerase chain reaction (PCR) of the ITS,

Figure 2 is a photoelectrophoresis after PCR for a variety of mycobacteria strains using a primer pair for ITS amplification comprising a portion of 16S rRNA and 23S rRNA of mycobacteria,

Figure 3 is a photoelectrophoresis after PCR for the various mycobacterial strains using primer pairs (ITSF and MYC2) to determine whether the mycobacteria,

4 is a schematic diagram illustrating multiplex PCR (identification of mycobacteria) and simultaneously identifying TB complex or non-tuberculosis mycobacteria (NTM) among mycobacteria;

FIG. 5 shows electrophoresis after performing multiplex PCR with primer pairs for identifying mycobacteria (ITSF and MYC2) and primer pairs for identifying complexes of mycobacteria (MTB2 and MYC2);

Figure 6 shows the electrophoresis after PCR for each mycobacterial strain using a species-specific primer pair prepared from the nucleotide sequence of the polymorphic region of each non-tuberculosis mycobacteria (NTM) strain,

Figure 7 is a picture of electrophoresis after PCR for a variety of mycobacteria strains using species-specific primer pairs prepared from the nucleotide sequence of the polymorphic region of each non-tuberculosis mycobacteria (NTM) strain.

The present invention is to provide a simple, high accuracy and efficient method that can replace the conventional bacterial culture and biochemical method identification method in consideration of the problems of the conventional mycobacteria identification and infection diagnosis method as described above.

That is, an object of the present invention is to determine whether a mycobacteria strain and TB among mycobacteria strains by providing a PCR primer or a probe for PCR, by providing a nucleotide sequence common to mycobacteria and a nucleotide sequence specific to each strain. It is intended to provide a method for identifying complexes or NTMs, that is, for accurately identifying mycobacteria genus and species.

In order to achieve the above object, in the present invention, Mycobacterium fortuitum ( Mycobacterium fortuitum ), Mycobacterium chelonae ( Mycobacterium chelonae ), Mycobacterium abscessus ( Mycobacterium abscessus) ), Mycobacterium vaccae , Mycobacterium flavescens , Mycobacterium asiaticum , Mycobacterium porcinum , Mycobacterium vaccae Solarium Oh provides DNA of ride sensor system (Mycobacterium acapulcensis) and Mycobacterium diethoxy other hopper Li (Mycobacterium diernhoferi) inside the transfer region of the gene (ITS, internal Transcribed Spacer region) .

In the present invention, as a primer for PCR or a hybridization probe,

Oligonucleotides used for identification of mycobacteria having the nucleotide sequence of SEQ ID NOs: 10-14;

Oligonucleotides used to distinguish between TB complex and non-tuberculosis mycobacteria among mycobacteria, having the nucleotide sequence of SEQ ID NOs: 15-23;

Oligonucleotides used for identification of MAC ( Mycobacterium avium and Mycobacterium intracellulare ) having the nucleotide sequence of SEQ ID NOs: 24-27;

Oligonucleotides used for identification of Mycobacterium fortuitum having the nucleotide sequence of SEQ ID NOs: 28 to 38;

Oligonucleotides used for identification of Mycobacterium chelonae having the nucleotide sequence of SEQ ID NOs: 39-46;

Oligonucleotides used for identification of Mycobacterium abscessus having the nucleotide sequence of SEQ ID NOs: 47 to 52;

Oligonucleotides used for identification of Mycobacterium vaccae having the nucleotide sequence of SEQ ID NOs: 53 to 64 ;

Oligonucleotides used for identification of Mycobacterium flavescens having the nucleotide sequence of SEQ ID NOs: 65-72;

Oligonucleotides used for identification of Mycobacterium gordonae having the nucleotide sequence of SEQ ID NOs: 73 to 77 ;

Oligonucleotides used for identification of Mycobacterium terrae having the nucleotide sequence of SEQ ID NOs: 78-100;

Oligonucleotides used for identification of Mycobacterium scroflaceum having the nucleotide sequence of SEQ ID NOs: 101-108;

Oligonucleotides used for identification of Mycobacterium kansasii having the nucleotide sequence of SEQ ID NOs: 109-112 ;

