KR101523877B1 - Methods for identification and detection of mycobacterium using real time polymerase chain reaction and melting curve analysis - Google Patents

Abstract

The present invention relates to a kit for identification of mycobacteria including a primer set for identifying mycobacteria, a primer set for identifying mycobacteria, and a real-time PCR method using the same and a method for identification of mycobacteria using a melting curve analysis. According to the above method, a variety of species of mycobacteria that can be identified can be provided, and a clinical diagnostic tool capable of identifying mycobacteria efficiently, easily, accurately, quickly, and inexpensively can be provided.

Description

Identification method of mycobacteria using real-time polymerase chain reaction method and melting curve analysis {METHODS FOR IDENTIFICATION AND DETECTION OF MYCOBACTERIUM USING REAL TIME POLYMERASE CHAIN REACTION AND MELTING CURVE ANALYSIS}

The present invention relates to a method for identifying mycobacteria using a real-time polymerase chain reaction method and melting curve analysis. More specifically, it relates to a mycobacteria identification kit including a primer set for mycobacteria identification, a mycobacteria identification primer set, and a mycobacteria identification method using real-time polymerase chain reaction method and melting curve analysis using the same.

Nontuberculous mycobacteria refers to all mycobacteria except Mycobacterium tuberculosis and Mycobacterium leprae. The mycobacterium tuberculosis was known not long after Koch discovered tuberculosis, but only recently began to be recognized as an important infectious bacteria.

Recently, the prevalence of tuberculosis in Korea has been decreasing due to the efforts of the health authorities, but the number of infections caused by the acidic non-tuberculosis bacteria is on the rise. According to reports, acidic non-Tuberculosis bacteria were isolated from 12% of tuberculosis smears and culture-positive samples. In addition, as the liquid tuberculosis culture method was covered by insurance in 2009, the number of laboratories that conduct liquid tuberculosis culture tests is increasing, and when tuberculosis is cultured using liquid medium, the detection of acidic non-Tuberculosis bacteria is increased compared to culture using conventional solid culture media.

In many countries in the United States and Europe, where the prevalence of Mycobacterium tuberculosis is low, the number of infections caused by Mycobacterium tuberculosis is increasing. According to a report from the United States, the detection of mycobacteria increased from one third of mycobacteria isolated in 1979 to 1980 to three quarters of mycobacteria isolated in 1992. In addition, in the United States, Canada, and Western Europe, it is known that 40-50% of the mycobacterium tuberculosis isolated from sputum causes lung disease.

Diagnosis of infectious diseases caused by mycobacteria traditionally uses a test method that isolates bacteria through culture and identifies them by biochemical methods. However, this method takes time to cultivate due to the slow growth due to the characteristics of mycobacteria, and it is not possible to accurately identify various mycobacterial species only by biochemical methods. Recently, methods for the identification of mycobacteria using molecular biological techniques are being introduced into laboratories. Commercial test kits such as Accuprobe, INNO-LiPA mycobacteria, GenoType Mycobacterium CM, and AS. These test kits have a relatively short test time and are sensitive, but have a limited number of mycobacterial species that can be identified, or there is a possibility of contamination in the process of hybridizing the amplified product after PCR reaction. Therefore, there is a need for a method for identifying mycobacteria that is simple, accurate, fast, inexpensive, and has many types of mycobacteria that can be identified.

An object of the present invention is to provide a primer set for mycobacteria identification that can accurately detect and identify mycobacteria as a specific species.

Another object of the present invention is to provide a kit for identification of mycobacteria comprising a set of primers for identification of mycobacteria capable of accurately detecting and identifying mycobacteria as a specific species.

Another object of the present invention is a double real-time polymerase chain reaction method using a kit for identification of mycobacteria including a set of primers for identification of mycobacteria that can accurately detect and identify mycobacteria as a specific species. ) And melting curve analysis.

In order to achieve the above object, a set of primers specific to the ITS (Internal transcribed spacer) gene of M. gastri including the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2; A primer set specific to the ITS gene of M. gastri comprising the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; A primer set specific to the ITS gene of M.xenopi comprising the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6; A primer set specific to the ITS gene of M. fortuitum comprising a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8; A primer set specific to the ITS gene of M. fortuitum comprising a set of forward primers of SEQ ID NO: 7 and reverse primers of SEQ ID NO: 9; A primer set specific to the IS6110 gene of M. tuberculosis complex, including the forward primer of SEQ ID NO: 10 and the reverse primer of SEQ ID NO: 11; A primer set specific to the 16S rRNA gene of Nontuberculous mycobacteria comprising the forward primer of SEQ ID NO: 12 and the reverse primer of SEQ ID NO: 13; A primer set specific to the ITS gene of M.abscessus comprising the forward primer of SEQ ID NO: 14 and the reverse primer of SEQ ID NO: 15; A primer set specific to the ITS gene of M. abscessus comprising the forward primer of SEQ ID NO: 16 and the reverse primer of SEQ ID NO: 17; A primer set specific to the 16S rRNA gene of M. intracellulare comprising a forward primer of SEQ ID NO: 18 and a reverse primer of SEQ ID NO: 19; A primer set specific to the ITS gene of M.szulgai comprising the forward primer of SEQ ID NO: 20 and the reverse primer of SEQ ID NO: 21; A primer set specific to the ITS gene of M. terrae comprising the forward primer of SEQ ID NO: 22 and the reverse primer of SEQ ID NO: 23; A primer set specific to the 16S rRNA gene of M. avium comprising the forward primer of SEQ ID NO: 24 and the reverse primer of SEQ ID NO: 25; A primer set specific to the ITS gene of M. chelonae comprising the forward primer of SEQ ID NO: 26 and the reverse primer of SEQ ID NO: 27; A primer set specific to the 16S rRNA gene of M. kansasii or M. gastri comprising a set of forward primers of SEQ ID NO: 28 and reverse primers of SEQ ID NO: 29; A primer set specific to the 16S rRNA gene of M. kansasii or M. gastri comprising a forward primer of SEQ ID NO: 30 and a reverse primer of SEQ ID NO: 31; A primer set specific to the ITS gene of M. gordonae comprising the forward primer of SEQ ID NO: 32 and the reverse primer of SEQ ID NO: 33; A primer set specific to the 16S rRNA gene of M. gordonae comprising the forward primer of SEQ ID NO: 34 and the reverse primer of SEQ ID NO: 35; A primer set specific to the ITS gene of M. marinum or M. ulcerans comprising a forward primer of SEQ ID NO: 36 and a reverse primer of SEQ ID NO: 37; A primer set specific to the ITS gene of M. simiae comprising a forward primer of SEQ ID NO: 38 and a reverse primer of SEQ ID NO: 39; A primer set specific to HSP65 (Heat shock protein 65) of M. ulcerans comprising a forward primer of SEQ ID NO: 40 and a reverse primer of SEQ ID NO: 41; A primer set specific to the ITS gene of M. septicum comprising the forward primer of SEQ ID NO: 42 and the reverse primer of SEQ ID NO: 43; A primer set specific to the ITS gene of M. smegmatis comprising a forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45; A primer set specific to the ITS gene of M. smegmatis comprising a forward primer of SEQ ID NO: 46 and a reverse primer of SEQ ID NO: 47; A primer set specific to the 16S rRNA gene of M.scrofulaceum comprising the forward primer of SEQ ID NO: 48 and the reverse primer of SEQ ID NO: 49; A primer set specific to the ITS gene of M. scrofulaceum comprising the forward primer of SEQ ID NO: 50 and the reverse primer of SEQ ID NO: 51; A primer set specific to the ITS gene of M. haemophilum comprising the forward primer of SEQ ID NO: 52 and the reverse primer of SEQ ID NO: 53; A primer set specific to the ITS gene of M. marinum or M. ulcerans comprising the forward primer of SEQ ID NO: 54 and the reverse primer of SEQ ID NO: 55; A primer set specific to the ITS gene of M. celatum comprising the forward primer of SEQ ID NO: 56 and the reverse primer of SEQ ID NO: 57; A primer set specific to the ITS gene of M. abscessus comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; A primer set specific to the 16S rRNA gene of M. abscessus or M. chelonae comprising a forward primer of SEQ ID NO: 60 and a reverse primer of SEQ ID NO: 61; A primer set specific to the ITS gene of M. peregrinum comprising a forward primer of SEQ ID NO: 62 and a reverse primer of SEQ ID NO: 63; A primer set specific to the ITS gene of M. mucogenicum comprising a forward primer of SEQ ID NO: 64 and a reverse primer of SEQ ID NO: 65; And it provides a primer set for the identification of mycobacteria comprising two or more primer sets selected from the group consisting of a primer set specific to the ITS gene of M. shimoidei comprising a forward primer of SEQ ID NO: 66 and a reverse primer of SEQ ID NO: 67.