Oligonucleotides used for identification of Mycobacterium szulgai having the nucleotide sequence of SEQ ID NOs: 113-116;

Oligonucleotides used for identification of Mycobacterium marinum and Mycobacterium ulcerans , having the nucleotide sequence of SEQ ID NOs: 117-119;

Oligonucleotides used for identification of Mycobacterium gastri having a nucleotide sequence of SEQ ID NOs: 120 to 123 ;

Oligonucleotides used for identification of Mycobacterium xenopi having the nucleotide sequence of SEQ ID NOs: 124-133;

Oligonucleotides used for identification of Mycobacterium genavense having the nucleotide sequence of SEQ ID NOs: 134-141;

Oligonucleotides used for identification of Mycobacterium malmoense having the nucleotide sequence of SEQ ID NOs: 142 to 146 ;

Oligonucleotides used for identification of Mycobacterium simiae having the nucleotide sequence of SEQ ID NOs: 147-153;

Oligonucleotides used for identification of Mycobacterium smegmatis having the nucleotide sequence of SEQ ID NOs: 154-165;

Oligonucleotides used for identification of Mycobacterium shimoidei having the nucleotide sequence of SEQ ID NOs: 166-172;

Oligonucleotides used for identification of Mycobacterium habana having the nucleotide sequence of SEQ ID NOs: 173 to 180;

Oligonucleotides used for identification of Mycobacterium farcinogen having the nucleotide sequence of SEQ ID NOs: 181 to 189 ;

Oligonucleotides used for identification of Mycobacterium asiaticum having the nucleotide sequence of SEQ ID NOs: 190 to 193;

Oligonucleotides used for identification of Mycobacterium porcinum having the nucleotide sequence of SEQ ID NOs: 194-205;

Oligonucleotides used for identification of Mycobacterium acapulcensis having the nucleotide sequence of SEQ ID NOs: 206 to 215 ;

Oligonucleotides used for identification of Mycobacterium diernhoferi having the nucleotide sequence of SEQ ID NOs: 216 to 227 ;

Oligonucleotides used for identification of Mycobacterium paratuberculosis , having the nucleotide sequence of SEQ ID NOs: 228-240; And

Provided is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 241 for use in identifying Mycobacteria sp .

In the conventional PCR method, only one or two strains could be identified. However, in the present invention, almost all human infectious mycobacterial strains can be identified simultaneously from the ITS sequence of mycobacteria. Primers and probes were designed. ITS is a region that has more polymorphic regions and conservative regions than 16S rRNA, which is often used as target DNA, and thus has high utility as a target DNA for genotyping (Gurtler, V., and VA Stanisich, 1996, New approaches). to typing and identification of bacteria using the 16S-23S rDNA spacer region.Microbiol. 142: 3-16).

Accordingly, in the present invention, Mycobacterium fortuitum ( Mycobacterium fortuitum ), Mycobacterium chelonae ( Mycobacterium chelonae ), Mycobacterium abscessus ), Mycobacterium vaccae , Mycobacterium flavescens , Mycobacterium asiaticum , Mycobacterium porcinum , Mycobacterium vaccae Solarium ah ride sensor system (Mycobacterium acapulcensis) and Mycobacterium diethoxy other hopper Li (Mycobacterium diernhoferi) reveal the ITS sequences of a gene, a probe and primers were designed to distinguish the species as possible by using them. In other strains, multi-alignment and blast analysis of nucleotide sequence information that has been published in the public (GenBank) is selected to select a site that can be discriminated theoretically by polymorphism of the same site. Probes or primers were designed and identified by strain specific hybridization. As a confirmation experiment, amplification by genus-specific and species-specific primers of the PCR reaction showed that identification was possible.

That is, the oligonucleotide probe of the present invention which hybridizes only to specific nucleic acid sequences present in the ITS of mycobacteria can be attached to a solid support to detect or identify mycobacteria species. In addition, it is possible to identify by amplification to a certain size after the PCR by primers prepared by the same sequence as these oligonucleotide probes that specifically detect each species. Using the probes or primers of the present invention prepared from these mycobacterial ITS sequences, it is possible to first distinguish whether they are mycobacteria, then to discriminate whether they are TB complexes or NTMs among mycobacteria, and to accurately distinguish species among NTMs Can be.

The present invention is explained in more detail by the following examples. However, these Examples are only illustrative to aid the understanding of the present invention, but the scope of the present invention is not limited to these.

Example 1 Culture of Mycobacteria and Isolation of Genomic DNA

Mycobacteria standard strains were obtained from Korean Collection for Type Culture (KCTC) and American Type Culture Collection (ATCC), and clinical strains were obtained from the Korean Tuberculosis Association, Tuberculosis Hospital, and Pusan National University Hospital. It was performed under the supervision of the professor of clinical pathology at the university hospital. The types of mycobacteria and general pathogenic bacteria used in the present invention are shown in Table 8 below.

Extraction of DNA from mycobacteria was carried out in the following manner: Mycobacteria were cultured in Ogawa medium, followed by 1 platinum centrifuge and placed into an InstaGene matrix (Bio-Rad Co.). After 200 μl of the suspension was suspended, the solution was held at 56 ° C. for 30 minutes, vortexed for 10 seconds, and then heat-treated at 100 ° C. for 8 minutes. After stirring for another 10 seconds (vortex) and centrifuged for 3 minutes at 12,000 rpm the supernatant was transferred to a new tube and stored at -20 ℃. 2 μl of each isolated DNA was used for the following PCR reaction.

Example 2 Preparation of Primer for ITS Amplification of Mycobacteria

The positions of the primers used for ITS of mycobacteria and PCR amplification of ITS are shown in FIG. 1. Primers for the amplification of mycobacteria were prepared by selecting some of the conserved regions of 16S rRNA and 23S rRNA of mycobacteria. In other words, the primers were designed by computer analysis of previously disclosed mycobacterial 16S and 23S rRNAs by multialignment and blast search, and the selected primers were combined with a portion of 16S rRNA and 23S rRNA. The ITS site is designed to be selectively amplified to about 500 bp in size and has the nucleotide sequences of SEQ ID NOs: 242 (ITSF) and 243 (ITSR). All primers used in the experiments, including the ITS amplification primers, were prepared at a concentration of 50 nmol in a Perkin-Elmer DNA synthesizer by BioBasic (Canada).

Example 3. PCR Reaction and Identification of Products

2 μl of DNA of each strain obtained in Example 1 was used for the PCR reaction. The composition of the reaction was as follows: 500 mM KCl, 100 mM Tris HCl (pH 9.0), 1% Triton X-100, 0.2 mM dNTP (dATP, dGTP, dTTP and dCTP), 1.5 mM MgCl ₂ , 1 pmol primer, 1U Taq DNA Polymerase from Bio Basic Inc. The mixture was reacted for 5 minutes at 94 ° C. to sufficiently denature, 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.

Figure 2 is a photoelectrophoresis after PCR for a variety of mycobacteria strains using a primer pair for ITS amplification comprising a portion of 16S rRNA and 23S rRNA of mycobacteria. Here, M is a molecular weight label with a 100 bp interval, C is a control, lane 1 is mycobacterium fortuitum , lane 2 mycobacterium cellone ( M. chelonae) ), Lane 3 is mycobacterium intracellularae , lane 4 is mycobacterium avium , lane 5 is mycobacterium tuberculosis ( M. Tb ), Lane 6 is M. agri , lane 7 is M. kansasii , lane 8 is M. gordonae , and lane 9 is M. agri Cobacterium tuberculosis ( M. Tb H37Rv), which represents the ITS amplification occurred in the mycobacteria of lanes 1-9 except for the control group C.

Example 4 DNA Sequence Analysis of Mycobacteria ITS

After confirmation of the PCR reaction, mycobacterium fortuitum ( M. fortuitum ), mycobacterium cellone ( M. chelonae ), mycobacterium epsus ( M. abscessus ), mycobacterium becé ( M. vaccae ), mycobacterium flavescens ( M. flavescens ), mycobacterium asiaticum , mycobacterium forcinum ( M. porcinum ), The ITS reactions of Mycobacterium acapulcensis and Mycobacterium Dietenhoferi were amplified by PCR and used for DNA sequencing.