In the primer set for identification of mycobacteria, M.gastri, M. xenopi , M. fortuitum , M. abscessus , M. avium , M. intracellulare , M.szulgai , M. terrae , M. chelonae , M. . kansasii, M. gordonae, M. marinum , M. simiae, M.ulcerans, M. septicum, M. smegmatis, M. scrofulaceum, M. haemophilum, M.celatum, M. peregrinum, M. mucogenicum, M. shimoidei Targets the 16S rRNA gene, hsp65 (Heat Shock Protein 65) gene, and ITS (internal transcribed spacer) gene, which is specific to each strain and has a large difference in melting temperature (Tm) of the melting curve of the real-time amplification products of each strain. It is designed to be

A primer set specific to the 16S rRNA gene of M. kansasii or M. gastri including a set of forward primers of SEQ ID NO: 28 and reverse primers of SEQ ID NO: 29 is specific to a strain of M. kansasii or M. gastri , in real time. When analyzing the polymerase chain reaction and melting curve, the two strains cannot be distinguished from each other because the base sequence is the same between the two strains, but it is designed to be distinguished from other mycobacterial strains.

A specific primer set specific to the 16S rRNA gene of M. kansasii or M. gastri comprising the forward primer of SEQ ID NO: 30 and the reverse primer of SEQ ID NO: 31 , specific to the strain of M.kansasii or M. gastri , real-time polymerase chain When analyzing the reaction and melting curve, the two strains cannot be distinguished from each other because the nucleotide sequence is the same between the two strains, but it is designed to be distinguished from other mycobacterial strains.

Specific primer sets on the ITS gene of M. marinum or M. ulcerans comprising a forward primer and a reverse primer of SEQ ID NO: 37 SEQ ID NO: 36 is specifically to the strain of the M.marinum or M. ulcerans, real-time polymerase chain When analyzing the reaction and melting curve, the two strains cannot be distinguished from each other because the nucleotide sequence is the same between the two strains, but it is designed to be distinguished from other mycobacterial strains.

Specific primer sets on the ITS gene of M. marinum or M. ulcerans comprising a forward primer and SEQ ID NO: 55 reverse primer of SEQ ID NO: 54 is specifically to the strain of the M.marinum or M. ulcerans, real-time polymerase chain When analyzing the reaction and melting curve, the two strains cannot be distinguished from each other because the nucleotide sequence is the same between the two strains, but it is designed to be distinguished from other mycobacterial strains.

16S rRNA-specific primer set for a gene of M. abscessus or M. chelonae comprising a reverse primer of SEQ ID NO: 60 and a reverse primer of SEQ ID NO: 61 is specifically to the strain of M.abscessus or M. chelonae, real-time polymerase In the case of chain reaction and melting curve analysis, the two strains cannot be distinguished from each other because the base sequence is the same between the two strains, but it is designed to be distinguished from other mycobacterial strains.

In the primer set for mycobacteria identification, Mycobacterium tuberculosis and mycobacterium tuberculosis are designed to have a large difference in melting temperature (Tm) of the melting curve of real-time amplification products between the strains while being specific for the IS6110 gene of Mycobacterium tuberculosis and the 16S rRNA of mycobacterium tuberculosis. will be.

The primer set was designed in consideration of the characteristic melting curve according to the base sequence, size and GC ratio of the real-time amplification product.

The forward primer of SEQ ID NO: 12 (5'-tktggtggaaagcttttgc-3') is a primer set comprising 5'-tgtggtggaaagcttttgc-3' (SEQ ID NO: 68) and 5'-tttggtggaaagcttttgc-3' (SEQ ID NO: 69). For example, 5'-tgtggtggaaagcttttgc-3' (SEQ ID NO: 68) and 5'-tttggtggaaagcttttgc-3' (SEQ ID NO: 69) may be a primer set including a 1:1 ratio.

The forward primer of SEQ ID NO: 18 (5'-ggtctaataccgrataggaccttta-3') is a primer set comprising 5'-ggtctaataccggataggaccttta-3' (SEQ ID NO: 70) and 5'-ggtctaataccgaataggaccttta-3' (SEQ ID NO: 71). For example, 5'-ggtctaataccggataggaccttta-3' (SEQ ID NO: 70) and 5'-ggtctaataccgaataggaccttta-3' (SEQ ID NO: 71) may be a primer set including a 1:1 ratio.

The reverse primer of SEQ ID NO: 19 (5'-cgcaaaagctttccaccwa-3') is a primer set comprising 5'-cgcaaaagctttccaccaa-3' (SEQ ID NO: 72) and 5'-cgcaaaagctttccaccta-3' (SEQ ID NO: 73). For example, 5'-cgcaaaagctttccaccaa-3' (SEQ ID NO: 72) and 5'-cgcaaaagctttccaccta-3' (SEQ ID NO: 73) may be a primer set including a 1:1 ratio.

In order to achieve the above other object, a primer set specific to the ITS (Internal transcribed spacer) gene of M. gastri including the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2; A primer set specific to the ITS (Internal transcribed spacer) gene of M. gastri including the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; A primer set specific to the ITS gene of M. xenopi comprising the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6; A primer set specific to the ITS gene of M. fortuitum comprising a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8; A primer set specific to the ITS gene of M. fortuitum comprising a set of forward primers of SEQ ID NO: 7 and reverse primers of SEQ ID NO: 9; A primer set specific to the IS6110 gene of M. tuberculosis complex, including the forward primer of SEQ ID NO: 10 and the reverse primer of SEQ ID NO: 11; A primer set specific to the 16S rRNA gene of Nontuberculous mycobacteria comprising the forward primer of SEQ ID NO: 12 and the reverse primer of SEQ ID NO: 13; A primer set specific to the ITS gene of M. abscessus comprising a forward primer of SEQ ID NO: 14 and a reverse primer of SEQ ID NO: 15; A primer set specific to the ITS gene of M.abscessus comprising the forward primer of SEQ ID NO: 16 and the reverse primer of SEQ ID NO: 17; A primer set specific to the 16S rRNA gene of M. intracellulare comprising a forward primer of SEQ ID NO: 18 and a reverse primer of SEQ ID NO: 19; A primer set specific to the ITS gene of M. szulgai comprising the forward primer of SEQ ID NO: 20 and the reverse primer of SEQ ID NO: 21; A primer set specific to the ITS gene of M. terrae comprising the forward primer of SEQ ID NO: 22 and the reverse primer of SEQ ID NO: 23; A primer set specific to the 16S rRNA gene of M. avium comprising the forward primer of SEQ ID NO: 24 and the reverse primer of SEQ ID NO: 25; A primer set specific to the ITS gene of M. chelonae comprising the forward primer of SEQ ID NO: 26 and the reverse primer of SEQ ID NO: 27; A primer set specific to the 16S rRNA gene of M.kansasii or M. gastri comprising a set of forward primers of SEQ ID NO: 28 and reverse primers of SEQ ID NO: 29; A primer set specific to the 16S rRNA gene of M. kansasii or M. gastri comprising a forward primer of SEQ ID NO: 30 and a reverse primer of SEQ ID NO: 31; A primer set specific to the ITS gene of M. gordonae comprising the forward primer of SEQ ID NO: 32 and the reverse primer of SEQ ID NO: 33; A primer set specific to the 16S rRNA gene of M. gordonae comprising the forward primer of SEQ ID NO: 34 and the reverse primer of SEQ ID NO: 35; A primer set specific to the ITS gene of M. marinum or M. ulcerans comprising a forward primer of SEQ ID NO: 36 and a reverse primer of SEQ ID NO: 37; A primer set specific to the ITS gene of M. simiae comprising a forward primer of SEQ ID NO: 38 and a reverse primer of SEQ ID NO: 39; A primer set specific to HSP65 (Heat shock protein 65) of M. ulcerans comprising a forward primer of SEQ ID NO: 40 and a reverse primer of SEQ ID NO: 41; A primer set specific to the ITS gene of M. septicum comprising the forward primer of SEQ ID NO: 42 and the reverse primer of SEQ ID NO: 43; A primer set specific to the ITS gene of M.smegmatis comprising a forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45; A primer set specific to the ITS gene of M. smegmatis comprising a forward primer of SEQ ID NO: 46 and a reverse primer of SEQ ID NO: 47; A primer set specific to the 16S rRNA gene of M. scrofulaceum comprising the forward primer of SEQ ID NO: 48 and the reverse primer of SEQ ID NO: 49; A primer set specific to the ITS gene of M.scrofulaceum comprising the forward primer of SEQ ID NO: 50 and the reverse primer of SEQ ID NO: 51; A primer set specific to the ITS gene of M. haemophilum comprising the forward primer of SEQ ID NO: 52 and the reverse primer of SEQ ID NO: 53; A primer set specific to the ITS gene of M. marinum or M. ulcerans comprising the forward primer of SEQ ID NO: 54 and the reverse primer of SEQ ID NO: 55; A primer set specific to the ITS gene of M. celatum comprising the forward primer of SEQ ID NO: 56 and the reverse primer of SEQ ID NO: 57; A primer set specific to the ITS gene of M. abscessus comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; A primer set specific to the 16S rRNA gene of M. abscessus or M. chelonae comprising a forward primer of SEQ ID NO: 60 and a reverse primer of SEQ ID NO: 61; A primer set specific to the ITS gene of M. peregrinum comprising the forward primer of SEQ ID NO: 62 and the reverse primer of SEQ ID NO: 63; A primer set specific to the ITS gene of M.mucogenicum comprising the forward primer of SEQ ID NO: 64 and the reverse primer of SEQ ID NO: 65; And a primer set for identification of mycobacteria comprising two or more primer sets selected from the group consisting of a primer set specific to the ITS gene of M. shimoidei comprising a forward primer of SEQ ID NO: 66 and a reverse primer of SEQ ID NO: 67. Provides a kit for identification of bacteria.