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

SEQ ID NOs: 1 to 9 are Mycobacterium fortuitum , Mycobacterium chelonae , Mycobacterium abscessus and Mycobacterium vaccae , respectively . , Mycobacterium flavescens , Mycobacterium asiaticum , Mycobacterium porcinum , Mycobacterium acapulcensis and Mycobacterium acapulcensis It shows the ITS sequence of Cobacterium diernhoferi ( Mycobacterium diernhoferi ).

Example 5. Selection and Synthesis of Oligonucleotide Primers

M. embodiment obtained in Example 4 Te Solarium Four tyuyi lithium (M. fortuitum), Mycobacterium cello four (M. chelonae), Mycobacterium epseo Versus (M. abscessus), Mycobacterium beche (M. vaccae ), Mycobacterium flavescens ( M. flavescens ), Mycobacterium Asiaticum ( M. asiaticum ), Mycobacterium forcinum ( M. porcinum ), Mycobacterium acapulsensis ( M. acapulcensis) and Mycobacterium diethoxy other hopper Li (after design primers of 20 bp in size, respectively from the ITS sequences of M. diernhoferi), the ITS sequences of a multi-alignment of the other mycobacteria obtained from GenBank (multi -alignment) and blast (blast) analysis.

Based on the above results, a conservative base sequence of mycobacteria ITS was selected as a primer or probe for identifying mycobacteria, and a strain specific primer or probe corresponds to a base sequence having a high polymorphism. The positions and the sequence numbers of the designed primers or probes in the ITS are shown in Tables 1-7.

Identification strain Probe Name Location in ITS Sequence number Mycobacteria MYC1MYC2MYC3MYC4MYC5 Depends on species 1011121314 TB complex MTB1MTB2MTB3MTB4MTB5MTB6MTB7MTB8MTB9 166-18565-8420-3941-6060-7981-100125-144139-158203-222 151617181920212223 M. avium-M. intracellularae (MAC) MAC1MAC2MAC3MAC4 241-260142-16192-111117-136 24252627 M. fortuitum FOR1FOR2FOR3FOR4FOR5FOR6FOR7FOR8FOR9FOR10FOR11 40-5944-6364-8378-9789-108109-128114-133134-153157-176246-265289-308 2829303132333435363738 M. chelonae CHE1CHE2CHE3CHE4CHE5CHE6CHE7CHE8 11-3029-4858-7778-97109-128132-151171-190246-265 3940414243444546

Identification strain Probe Name Location in ITS Sequence number M. abscessus ABC1ABC2ABC3ABC4ABC5ABC6 37-5655-74247-266263-282270-289261-280 474849505152 M. vaccae VAC1VAC2VAC3VAC4VAC5VAC6VAC7VAC8VAC9VAC10VAC11VAC12 18-3738-5758-77118-137138-157158-177178-197199-218219-238265-284298-317321-340 535455565758596061626364 M. flavescens FLA1FLA2FLA3FLA4FLA5FLA6FLA7FLA8 12-3132-5152-7172-91105-124125-144173-192278-297 6566676869707172 M. gordonae GOR1GOR2GOR3GOR4GOR5 84-103249-268216-235201-220223-242 7374757677

Identification strain Probe Name Location in ITS Sequence number M. terrae TER1TER2TER3TER4TER5TER6TER7TER8TER9TER10TER11TER12TER13TER14TER15TER16TER17TER18TER19TER20TER21TER22TER23 178-197237-25624-4370-8989-108102-121122-141142-161162-181182-201202-221222-241238-257307-326322-341342-361362-381382-40112-2131-5052-7172-9182-101 78798081828384858687888990919293949596979899100 M. scroflaceum SCO1SCO2SCO3SCO4SCO5SCO6SCO7SCO8 118-137131-150210-22984-103152-171200-219221-240241-260 101102103104105106107108 M. kansasii KAN1KAN2KAN3KAN4 35-54238-25783-102214-233 109110111112

Identification strain Probe Name Location in ITS Sequence number M. szulgai SZU1SZU2SZU3SZU4 124-143209-228227-246247-166 113114115116 M.marinum and M. ulcerans MAR-ULC1MAR-ULC2MAR-ULC3 85-104128-147224-243 117118119 M. gastri GAS1GAS2GAS3GAS4 85-104145-164133-152239-258 120121122123 M. xenopi XEN1XEN2XEN3XEN4XEN5XEN6XEN7XEN8XEN9XEN10 190-2091-2021-4041-6061-8081-100121-140141-160201-220221-240 124125126127128129130131132133 M. genavense GEN1GEN2GEN3GEN4GEN5GEN6GEN7GEN8 190-20985-104131-150147-166186-205206-225226-245240-265 134135136137138139140141 M. malmoense MAL1MAL2MAL3MAL4MAL5 203-22229-48136-155222-241242-261 142143144145146 M. simiae SIM1SIM2SIM3SIM4SIM5SIM6SIM7 83-102129-148208-22722-4180-99136-155241-260 147148149150151152153

Identification strain Probe Name Location in ITS Sequence number M. smegmatis SMEG1SMEG2SMEG3SMEG4SMEG5SMEG6SMEG7SMEG8SMEG9SMEG10SMEG11SMEG12 17-3637-5657-7677-9697-116117-136137-156157-176177-196193-21261-80112-131 154155156157158159160161162163164165 M. shimoidei SHI1SHI2SHI3SHI4SHI5SHI6SHI7 89-10820-3970-8997-116135-154224-243244-263 166167168169170171172 M. habana HAB1HAB2HAB3HAB4HAB5HAB6HAB7HAB8 86-10517-3651-7081-100134-153175-194200-219242-261 173174175176177178179180 M. farcinogen FAR1FAR2FAR3FAR4FAR5FAR6FAR7FAR8FAR9 122-141111-13022-4148-6776-95108-127114-133275-294295-314 181182183184185186187188189

Identification strain Probe Name Location in ITS Sequence number M. asiaticum ASI1ASI2ASI3ASI4 82-101145-164189-208274-293 190191192193 M. porcinum POR1POR2POR3POR4POR5POR6POR7POR8POR9POR10POR11POR12 45-6413-3267-8691-110115-134137-156164-183194-213221-240273-292298-317325-344 194195196197198199200201202203204205 M. acapulcensis ACA1ACA2ACA3ACA4ACA5ACA6ACA7ACA8ACA9ACA10 66-85112-131132-151178-197198-217219-238242-261262-281318-337350-369 206207208209210211212213214215 M. diernhoferi DIE1DIE2DIE3DIE4DIE5DIE6DIE7DIE8DIE9DIE10DIE11DIE12 16-3536-5562-81103-122154-173175-194195-214232-251261-280282-301304-323344-363 216217218219220221222223224225226227

Identification strain Probe Name Location in ITS Sequence number M. para- tuberculosis PARA1PARA2PARA3PARA4PARA5PARA6PARA7PARA8PARA9PARA10PARA11PARA12PARA13 7-2630-4940-5950-6971-9083-102103-122135-154157-176178-197198-217219-238241-260 228229230231232233234235236237238239240 M. sp SP1 225-244 241

Example 6 RCR Results by Primer for Identification of Mycobacteria

The conservative sequences present in mycobacteria were selected and constructed as genus-specific primers that amplify microbacteria only. That is, a conservative base sequence was selected from the ITS sequences of about 270-350 bp in size and prepared as a primer. Among them, PCR reaction was performed using primer pairs of ITSF (SEQ ID NO: 242) and MYC2 (SEQ ID NO: 11). As a result, it was amplified to about 350 bp for the mycobacterial strain, but other pathogenic bacteria Staphylococcus aureus , Enterococcus faecium , and Serratia marquessen marcescens ) and the like. Therefore, it can be seen that these primers can be used for identifying mycobacteria.