The kit for identification of mycobacteria may include a reagent for amplifying DNA through a real-time polymerase chain reaction. Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like. The kit for identification of mycobacteria may include a primer designed to have a large difference in melting temperature while targeting a gene specific to a specific strain in order to identify each strain as a specific bacteria so as to reliably distinguish the mycobacteria. . The primer set was designed in consideration of the characteristic melting curve according to the base sequence, size and GC ratio of the real-time amplification product.

In the kit for detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis according to an embodiment of the present invention, a fluorescent dye such as SYBR Green lowers the polymerase chain reaction at a high concentration, and redistribution of the fluorescent dye occurs during the melting process, resulting in a high resolution melting curve ( High-resolution melting curve) analysis may be unfavorable, so fluorescent dyes such as SYTO 9, EvaGreen, and LCGreen may be included. In other words, the reaction solution for real-time polymerase chain reaction method and melting curve analysis may contain fluorescent dyes such as SYTO 9, EvaGreen, and LCGreen. For example, the reaction solution for polymerase chain reaction method and melting curve analysis may contain EvaGreen fluorescent dye. In one embodiment of the present invention, a real-time-polymerase chain reaction was performed using a Type-it HRM PCR kit (QIAGEN Inc., Germantown, MD, USA). The Type-it HRM PCR kit contains EvaGreen as a fluorescent dye.

According to an embodiment of the present invention, the kit for identification of mycobacteria is a primer set specific to the ITS gene of M. gastri comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, a forward primer of SEQ ID NO: 5 and SEQ ID NO: 6, specific primers sets the ITS gene of M. xenopi, which contains the reverse primer of SEQ ID NO: 7 and SEQ ID NO: forward primer specific for a primer set for a gene of the ITS M.fortuitum comprising a reverse primer of 8, 16S rRNA gene of acidic non-Tuberculosis bacteria comprising a primer set specific to the IS6110 gene of Mycobacterium tuberculosis including a forward primer of SEQ ID NO: 10 and a reverse primer of SEQ ID NO: 11, and a forward primer of SEQ ID NO: 12 and a reverse primer of SEQ ID NO: 13 A first primer set for identification of mycobacteria comprising a primer set specific to the group; A primer set specific for the ITS gene of M. abscessus comprising a forward primer of SEQ ID NO: 14 and a reverse primer of SEQ ID NO: 15, a forward primer of SEQ ID NO: 18, and a 16S rRNA of M. intracellulare comprising a reverse primer of SEQ ID NO: 19. A primer set specific for the gene, a primer set specific for the ITS gene of M. szulgai comprising a forward primer of SEQ ID NO: 20 and a reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: 23. A second primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. terrae containing; ITS of M. chelonae comprising a primer set specific to the 16S rRNA gene of M.avium comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25, a forward primer of SEQ ID NO: 26, and a reverse primer of SEQ ID NO: 27. A primer set specific for the gene-specific primer set, a primer set specific for the 16S rRNA gene of M. kansasii or M. gastri comprising a set of a forward primer of SEQ ID NO: 28 and a reverse primer of SEQ ID NO: 29, and a forward primer and sequence of SEQ ID NO: 32 A third primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. gordonae comprising the reverse primer of No. 33; M. containing the reverse primer of the primer set specific for SEQ ID NO: 38 of the forward primer and SEQ ID NO: 39 to the ITS gene of M. marinum or M. ulcerans containing SEQ ID NO: 36 of the forward primer and a reverse primer of SEQ ID NO: 37 simiae ITS gene-specific primer set, a primer set specific to HSP65 of M.ulcerans comprising a forward primer of SEQ ID NO: 40 and a reverse primer of SEQ ID NO: 41, a forward primer of SEQ ID NO: 42, and a reverse primer of SEQ ID NO: 43 Identification of a fourth mycobacteria comprising a primer set specific to the ITS gene of M. septicum and a primer set specific to the ITS gene of M. smegmatis comprising a forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45. Primer set for; ITS of M. scrofulaceum comprising a primer set specific to the 16S rRNA gene of M. scrofulaceum comprising a forward primer of SEQ ID NO: 48 and a reverse primer of SEQ ID NO: 49, a forward primer of SEQ ID NO: 50, and a reverse primer of SEQ ID NO: 51. A primer set specific to the ITS gene of M.haemophilum comprising a gene-specific primer set, a forward primer of SEQ ID NO: 52 and a reverse primer of SEQ ID NO: 53, and a forward primer of SEQ ID NO: 56 and a reverse primer of SEQ ID NO: 57 A fifth mycobacteria identification primer set comprising a primer set specific to the ITS gene of M. celatum; And of SEQ ID NO: M. peregrinum containing specific primer sets, the forward primer of SEQ ID NO: 62 and a reverse primer of SEQ ID NO: 63 to the ITS gene of M.abscessus containing 58 forward primer and a reverse primer of SEQ ID NO: 59 ITS A primer set specific to the gene, a primer set specific to the ITS gene of M. mucogenicum comprising a forward primer of SEQ ID NO: 64 and a reverse primer of SEQ ID NO: 65, and a forward primer of SEQ ID NO: 66 and a reverse primer of SEQ ID NO: 67 The sixth mycobacteria identification primer set including a primer set specific to the ITS gene of M. shimoidei may be used.

According to an embodiment of the present invention, the kit for identification of mycobacteria is a set of primers specific to the ITS gene of M. gastri including a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, and a forward primer of SEQ ID NO: 5 and SEQ ID NO: 6, specific primers sets the ITS gene of M. xenopi, which contains the reverse primer of SEQ ID NO: 7 and SEQ ID NO: forward primer specific for a primer set for a gene of the ITS M.fortuitum comprising a reverse primer of 8, 16S rRNA gene of acidic non-Tuberculosis bacteria comprising a primer set specific to the IS6110 gene of Mycobacterium tuberculosis including a forward primer of SEQ ID NO: 10 and a reverse primer of SEQ ID NO: 11, and a forward primer of SEQ ID NO: 12 and a reverse primer of SEQ ID NO: 13 A first primer set for identification of mycobacteria comprising a primer set specific to the group; A primer set specific for the ITS gene of M. abscessus comprising a forward primer of SEQ ID NO: 16 and a reverse primer of SEQ ID NO: 17, a forward primer of SEQ ID NO: 18, and a 16S rRNA of M. intracellulare comprising a reverse primer of SEQ ID NO: 19. A primer set specific for the gene, a primer set specific for the ITS gene of M. szulgai comprising a forward primer of SEQ ID NO: 20 and a reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: 23. A second primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. terrae containing; ITS of M. chelonae comprising a primer set specific to the 16S rRNA gene of M.avium comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25, a forward primer of SEQ ID NO: 26, and a reverse primer of SEQ ID NO: 27. A primer set specific for the gene-specific primer set, a primer set specific for the 16S rRNA gene of M. kansasii or M. gastri comprising a set of a forward primer of SEQ ID NO: 28 and a reverse primer of SEQ ID NO: 29, and a forward primer and sequence of SEQ ID NO: 32 A third primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. gordonae comprising the reverse primer of No. 33; A primer set specific for the ITS gene of M. simiae comprising a forward primer of SEQ ID NO: 38 and a reverse primer of SEQ ID NO: 39, a forward primer of SEQ ID NO: 40 and a reverse primer of SEQ ID NO: 41 to HSP65 of M. ulcerans comprising a reverse primer of SEQ ID NO: 41 A specific primer set, a primer set specific to the ITS gene of M. septicum comprising a forward primer of SEQ ID NO: 42 and a reverse primer of SEQ ID NO: 43, and M comprising a forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45 the fourth mycobacteria identifying primer set comprising a set of primers specific to the gene of the ITS smegmatis; ITS of M. haemophilum comprising a primer set specific for the 16S rRNA gene of M. scrofulaceum comprising a forward primer of SEQ ID NO: 48 and a reverse primer of SEQ ID NO: 49, a forward primer of SEQ ID NO: 52, and a reverse primer of SEQ ID NO: 53. A primer set specific to the ITS gene of M.marinum or M. ulcerans comprising a primer set specific to the gene, a forward primer of SEQ ID NO: 54 and a reverse primer of SEQ ID NO: 55; A fifth mycobacterial identification primer set comprising a primer set specific to the ITS gene of M. celatum comprising the forward primer of SEQ ID NO: 56 and the reverse primer of SEQ ID NO: 57; And a primer set specific for the 16S rRNA gene of M. abscessus or M. chelonae comprising a forward primer of SEQ ID NO: 60 and a reverse primer of SEQ ID NO: 61, a forward primer of SEQ ID NO: 62, and a reverse primer of SEQ ID NO: 63. A primer set specific to the ITS gene of M. peregrinum , a primer set specific to the ITS gene of M. mucogenicum comprising a forward primer of SEQ ID NO: 64 and a reverse primer of SEQ ID NO: 65, and a forward primer of SEQ ID NO: 66 and a forward primer of SEQ ID NO: 67 It may consist of a primer set for identification of the sixth mycobacteria comprising a primer set specific to the ITS gene of M. shimoidei containing the reverse primer of.