Figure 3 is a photograph of electrophoresis after PCR for a variety of mycobacteria strains using primer pairs (ITSF and MYC2) to determine whether the mycobacteria. Here, M has a molecular weight label 100 bp intervals, C is a control, lane 1 is Mycobacterium Epsus ( M. abscessus ATCC 19977), lane 2 is Mycobacterium agri ( M. agri ATCC) 27406), lane 3 is mycobacterium asiaticum ( M. asiaticum ATCC 25276), lane 4 is mycobacterium austrian africanum ( M. austroafricanum ATCC 33464), lane 5 is mycobacterium avium ( M avium ATCC 25291), lane 6 is Mycobacterium bovis ( M. bovis ATCC 19210), lane 7 is Mycobacterium cellone ( M. chelonae ATCC 35752), lane 8 is Mycobacterium flavesense ( M. flavescens ATCC 14474), lane 9 is Mycobacterium fortuitium ( M. fortuitum ATCC 6841), lane 10 is Mycobacterium Godne ( M. gordonae ATCC 14470 ), lane 11 is Mycobacterium intra Cellulare ( M. intracellularae ATCC 13950), lane 12 is mycobacterium Kansashi ( M. kansasii ATCC 12478), lane 13 is mycobacterium play ( M. phlei ATCC 354), lane 14 is mycobacterium scrablasium ( M. scroflaceum ATCC 19981), lane 15 is mycobacterium smegmatis ( M. smegmatis ATCC 21701) Lane 16 is M. szulgai ATCC 35799, lane 17 is M. terrae ATCC 15755, lane 18 is Mycobacterium triviale ( M. triviale ATCC) 23292), lane 19 represents mycobacterium tuberculosis H37Rv, and lane 20 represents mycobacterium becche ( M. vaccae ATCC 15483), except for the control group lanes 1 to ITS amplification can be found in all 20 mycobacteria.

Table 8 shows the amplification after PCR with primer pairs (ITSF and MYC2) to confirm whether the mycobacteria, it can be seen that the amplification did not occur in strains other than mycobacteria.

Strain name result Strain name result M. tuberculosis H37Rv + M. phlei ATCC 354 + M. bovis ATCC 19210 + Aeromonas hydrophila - M. avium ATCC 25291 + Burkholderia cepacia - M. intracellulare ATCC 13950 + Candida albicans - M. abscessus ATCC 19977 + Citrobacter freundii - M. chelonae ATCC 35752 + Enterobacter aerogenes - M. flavescens ATCC 14474 + Enterobacter cloacae - M. fortuitum ATCC 6841 + Enterococcus faecalis - M. gastri ATCC 15754 + Enterococcus faecium - M. genavense ATCC 51233 + Enterococcus raffinosis - M. gordonae ATCC 14470 + Escherichia coli - M. kansasii ATCC 12478 + Klebsiella pneumoniae - M. malmoense ATCC 29571 + Plesiomonas shigelloides - M. scroflaceum ATCC 19981 + Proteus mirabilis - M. simiae ATCC 25275 + Proteus vulgaris - M. smegmatis ATCC 21701 + Providencia rettgeri - M. szulgai ATCC 35799 + Pseudomonas aeruginosa - M. terrae ATCC 15755 + Rahnella aquatilis - M. vaccae ATCC 15483 + Salmonella spp. - M. xenopi ATCC 19250 + Serratia marcescens - M. marinum ATCC 927 + Shewanella putrefaciens - M. ulcerance ATCC 19423 + Shigella flexneri - M. porcinum ATCC 33776 + Shigella sonnei - M. asiaticum ATCC 25276 + Staphylococcus epidermidis - M. acapulcensis ATCC 14473 + Staphylococcus aureus - M. diernhoferi ATCC 19340 + Streptococcus agalactiae - M. agri ATCC 27406 + Streptococcus intermidius - M. austroafricanum ATCC 33464 + Streptococcus pneumoniae - M. triviale ATCC 23292 + Vibrio parahemolyticus -

Example 7. PCR Results by TB Complex Identification Primer

An oligonucleotide according to the present invention was prepared as a primer for PCR, and the experiments for the identification of each strain specificity were confirmed by amplification in PCR. First, in order to identify TB complex, multiplex PCR (primer pair) (ITSF and MYC2) to identify mycobacteria and primer pair (MTB2 and MYC2; SEQ ID NO: 16 and 11) to identify TB complex ) Was performed. FIG. 4 is a schematic diagram illustrating multiplex PCR confirming whether or not the mycobacteria, and at the same time identify whether the TB complex or non-tuberculosis mycobacteria (NTM) among the mycobacteria.

Figure 5 is an electrophoresis after performing a multiplex PCR with a primer pair (ITSF and MYC2) to identify the mycobacteria and primer pairs for identifying the TB complex (MTB2 and MYC2) of mycobacteria. Here, M has a 100 bp interval with a molecular weight label, C is a control group, lanes 1 and 2 are TB complexes Mycobacterium tuberculosis ( M. Tb H37Rv) and Mycobacterium bovis ( M. bovis ), lanes 3 to 10 represent Mycobacterium avium , Mycobacterium intracellularae , Mycobacterium fortuitum and Mycobacterium M. Te Leeum four cellos (M. chelonae), Mycobacterium altitude four (M. gordonae), Mycobacterium seujul Guy (M. szulgai), Mycobacterium terephthalate (M. terrae) and Mycobacterium Scroflashium ( M. scroflaceum ATCC 19981), which shows that in the lanes 1 and 2 of the TB complexes, two bands were formed by the primers for identifying the mycobacteria and the primers for the TB complex. Only one band is formed in the remaining NTM strains. We can see that a single amplification occurs.

Example 8. PCR results by primers for NTM species identification

In order to identify each species of non-tuberculosis mycobacteria (NTM), PCR was carried out with a species-specific primer prepared from the nucleotide sequence of the polymorphic site of each strain.

That is, SEQ ID NOs: 16 and 21 specific to Mycobacterium tuberculosis H37Rv and Mycobacterium bovis , and Mycobacterium avium and Mycobac. SEQ ID NOs: 24 and 27 specific for M. intracellularae , SEQ ID NOs: 29 and 37 specific for M. fortuitum , and M. chelonae Specific sequences 41 and 44, sequences 48 and 49 specific for M. abscessus , sequences 55 and 63 specific to M. vaccae , mycobacterium flave SEQ ID NOs: 66 and 72 specific for M. flavescens ; SEQ ID NOs: 73 and 75 specific for M. gordonae ; SEQ ID NOs: 88 and 96 specific for M. terrae , Mycobacterium disk by flash Titanium-specific sequence 102 and 103 for (M. scroflaceum), Mycobacterium kansa (M. kansasii) specific sequences 109 and 110, Mycobacterium seujul this specific sequence and 117 to 119 (M. szulgai) specific sequences 113 and 114, Mycobacterium grains num (M. marinum) in the , Mycobacterium ulse lance (M. ulcerans) specific sequences 117 and 119, Mycobacterium gas tree (M. gastri) specific sequences 120 and 123, Mycobacterium gen blood (M. xenopi) in the SEQ ID NOs: 128 and 132, which are specific for M. genavense , SEQ ID NOs: 135 and 141, which are specific for M. malmoense , SEQ ID NOs: 143 and 145, which are specific for Mycobacterium malmoense . By selecting the nucleotide sequences of SEQ ID NO: 147 and 149 specific to M. simiae and the sequences 154 and 159 specific to Mycobacterium smegmatis , respectively, the sense strand of the first nucleotide sequence ( primer with sense strand and antisense strand of second sequence PCR was performed with a pair of primers having an isense strand), and the results were confirmed by electrophoresis after PCR reaction with the mycobacterial strains.