According to an embodiment of the present invention, the kit for identification of mycobacteria is a set of primers specific to the ITS gene of M. xenopi including a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, and a forward primer of SEQ ID NO: 62. And a primer set specific to the ITS gene of M. peregrinum comprising a reverse primer of SEQ ID NO: 63, a forward primer of SEQ ID NO: 10, and a reverse primer of SEQ ID NO: 11; A first set of primers for identification of mycobacteria comprising a primer set specific to the 16S rRNA gene of an acidic non-Tuberculosis bacteria including the forward primer of 12 and the reverse primer of SEQ ID NO: 13; A primer set specific for the ITS gene of M. abscessus comprising a forward primer of SEQ ID NO: 16 and a reverse primer of SEQ ID NO: 17, a forward primer of SEQ ID NO: 18, and a 16S rRNA of M. intracellulare comprising a reverse primer of SEQ ID NO: 19. A primer set specific for the gene, a primer set specific for the ITS gene of M. szulgai comprising a forward primer of SEQ ID NO: 20 and a reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: 23. A second primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. terrae containing; ITS of M. chelonae comprising a primer set specific to the 16S rRNA gene of M. avium comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25, a forward primer of SEQ ID NO: 26, and a reverse primer of SEQ ID NO: 27. A primer set specific to the gene, a primer set specific to the 16S rRNA gene of M. kansasii or M.gastri comprising a forward primer of SEQ ID NO: 30 and a reverse primer of SEQ ID NO: 31, and a forward primer of SEQ ID NO: 7 and SEQ ID NO: 9 A third primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. fortuitum comprising a set of reverse primers of; A primer set specific for the ITS gene of M. simiae comprising a forward primer of SEQ ID NO: 38 and a reverse primer of SEQ ID NO: 39, a forward primer of SEQ ID NO: 40 and a reverse primer of SEQ ID NO: 41 to HSP65 of M. ulcerans comprising a reverse primer of SEQ ID NO: 41 A specific primer set, a primer set specific to the ITS gene of M. septicum comprising a forward primer of SEQ ID NO: 42 and a reverse primer of SEQ ID NO: 43, and M comprising a forward primer of SEQ ID NO: 34 and a reverse primer of SEQ ID NO: 35 the fourth mycobacteria identifying primer set comprising a primer set specific for the 16S rRNA gene of gordonae; A primer set specific to the 16S rRNA gene of M. scrofulaceum comprising the forward primer of SEQ ID NO: 48 and the reverse primer of SEQ ID NO: 49 , M. marinum or M comprising the forward primer of SEQ ID NO: 54 and the reverse primer of SEQ ID NO: 55 fifth mycobacteria identifying primer set comprising a primer set specific for the ITS gene of M. smegmatis containing the specific primer set and the forward primer of SEQ ID NO: 46 and a reverse primer of SEQ ID NO: 47 to the ITS gene of ulcerans; And of SEQ ID NO: M. mucogenicum containing three of the forward primer, and specific primers, a forward primer of SEQ ID NO: 64 and a reverse primer of SEQ ID NO: 65 to the ITS gene of M.gastri comprising a reverse primer of SEQ ID NO: 4 ITS It may consist of a primer set specific for a gene and a primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. celatum including a forward primer of SEQ ID NO: 56 and a reverse primer of SEQ ID NO: 57.

According to an embodiment of the present invention, the kit for identification of mycobacteria is a set of primers specific to the ITS gene of M. xenopi including a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, and a forward primer of SEQ ID NO: 62. And a primer set specific to the ITS gene of M. peregrinum comprising a reverse primer of SEQ ID NO: 63, a forward primer of SEQ ID NO: 10, and a reverse primer of SEQ ID NO: 11; A first set of primers for identification of mycobacteria comprising a primer set specific to the 16S rRNA gene of an acidic non-Tuberculosis bacteria including the forward primer of 12 and the reverse primer of SEQ ID NO: 13; A primer set specific for the ITS gene of M. abscessus comprising a forward primer of SEQ ID NO: 16 and a reverse primer of SEQ ID NO: 17, a forward primer of SEQ ID NO: 18, and a 16S rRNA of M. intracellulare comprising a reverse primer of SEQ ID NO: 19. A primer set specific for the gene, a primer set specific for the ITS gene of M. szulgai comprising a forward primer of SEQ ID NO: 20 and a reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: 23. A second primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. terrae containing; ITS of M. chelonae comprising a primer set specific to the 16S rRNA gene of M. avium comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25, a forward primer of SEQ ID NO: 26, and a reverse primer of SEQ ID NO: 27. A primer set specific to the gene, a primer set specific to the 16S rRNA gene of M. kansasii or M.gastri comprising a forward primer of SEQ ID NO: 30 and a reverse primer of SEQ ID NO: 31, and a forward primer of SEQ ID NO: 7 and SEQ ID NO: 9 A third primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. fortuitum comprising a set of reverse primers of; A primer set specific for the ITS gene of M. simiae comprising a forward primer of SEQ ID NO: 38 and a reverse primer of SEQ ID NO: 39, a forward primer of SEQ ID NO: 40 and a reverse primer of SEQ ID NO: 41 to HSP65 of M. ulcerans comprising a reverse primer of SEQ ID NO: 41 A specific primer set, a primer set specific to the ITS gene of M. septicum comprising a forward primer of SEQ ID NO: 42 and a reverse primer of SEQ ID NO: 43, and M comprising a forward primer of SEQ ID NO: 34 and a reverse primer of SEQ ID NO: 35 the fourth mycobacteria identifying primer set comprising a primer set specific for the 16S rRNA gene of gordonae; A primer set specific to the 16S rRNA gene of M. scrofulaceum comprising the forward primer of SEQ ID NO: 48 and the reverse primer of SEQ ID NO: 49 , M. marinum or M comprising the forward primer of SEQ ID NO: 54 and the reverse primer of SEQ ID NO: 55 fifth mycobacteria identifying primer set comprising a primer set specific for the ITS gene of M. smegmatis containing the specific primer set and the forward primer of SEQ ID NO: 46 and a reverse primer of SEQ ID NO: 47 to the ITS gene of ulcerans; And of SEQ ID NO: M. mucogenicum containing three of the forward primer, and specific primers, a forward primer of SEQ ID NO: 64 and a reverse primer of SEQ ID NO: 65 to the ITS gene of M.gastri comprising a reverse primer of SEQ ID NO: 4 ITS It may consist of a primer set specific for a gene and a primer set for identification of mycobacteria comprising a primer set specific to the ITS gene of M. celatum including a forward primer of SEQ ID NO: 56 and a reverse primer of SEQ ID NO: 57.

According to an embodiment of the present invention, the first primer set for mycobacterial identification to the sixth primer set for mycobacterial identification may be separated by a separate storage means. For example, the first primer set for mycobacteria identification to the sixth primer set for mycobacteria identification may be separated by a separate tube.

In order to achieve the above other object, the step of separating DNA from the specimen; Subjecting the DNA to a real-time polymerase chain reaction using the kit for identification of mycobacteria; Obtaining a melting curve by melting the amplified product amplified by the real-time polymerase chain reaction while changing the temperature; And it provides a mycobacteria identification method comprising the step of checking the melting temperature of the melting curve.

The melting curve analysis according to an embodiment of the present invention may be performed by measuring a fluorescence wavelength of 510 nm.

The present invention is a mycobacteria identification primer set capable of accurately detecting and identifying mycobacteria as a specific species, a mycobacteria identification kit including the same, and a real-time polymerase chain reaction method using the same and a mycobacteria identification method using the melting curve analysis Provides. According to the above method, there are many types of mycobacteria that can be identified, and it is possible to provide a clinical diagnosis means capable of efficiently identifying mycobacteria at a simple, accurate, fast, and low cost test.

1 to 5 are amplification products obtained by performing real-time polymerase chain reaction of M.fortuitum , M.gastri , Nontuberculous mycobacteria, M.tuberculosis complex, and M.xenopi using the primer mix of Tube I-A, respectively. ) Shows the results of melting curve analysis. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
6 to 9 are the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M.abscessus , M.intracellulare , M. szulgai, and M.terrae using the primer mix of Tube II-A, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
10 to 13 are the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M. avium , M.chelonae , M. gordonae and M. kansasii using the primer mix of Tube III-A, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
14 to 18 are amplification products obtained by performing real-time polymerase chain reaction of M. marinum , M.septicum , M. simiae , M. smegmatis and M. ulcerans using the primer mix of Tube IV-A, respectively. ) Shows the results of melting curve analysis. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
19 to 21 show the results of melting curve analysis of the amplified products obtained by performing real-time polymerase chain reaction of M. celatum , M. haemophilum and M. scrofulaceum using the primer mix of Tube V-A, respectively. . In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
22 to 25 are the melting curves of the amplicons obtained by performing real-time polymerase chain reactions of M.abscessus , M. mucogenicum , M. peregrinum and M. shimoidei using the primer mix of Tube VI-A, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
26 to 30 show an amplification product obtained by performing real-time polymerase chain reaction of M.fortuitum , M. gastri , Nontuberculous mycobacteria, M. tuberculosis complex and M.xenopi using the primer mix of Tube I-B, respectively. ) Shows the results of melting curve analysis. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
31 to 34 show the melting curves of the amplicons obtained by performing real-time polymerase chain reactions of M.abscessus , M. intracellulare , M. szulgai and M. terrae using the primer mix of Tube II-B, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
35 to 38 show melting curves of an amplicon obtained by performing real-time polymerase chain reaction of M. avium , M.chelonae , M. gordonae and M. kansasii using the primer mix of Tube III-A, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
39 to 42 are the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M.septicum , M. simiae , M. smegmatis and M. ulcerans using the primer mix of Tube IV-B, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
43 to 47 show amplification products obtained by performing real-time polymerase chain reaction of M. celatum , M.haemophilum , M. scrofulaceum , M. marinum, and M. ulcerans using the primer mix of Tube V-B, respectively. ) Shows the results of melting curve analysis. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
48 to 52 show amplification products obtained by performing real-time polymerase chain reaction of M. abscessus , M. chelonae , M. mucogenicum , M. peregrinum and M. shimoidei using the primer mix of tube VI-B, respectively. ) Shows the results of melting curve analysis. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
53 to 56 are melting curves of the amplification products obtained by performing real-time polymerase chain reaction of M. xenopi , M. peregrinum, Nontuberculous mycobacteria and M. tuberculosis complex using the primer mix of Tube I-C, respectively The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
57 to 60 show the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M.abscessus , M. intracellulare , M. szulgai and M. terrae using the primer mix of Tube II-B, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
61 to 64 show the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M. avium , M.chelonae , M. fortuitum and M. kansasii using the primer mix of Tube III-B, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
Figures 65 to 68 are the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M.septicum , M. simiae , M. gordonae and M. ulcerans using the primer mix of Tube IV-C, respectively. The analysis result is shown. In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
69 to 71 show the results of melting curve analysis of an amplicon obtained by performing real-time polymerase chain reaction of M.scrofulaceum , M. marinum, and M. smegmatis using the primer mix of Tube V-B, respectively. . In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).
72 to 74 show the results of melting curve analysis of an amplicon obtained by performing real-time polymerase chain reaction of M. gastri , M. mucogenicum and M. celatum using the primer mix of Tube VI-B, respectively. . In the melting curve, the x-axis is the temperature (°C) and the y-axis is the change in fluorescence per unit time (dF/dT).

Hereinafter, the present invention will be described in detail by examples, but the following examples are only illustrative of the present invention, and the content of the present invention is not limited thereto.

Example: Double real-time polymerase chain reaction method and mycobacteria identification method using melting curve analysis

1. Design of detection target site and primer

end. Gene to be detected

16S rRNA gene, hsp65 (Heat Shock Protein 65) gene, ITS (internal transcribed spacer) gene sequence data of mycobacteria were analyzed by Sequencher 4.10 and analyzed with M.gastri, M.xenopi , M.fortuitum , M.abscessus , M. avium , M.intracellulare , M.szulgai , M.terrae , M.chelonae , M.kansasii , M.gordonae , M.marinum , M.simiae , M.ulcerans , M.septicum , M.smegmatis , M.scrofulaceum , Primers specific to M.haemophilum, M.celatum, M.peregrinum , M.mucogenicum , and M.shimoidei were designed using the primer3 program and manual techniques.

The nucleotide sequence data were obtained from the database of NCBI (National Center for Biotechnology Information), and the nucleotide sequence data used for designing primers specific to the mycobacteria were as follows.

M. gastri (GU142918.1, AF547836.1, AY299182.1, Y14182.1), M.xenopi (AJ536033.1, AY082373.1, AF547891.1, AJ243481.1, L15624.1, Y14192.1), M.fortuitum (AY458072.1, AY299168.1, AY299167.1, AY457066.1, AF480580.1, GU142933.1, AF144326.1, AM709726.1), M.abscessus (AJ419970.1, AJ416940.1, AJ536038 ,AM709728.1, AF163815.1, FM955485.1, FM955486.1, AY458075.1, EF486338.1), M.avium (NR_025584.1, AJ536037.1, EF521892.1, GU362530.1, HM056131.1, AB0266901.1, AF410479.1, L15620.1, X74054.1, Z46421.1), M.intracellulare (AY652958.1, AJ536036.1, X52927.1, M61684.1, AB026691.1, AJ306711.1, AJ314865 .1, AM709724.1, Z46423.1, GQ153290.1, EU239784.1), M.szulgai (X52926.1, AY299141.1, AY299173.1, AF547878.1, AB026704.1, X99220.1), M .terrae (NR_029168.1, AY299142.1, AY550211.1, HM584725.1, Z46427.1), M.chelonae (AM884324.1, AJ419969.1, AF144327.1, AY458082.1, AY299148.1), M .kansasii (M29575.1, X15916.1, AB026695.1, AB232369.1, AM709725.1), M. gordonae (GU142923.1, AB026692.1, AJ315574.1, AY604571.1), M.marinum (AF456238.1, AY513243.1, AB026701.1, GU827996.1, Y14185.1), M.simiae (GQ153280.1,AB026694.1, Y14186.1, Z46426.1), M.ulcerans (Z13990.1) ,X99217.1), M. septicum (AY457070.1, AM902922.1), M.smegmatis (NR_025311.1, Y08453.1), M.scrofulaceum (GQ153271.1, AB026702.1, L15622.1), M . haemophilum (V06638.1, AY579398.1, AY579399.1 , DQ235664.1), M.celatum (L08169.1, AF375990.1, FJ754638.1), M.peregrinum (AY457069.1, AM396454.1, AM421288 .1), M.mucogenicum (AF480585.1, AM902932.1, AY504951.1, DQ235663.1), M. shimoidei (X82459.1, AJ005005.1, X99219.1)

I. primer

1) M. gastri 1

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gggcttgtcttggactcgt-3′ (SEQ ID NO: 1)

Reverse primer: 5′-tggtgggacaacactcttgg-3′ (SEQ ID NO: 2)

Amplification product size: 39bp

Amplification product melting temperature (Tm): 75.06℃

2) M. gastri 2

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gcaacactcgggcttgtctt-3′ (SEQ ID NO: 3)

Reverse primer: 5′-atggtgggacaacactcttgg-3′ (SEQ ID NO: 4)

Amplification product size: 49bp

Amplification product melting temperature (Tm): 78.10℃

3) M. xenopi

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gttgggcagcaggcagta-3′ (SEQ ID NO: 5)

Reverse primer: 5′-gttgcctcaaaacccaacag-3′ (SEQ ID NO: 6)

Amplification product size: 50bp

Amplification product melting temperature (Tm): 80.86℃

4) M. fortuitum 1

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-cccgagccgtgaggaac-3′ (SEQ ID NO: 7)

Reverse primer: 5′-caatagtgtgtctggcagtcaaaa-3′ (SEQ ID NO: 8)

Amplification product size: 82bp

Amplification product melting temperature (Tm): 81.70℃

5) M. fortuitum 2

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-cccgagccgtgaggaac-3′ (SEQ ID NO: 7)

Reverse primer: 5′-cgacacccgcaacaggat-3′ (SEQ ID NO: 9)

Amplification product size: 141bp

Amplification product melting temperature (Tm): 85.50℃

6) M. tuberculosis complex

Gene to be detected: IS6110

Forward primer: 5′-cgaactcaaggagcacatcag-3′ (SEQ ID NO: 10)

Reverse primer: 5′-cagggttagccacactttgc-3′ (SEQ ID NO: 11)

Amplification product size: 79bp

Amplification product melting temperature (Tm): 84.16℃

7) Nontuberculous mycobacteria

Gene to be detected: 16S rRNA

Forward primer: 5′-tktggtggaaagcttttgc-3′ (SEQ ID NO: 12)

Reverse primer: 5′-cgtaggagtctgggccgta-3′ (SEQ ID NO: 13)

Amplification product size: 146bp

Amplification product melting temperature (Tm): 87.54℃

8) M. abscessus 1

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-atgaactagggaacataaagtatgca-3′ (SEQ ID NO: 14)

Reverse primer: 5′-aggatttacaaaacatattcaccaagt-3′ (SEQ ID NO: 15)

Amplification product size: 69bp

Amplification product melting temperature (Tm): 71.70℃

9) M. abscessus 2

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-tcgaatgaactagggaacataaagta-3′ (SEQ ID NO: 16)

Reverse primer: 5′-gatttacaaaacatattcaccaagtaga-3′ (SEQ ID NO: 17)

Amplification product size: 71bp

Amplification product melting temperature (Tm): 72.10℃

10) M. intracellulare

Gene to be detected: 16S rRNA

Forward primer: 5′-ggtctaataccgrataggaccttta-3′ (SEQ ID NO: 18)

Reverse primer: 5′-cgcaaaagctttccaccwa-3′ (SEQ ID NO: 19)