Figure 6 is a photograph of the electrophoresis after PCR for each mycobacteria strain using a species-specific primer pair prepared from the nucleotide sequence of the polymorphic region of each non-tuberculosis mycobacteria (NTM) strain. Here, M has a 100 bp interval with a molecular weight label, C is a control, lanes 1 and 2 are mycobacterium tuberculosis (TB complex)M. tuberculosisH37Rv) and Mycobacterium Vorbis (M. bovisATCC 19210), lanes 3 and 4 Mycobacterium Abium (M. aviumATCC 25291) and Mycobacterium IntracellularM. intracellularaeATCC 13950), Lane 5 shows Mycobacterium Potitiumium (M. fortuitumATCC 6841), Lane 6 is the Mycobacterium Cellone (M. chelonaeATCC 35752), Lane 7 is Mycobacterium Epsus (M. abscessusATCC 19977), Lane 8 shows Mycobacterium Becce (M. vaccaeATCC 15483), lane 9 is Mycobacterium flavesense (M. flavescensATCC 14474), Lane 10 is Mycobacterium Godone (M. gordonaeATCC 14470), Lane 11 is Mycobacterium Tere (M. terraeATCC 15755), Lane 12 is Mycobacterium Scroflashum (M. scroflaceumATCC 19981), lane 13 shows the mycobacterium Kansashi (M. kansasiiATCC 12478), Lane 14 is Mycobacterium Schluggai (M. szulgaiATCC 35799), lane 15 shows Mycobacterium marinum (M. marinumATCC 927, lane 16 is Mycobacterium Ulcerans (M. ulceransATCC 19423), Lane 17 is Mycobacterium Gastri (M. gastriATCC 15754), Lane 18 is Mycobacterium Genophy (M. xenopiATCC 19250, lane 19 is Mycobacterium Jennavens (M. genavenseATCC 1233), Lane 20 is Mycobacterium Malmoens (M. malmoenseATCC 29571), Lane 21 is Mycobacterium Shimie (M. simiaeATCC 25275), and lane 22 is Mycobacterium smegmatis (M. smegmatisATCC 21701), the species-specific amplification of each mycobacterial strain in lanes 1 to 22 can be seen.

In addition, Figure 7 is a picture of the electrophoresis after PCR for a variety of mycobacteria strains using species-specific primer pairs prepared from the nucleotide sequence of the polymorphic region of each non-tuberculosis mycobacteria (NTM) strain. Here, in the primer pairs of the first stage specific to the TB complex (ITSF and MYC2), Mycobacterium tuberculosis of lanes 1 and 2 (M. tuberculosisH37Rv) and Mycobacterium Vorbis (M. bovis) Is amplified, Mycobacterium Abium (M. avium) And Mycobacterium IntracellularM. intracellularaeIn the second pair of primer pairs (MAC1 and MAC4) specific to the mycobacterium avium (lanes 3 and 4)M. avium) And Mycobacterium IntracellularM. intracellularae) Is amplified and mycobacterium fortuitium (M. fortuitumIn the third primer pair (FOR2 and FOR10) specific to), the mycobacterium fortuitium of lane 5 (M. fortuitum) Is amplified, and Mycobacterium Cellone (M. chelonae), The fourth pair of primers (CHE3 and CHE6) are specific to the mycobacterium cellone (lane 6).M. chelonae) Is amplified, and Mycobacterium Godone (M. gordonaeIn the fifth primer pair (GOR1 and GOR3) specific toM. gordonae) Is amplified, and mycobacterium schullai (M. szulgaiIn the 6-stage primer pairs (SZU1 and SZU2) specific to Mycobacterium strugai (lane 8)M. szulgai) Is amplified, and Mycobacterium scroflashium (M. scroflaceumIn the seventh-stage primer pairs (SCO2 and SCO3), the Mycobacterium scroplasmium (lane 10)M. scroflaceum) Only amplified and no amplification in other species. Lane 9 is Mycobacterium Tere (M. terrae), No amplification occurred in any of these species specific primer pairs.

Therefore, it can be seen that each species of mycobacteria can be specifically identified by PCR reaction with each species-specific primer pair.

As described above, Mycobacterium fortuitum , Mycobacterium chelonae , Mycobacterium abscessus , Mycobacterium, which are a type of non-tuberculosis mycobacteria Mycobacterium vaccae , Mycobacterium flavescens , Mycobacterium asiaticum , Mycobacterium porcinum , Mycobacterium acapulsensis ( Mycobacterium acapulcensis ), and the nucleotide sequence of the Internal Transcribed Spacer region (ITS) of the Mycobacterium diernhoferi , and using this to identify the genus Mycobacterium and each Oligonucleotide probes or primers can be designed that allow for identification of the Leeum species. The PCR primers or hybridization reaction probes thus obtained can quickly and effectively obtain the identification results of these species, and can distinguish between TB complex and NTM species, so that the correct treatment method can be selected according to the result and simultaneous infection. It can also diagnose effectively.

Claims (2)

DNA of the Internal Transcribed Spacer region (ITS) of the Mycobacterium vaccae gene described in SEQ ID NO: 4. Oligonucleotide used for the identification of Mycobacterium vaccae which has a base sequence of the sequence of 53-64 .