Amplification product size: 56bp

Amplification product melting temperature (Tm): 75.40℃ or 76.90℃

11) M. szulgai

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-ggtcctgaggcaacactca-3′ (SEQ ID NO: 20)

Reverse primer: 5′-ccaagatggtgggacaacag-3′ (SEQ ID NO: 21)

Amplification product size: 52bp

Amplification product melting temperature (Tm): 79.66℃

12) M. terrae

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gattcccccgtacctcacat-3′ (SEQ ID NO: 22)

Reverse primer: 5′-accaccgaccacccactac-3′ (SEQ ID NO: 23)

Amplification product size: 80bp

Amplification product melting temperature (Tm): 82.86℃

13) M. avium

Gene to be detected: 16S rRNA

Forward primer: 5′-gacctcaagacgcatgtcttc-3′ (SEQ ID NO: 24)

Reverse primer: 5′-cgcaaaagctttccaccag-3′ (SEQ ID NO: 25)

Amplification product size: 39bp

Amplification product melting temperature (Tm): 73.50℃

14) M. chelonae

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-tgtccaccccgtggata-3′ (SEQ ID NO: 26)

Reverse primer: 5′-gtgccagcgtttcaattcta-3′ (SEQ ID NO: 27)

Amplification product size: 65bp

Amplification product melting temperature (Tm): 79.00℃

15) M. kansasii / M. gastri 1

Gene to be detected: 16S rRNA

Forward primer: 5′-gcaatctgccctgcacac-3′ (SEQ ID NO: 28)

Reverse primer: 5′-ccacaaggcatgctccaa-3′ (SEQ ID NO: 29)

Amplification product size: 80bp

Amplification product melting temperature (Tm): 81.16℃

16) M. kansasii / M. gastri 2

Gene to be detected: 16S rRNA

Forward primer: 5′-cggaaaggtctcttcggagac-3′ (SEQ ID NO: 30)

Reverse primer: 5′-tttcccaggcttatcctggt-3′ (SEQ ID NO: 31)

Amplification product size: 88bp

Amplification product melting temperature (Tm): 83.36℃

17) M. gordonae 1

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-tgcaagccttgagtggtca-3′ (SEQ ID NO: 32)

Reverse primer: 5′-ggggacagcaccagagg-3′ (SEQ ID NO: 33)

Amplification product size: 111bp

Amplification product melting temperature (Tm): 83.80℃

18) M. gordonae 2

Gene to be detected: 16S rRNA

Forward primer: 5′-taacacatgcaagtcgaaaggt-3′ (SEQ ID NO: 34)

Reverse primer: 5′-ctttccaccacaggacatgt-3′ (SEQ ID NO: 35)

Amplification product size: 156bp

Amplification product melting temperature (Tm): 83.74℃

19) M. marinum / M. ulcerans 1

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gggtcctgaggcaacatct-3′ (SEQ ID NO: 36)

Reverse primer: 5′-caacatcccgaaaccaacag-3′ (SEQ ID NO: 37)

Amplification product size: 39bp

Amplification product melting temperature (Tm): 74.60℃

20) M. simiae

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-ctcggccgacttcggtt-3′ (SEQ ID NO: 38)

Reverse primer: 5′-agatggagggacaccacttca-3′ (SEQ ID NO: 39)

Amplification product size: 37bp

Amplification product melting temperature (Tm): 76.40℃

21) M. ulcerans

Gene to be detected: HSP65 (Heat shock protein 65)

Forward primer: 5′-accgagaccctgctcaaa-3′ (SEQ ID NO: 40)

Reverse primer: 5′-gctccttggtctcgacctct-3′ (SEQ ID NO: 41)

Amplification product size: 46bp

Amplification product melting temperature (Tm): 79.10℃

22) M. septicum

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-atggcctcgcacctgtag-3′ (SEQ ID NO: 42)

Reverse primer: 5′-ccaatagtgtgtctggcagttcta-3′ (SEQ ID NO: 43)

Amplification product size: 72bp

Amplification product melting temperature (Tm): 81.70℃

23) M. smegmatis 1

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gagctggagcgctgtagtg-3′ (SEQ ID NO: 44)

Reverse primer: 5′-gaaacagcgtttcccacac-3′ (SEQ ID NO: 45)

Amplification product size: 73bp

Amplification product melting temperature (Tm): 84.20℃

24) M. smegmatis 2

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gagccgtgaggagctgga-3′ (SEQ ID NO: 46)

Reverse primer: 5′-accgcatctcaacgtttgc-3′ (SEQ ID NO: 47)

Amplification product size: 66bp

Amplification product melting temperature (Tm): 84.36℃

25) M. scrofulaceum 1

Gene to be detected: 16S rRNA

Forward primer: 5′-accatcgacgaaggctcac-3′ (SEQ ID NO: 48)

Reverse primer: 5′-cacctaccgtcaacccaca-3′ (SEQ ID NO: 49)

Amplification product size: 40bp

Amplification product melting temperature (Tm): 77.50℃

26) M. scrofulaceum 2

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gcaacactcggctcgttct-3′ (SEQ ID NO: 50)

Reverse primer: 5′-agatggagggacaccactca-3′ (SEQ ID NO: 51)

Amplification product size: 38bp

Amplification product melting temperature (Tm): 77.50℃

27) M. haemophilum

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gcacaacagcaaatgaatcg-3′ (SEQ ID NO: 52)

Reverse primer: 5′-acatgggacaagcctgagt-3′ (SEQ ID NO: 53)

Amplification product size: 69bp

Amplification product melting temperature (Tm): 80.76℃

28) M. marinum / M. ulcerans 2

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gggtcctgaggcaacatc-3′ (SEQ ID NO: 54)

Reverse primer: 5′-acagaacacgccaccaaa-3′ (SEQ ID NO: 55)

Amplification product size: 133bp

Amplification product melting temperature (Tm): 83.56℃

29) M. celatum

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-cacgaaaaacactccgcatc-3′ (SEQ ID NO: 56)

Reverse primer: 5′-gcgatttttcccatttgttg-3′ (SEQ ID NO: 57)

Amplification product size: 100bp

Amplification product melting temperature (Tm): 85.84℃

30) M. abscessus 3

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-catttcccagtcgaatgaacta-3′ (SEQ ID NO: 58)

Reverse primer: 5′-ggatttacaaaacatattcaccaagtagatac-3′ (SEQ ID NO: 59)

Amplification product size: 80bp

Amplification product melting temperature (Tm): 75.10℃

31) M. abscessus / M. chelonae

Gene to be detected: 16S rRNA

Forward primer: 5′-ctaataccggataggaccacaca-3′ (SEQ ID NO: 60)

Reverse primer: 5′-gcaaaagctttgcaccact-3′ (SEQ ID NO: 61)

Amplification product size: 52bp

Amplification product melting temperature (Tm): 76.40℃

32) M. peregrinum

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-attcgttggatggcctcac-3′ (SEQ ID NO: 62)

Reverse primer: 5′-ccacgccaagtttgttgag-3′ (SEQ ID NO: 63)

Amplification product size: 65bp

Amplification product melting temperature (Tm): 81.70℃

33) M. mucogenicum

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-catttacatgccctgatcca-3′ (SEQ ID NO: 64)

Reverse primer: 5′-cgacgacaatcccaacca-3′ (SEQ ID NO: 65)

Amplification product size: 76bp

Amplification product melting temperature (Tm): 83.50℃

34) M. shimoidei

Gene to be detected: ITS (Internal transcribed spacer)

Forward primer: 5′-gaagtcgagccgtgagga-3′ (SEQ ID NO: 66)

Reverse primer: 5′-accaccaaagatgaggcaac-3′ (SEQ ID NO: 67)

Amplification product size: 142bp

Amplification product melting temperature (Tm): 86.96℃

2. Real time Polymerase chain reaction And melting curve analysis

end. DNA Separation of

100 strains of anti-acid non-tuberculous bacteria isolated from clinical specimens and 68 strains of mycobacteria standard strain were used. The standard strains used were M. abscessus ATCC 19977 , M. acapulcensis KCTC 9501, M. africanum ATCC 25420, M. agri KCTC 9502, M. alvei KCTC 19709, M. asiaticum KCTC 9503, M. aurum KCTC 19457, M. austroafricanum KCTC 9504, M. avium ATCC 25291, M. bolletii KCTC 19281, M. botniense KCTC 19646, M. bovis ATCC 19210, M. brumae KCTC 19711, M. celatum ATCC 51131, M. chelonae subsp chelonae KCTC 9505, M. chlorophenolicum KCTC 19089, M. chubuense KCTC 19712, M. diernhoferi KCTC 9506, M. fallax KCTC 9508, M. flavescens ATCC 14474, M. fortuitum ATCC 6841, M. frederiksbergense KCTC 19100, M. gadium gadium ATCC 27726, M. gastri ATCC 15754, M. gilvum KCTC 19423, M. goodii ATCC BAA-955, M. gordonae KCTC 9513, M. haemophilum ATCC 29548, M. hassiacum ATCC 700660, M. interjectum ATCC 51457, M. intermedium ATCC 51848, M. intracellulare ATCC 13950, M. intracellulare KCTC 9514, M. kansasii ATCC 12478, M. lentiflavum KMRC 70087, M. malmoense ATCC 29571 , M. mantobense KCTC 9977, M. marinum ATCC 927, M. massiliense KCTC 19086, M. microti ATCC 19422, M. moriokaense KCTC 9516, M. mucogenicum KCTC 19088, M. neoaurum KCTC 19096, M. nonchromogenicum ATCC 19530, M. obuense KCTC 19097, M. parascrofulaceum KCTC 9979, M. peregrinum KCTC 9615 , KMRC 75002, M. phlei KCTC 9689 , M. porcinum KCTC 9517, M. pulveris KCTC 9518, M. scrofulaceum ATCC 19981, M. septicum ATCC 700731, M. simiae ATCC 25275, M. shimoidei ATCC 27962, M. smegmatis KCTC 9108, M. szulgai KCTC 9520 , KMRC 31125, M. terrae KCTC 9614, M. triplex ATCC 700071, M. triviale KMRC 70093, M. tuberculosis ATCC 25177, ATCC 27294, M. ulcerans ATCC 19423, M. vaccae KCTC 19087, M. vanbaalenii KCTC 9966, M . wolinskyi ATCC 700010, M. was xenopi KMRC 42001.

100 anti-acid non-Tuberculosis bacteria isolated from clinical specimens were strains detected by culturing in liquid medium (MGIT mycobacteria medium) or solid (Ogawa medium) medium. ATCC and KCTC strains were cultured and used in a liquid medium, and KMRC strains were cultured and used in a solid medium.

The DNA of mycobacteria grown in liquid medium was extracted as follows. After the MGIT mycobacteria culture tube in which the bacteria were cultured was well mixed, 500 µl of the liquid medium was taken, put into a 1.5 ml tube, and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 µl of sterile distilled water was added to the remaining saliva portion and heated in a boiling water for 10 minutes in a bath. After heating in a bath, centrifugation was performed at 14,000 rpm for 5 minutes, and the supernatant was used as a template DNA for polymerase chain reaction.

DNA of mycobacteria grown in solid medium was extracted as follows. 500 µl of sterile distilled water was placed in a 1.5 ml tube, and 1 platinum was taken from the solid medium and dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged for 5 minutes at 14,000 rpm, and the supernatant was used as a template DNA for the polymerase chain reaction.

I. primer Mix( Primers mix )

Six types of primer mix tubes were prepared. Each primer mix tube contains the same amount of forward and reverse primers specific to the mycobacteria.

1) TubeⅠ

a. Tube I-A

M. gastri 1 primer 10pmole/µl

M. xenopi primer 9pmole/µl

M. fortuitum 1 primer 6pmole/µl

M. tuberculosis complex primer 14pmole/µl

Nontuberculous mycobacteria primer 8pmole/µl

b. Tube I-B

M. gastri 2 primer 8pmole/µl

M. xenopi primer 9pmole/µl

M. fortuitum 1 primer 6pmole/µl

M. tuberculosis complex primer 14pmole/µl

Nontuberculous mycobacteria primer 8pmole/µl

c. Tube I-C

M. xenopi primer 9pmole/µl

M. peregrinum primer 12pmole/µl

M. tuberculosis complex primer 14pmole/µl

Nontuberculous mycobacteria primer 8pmole/µl

2) TubeⅡ

a. Tube II-A

M. abscessus 1 primer 10pmole/µl

M. intracellulare primer 10pmole/µl

M. szulgai primer 10pmole/µl

M. terrae primer 10pmole/µl

b. TubeⅡ-B

M. abscessus 2 primer 12pmole/µl

M. intracellulare primer 10pmole/µl

M. szulgai primer 10pmole/µl

M. terrae primer 10pmole/µl

3) TubeⅢ

a. TubeⅢ-A

M. avium primer 10pmole/µl

M. chelonae primer 14pmole/µl

M. kansasii / M. gastri 1 primer 8pmole/µl

M. gordonae 1 primer 10pmole/µl

b. TubeⅢ-B

M. avium primer 10pmole/µl

M. chelonae primer 14pmole/µl

M. kansasii / M. gastri 2 primer 8pmole/µl

M. fortuitum 2 primer 10pmole/µl

4) Tube IV

a. Tube IV-A

M. marinum / M. ulcerans 1 primer 10pmole/µl

M. simiae primer 10pmole/µl

12pmole/µl of M. ulcerans primer

M. septicum primer 10pmole/µl

M. smegmatis 1 primer 8pmole/µl

b. Tube IV-B

M. simiae primer 10pmole/µl

12pmole/µl of M. ulcerans primer

M. septicum primer 10pmole/µl

M. smegmatis 1 primer 8pmole/µl

c. Tube IV-C

M. simiae primer 10pmole/µl

12pmole/µl of M. ulcerans primer

M. septicum primer 10pmole/µl

M. gordonae 2 primer 10pmole/µl

5) Tube V

a. Tube V-A

M. scrofulaceum 1 primer 12pmole/µl

M. scrofulaceum 2 primer 12pmole/µl

M. haemophilum primer 10pmole/µl

M. celatum primer 8pmole/µl

b. Tube V-B

M. scrofulaceum 1 primer 12pmole/µl

M. haemophilum primer 10pmole/µl

M. marinum / M. ulcerans 2 primer 10pmole/µl

M. celatum primer 8pmole/µl

c. Tube V-C

M. scrofulaceum 1 primer 12pmole/µl

M. marinum / M. ulcerans 2 primer 10pmole/µl

M. smegmatis 2 primer 10pmole/µl

6) Tube VI

a. Tube VI-A

M. abscessus 3 primer 10pmole/µl

M. peregrinum primer 10pmole/µl

M. mucogenicum primer 10pmole/µl

M. shimoidei primer 4pmole/µl

b. Tube VI-B

M. abscessus / M. chelonae primer 10pmole/µl

M. peregrinum primer 10pmole/µl

M. mucogenicum primer 10pmole/µl

M. shimoidei primer 4pmole/µl

c. Tube VI-C

M. gastri 2 primer 10pmole/µl

M. mucogenicum primer 10pmole/µl

M. celatum primer 8pmole/µl

All. Double real time Polymerase chain reaction method And melting curve analysis

Real-time polymerase chain reaction was performed using a Type-it HRM PCR kit (QIAGEN Inc., Germantown, MD, USA). The Type-it HRM PCR kit contains EvaGreen as a fluorescent dye. Real-time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed by performing a denaturation process at about 95°C for about 5 minutes, 1 cycle, denaturation at about 95°C for about 10 seconds, annealing and extension processes at about 60°C for about 15 seconds in 1 cycle. After the final polymerase chain reaction was completed, the melting curve was analyzed by measuring the fluorescence wavelength of 510 nm while increasing the temperature at a rate of about 0.2° C. per second from about 68° C. to about 93° C. At this time, the composition of the reactants for performing the double real-time polymerase chain reaction are shown in Table 1 below.

ingredient Volume (µl) density 2X HRM PCR Master Mix 5 1X Fryer mix 0.5 See Table 2 Nuclease free water 3.5 - DNA template One - all 10

Primer amount (pmole/µl) Concentration in reaction solution (μM) 4 0.2 6 0.3 8 0.4 9 0.45 10 0.5 12 0.6 14 0.7

< Example 1>

This is a method for identifying mycobacteria using the primer mix of Tube I-A, Tube II-A, Tube III-A, Tube IV-A, Tube V-A, and Tube VI-A. Mycobacteria were identified according to the above-described real-time polymerase chain reaction and melting curve analysis.

< Example 2>

This is a method for identifying mycobacteria using a primer mix using the primer mixes of Tube I-B, Tube II-B, Tube III-A, Tube IV-B, Tube V-B, and Tube VI-B. Mycobacteria were identified according to the above-described real-time polymerase chain reaction and melting curve analysis.

< Example 3>

This is a method for identifying mycobacteria using a primer mix of Tube I-C, Tube II-B, Tube III-B, Tube IV-C, Tube V-C, and Tube VI-C. Mycobacteria were identified according to the above-described real-time polymerase chain reaction and melting curve analysis.

la. real time Polymerase chain reaction method And melting curve analysis results

< Example 1>

1 to 5 are an amplification product obtained by performing real-time polymerase chain reaction of M. fortuitum , M.gastri , Nontuberculous mycobacteria, M. tuberculosis complex, and M. xenopi using the primer mix of Tube I-A, respectively. ) Shows the results of melting curve analysis.

6 to 9 are the melting curves of the amplicons obtained by performing real-time polymerase chain reactions of M. abscessus , M.intracellulare , M. szulgai and M. terrae using the primer mix of Tube II-A, respectively. The analysis result is shown.

10 to 13 are the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M. avium , M.chelonae , M. gordonae and M. kansasii using the primer mix of Tube III-A, respectively. The analysis result is shown.

14 to 18 are amplification products obtained by performing real-time polymerase chain reaction of M. marinum , M.septicum , M. simiae , M. smegmatis and M. ulcerans using the primer mix of Tube IV-A, respectively. ) Shows the results of melting curve analysis.

19 to 21 show the results of melting curve analysis of the amplified products obtained by performing real-time polymerase chain reaction of M. celatum , M. haemophilum and M. scrofulaceum using the primer mix of Tube V-A, respectively. .

22 to 25 are the melting curves of the amplicons obtained by performing real-time polymerase chain reactions of M.abscessus , M. mucogenicum , M. peregrinum and M. shimoidei using the primer mix of Tube VI-A, respectively. The analysis result is shown.

1 to 25, it was confirmed that the peak corresponding to the melting temperature (Tm) of the amplification product corresponding to the primer designed according to each mycobacterial strain was shown.

1 and 2, since real-time polymerase chain reaction is performed using the primer mix of Tube I-A, M. The amplification product melting temperature (Tm) peaks corresponding to fortuitum (about 81.70°C) and M.gastri (about 75.06°C) appeared. However, for M.xenopi, for the M. tuberculosis but wherein the acid rain were only amplified product melting temperature (Tm) peak (about 80.86 ℃), corresponding to M. xenopi (see Fig. 5).

Referring to Figure 18, because by using a primer mix of the tube-A Ⅳ perform real-time polymerase chain reaction, in the case of a strain of M. ulcerans, M. marinum / M. ulcerans amplification product melting temperature for the 1 ( Tm) peak (about 74.60 °C) and the amplification product melting temperature (Tm) peak (about 79.10 °C) corresponding to M. ulcerans appeared.

Referring to FIG. 21, because by using a primer mix of the tube Ⅴ-A (including the primer set of the M. scrofulaceum M.scrofulaceum 1 and 2) to perform a real-time polymerase chain reaction, in the case of a strain of M. scrofulaceum amplification product The melting temperature (Tm) peak (about 77.50°C) appeared, and the amplification product melting temperature (Tm) peak corresponding to the primer sets of M. scrofulaceum 1 and M. scrofulaceum 2 all corresponded to about 77.50° C., so the peak was one. .

Tube in Example 1 Ⅱ-A (M. abscessus 1 ) and the tube Ⅵ-A (M. abscessus 3) to be equipped with a primer set that can be distinguished by detecting the M.abscessus, M. effective in clinical specimens It was confirmed that abscessus could be identified. In addition, with a M. scrofulaceum M. scrofulaceum 1 and 2 the tube Ⅴ-A, it was confirmed that it is possible to identify the M.scrofulaceum from clinical specimens.

Through Example 1, whether Mycobacterium tuberculosis or mycobacterium tuberculosis, among the Mycobacterium tuberculosis, M. gastri , M. xenopi , M. fortuitum , M. abscessus , M. avium , M. intracellulare , M.szulgai , M. terrae , M. chelonae , M. kansasii , M. gordonae , M. marinum , M. simiae , M.ulcerans , M. septicum , M. smegmatis , M. scrofulaceum , M. haemophilum , M.celatum , M. peregrinum , M. mucogenicum And M. shimoidei .

< Example 2>

26 to 30 show an amplification product obtained by performing real-time polymerase chain reaction of M.fortuitum , M. gastri , Nontuberculous mycobacteria, M. tuberculosis complex and M.xenopi using the primer mix of Tube I-B, respectively. ) Shows the results of melting curve analysis.

31 to 34 show the melting curves of the amplicons obtained by performing real-time polymerase chain reactions of M.abscessus , M. intracellulare , M. szulgai and M. terrae using the primer mix of Tube II-B, respectively. The analysis result is shown.

35 to 38 show melting curves of an amplicon obtained by performing real-time polymerase chain reaction of M. avium , M.chelonae , M. gordonae and M. kansasii using the primer mix of Tube III-A, respectively. The analysis result is shown.

39 to 42 are the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M.septicum , M. simiae , M. smegmatis and M. ulcerans using the primer mix of Tube IV-B, respectively. The analysis result is shown.

43 to 47 show amplification products obtained by performing real-time polymerase chain reaction of M. celatum , M.haemophilum , M. scrofulaceum , M. marinum, and M. ulcerans using the primer mix of Tube V-B, respectively. ) Shows the results of melting curve analysis.

48 to 52 show amplification products obtained by performing real-time polymerase chain reaction of M. abscessus , M. chelonae , M. mucogenicum , M. peregrinum and M. shimoidei using the primer mix of tube VI-B, respectively. ) Shows the results of melting curve analysis.

26 to 52, it was confirmed that the peak corresponding to the melting temperature (Tm) of the amplification product corresponding to the primer designed according to each mycobacterial strain was shown.

Referring to Figures 26 and 27, since real-time polymerase chain reaction is performed using the primer mix of Tube I-B, M. The amplification product melting temperature (Tm) peaks corresponding to fortuitum (about 81.70°C) and M. gastri (about 78.10°C) appeared. However, in the case of M. xenopi, Mycobacterium tuberculosis, but that the anti-acid rain were only amplified product melting temperature (Tm) peak (about 80.86 ℃), corresponding to M. xenopi (see Fig. 30).

46 and 47, real-time polymerase chain reaction was performed using the primer mix of Tube V-B, and the amplification product melting temperature (Tm) peak (about 83.56) corresponding to M. marinum / M. ulcerans 2 °C), M. marinum or M. ulcerans all exhibited a melting temperature (Tm) peak corresponding to about 83.56 °C. In this case, when compared to the results of using a primer mix of the tube Ⅵ-B perform the polymerase chain reaction (PCR), and performing a melting curve analysis, amplified product melting temperature (Tm) peak corresponding to M. ulcerans (about 79.10℃) , the strain can be identified as M. ulcerans , and if the peak does not appear, the strain can be identified as M. marinum. In addition, by performing real-time polymerase chain reaction using the primer mix of Tube V-B, the amplification product melting temperature (Tm) peak (about 83.56°C) corresponding to M. marinum / M. ulcerans 2 appears, and tube VI- If the amplification product melting temperature (Tm) peak (about 79.10°C) corresponding to M.ulcerans appears by performing a polymerase chain reaction using the primer mix of B, and performing a melting curve analysis, M. marinum and M. marinum in the sample. It can be identified as having M. ulcerans.

In Example 2, tube II-B ( M. abscessus 2) and tube III-A ( M. chelonae ) were provided with primer sets capable of detecting and distinguishing M. abscessus and M. chelonae, respectively, and tube VI- to B can not distinguish M. abscessus and M. chelonae, but provided with a high specificity M. abscessus / M. chelonae for M. abscessus or M. chelonae, M. chelonae effectively identify M. abscessus or from clinical specimens I was able to confirm that I could do it.

Through Example 2, whether Mycobacterium tuberculosis or mycobacterium tuberculosis, among the Mycobacterium tuberculosis, M. gastri , M. xenopi , M. fortuitum , M. abscessus , M. avium , M. intracellulare , M.szulgai , M. terrae , M. chelonae , M. kansasii , M. gordonae , M. marinum , M. simiae , M.ulcerans , M. septicum , M. smegmatis , M. scrofulaceum , M. haemophilum , M.celatum , M. peregrinum , M. It was confirmed that any strain of mucogenicum and M. shimoidei could be effectively identified.

< Example 3>

53 to 56 are melting curves of the amplification products obtained by performing real-time polymerase chain reaction of M. xenopi , M. peregrinum, Nontuberculous mycobacteria and M. tuberculosis complex using the primer mix of Tube I-C, respectively The analysis result is shown.

57 to 60 show the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M.abscessus , M. intracellulare , M. szulgai and M. terrae using the primer mix of Tube II-B, respectively. The analysis result is shown.

61 to 64 show the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M. avium , M.chelonae , M. fortuitum and M. kansasii using the primer mix of Tube III-B, respectively. The analysis result is shown.

Figures 65 to 68 are the melting curves of the amplicons obtained by performing real-time polymerase chain reaction of M.septicum , M. simiae , M. gordonae and M. ulcerans using the primer mix of Tube IV-C, respectively. The analysis result is shown.

69 to 71 show the results of melting curve analysis of an amplicon obtained by performing real-time polymerase chain reaction of M.scrofulaceum , M. marinum, and M. smegmatis using the primer mix of Tube V-B, respectively. .

72 to 74 show the results of melting curve analysis of an amplicon obtained by performing real-time polymerase chain reaction of M. gastri , M. mucogenicum and M. celatum using the primer mix of Tube VI-B, respectively. .

53 to 74, it was confirmed that the peak corresponding to the melting temperature (Tm) of the amplification product corresponding to the primer designed according to each mycobacterial strain was shown.

Referring to FIG. 54, since the polymerase chain reaction is performed in real time using the primer mix of Tube I-C, the peak corresponding to Nontuberculous mycobacteria (about 87.54° C.) and M. peregrinum (about 81.70° C.) The amplification product melting temperature (Tm) peak corresponding to) appeared. However, in the case of M. xenopi , the amplification product melting temperature (Tm) peak (approximately 80.86°C) corresponding to M.xenopi, although corresponding to the acidic non-tuberculous bacteria, appeared (see FIG. 53).

Through Example 3, whether Mycobacterium tuberculosis or mycobacterium tuberculosis, among mycobacterium tuberculosis, M. gastri , M. xenopi , M. fortuitum , M. abscessus , M. avium , M. intracellulare , M.szulgai , M. terrae , Any of M. chelonae , M. kansasii , M. gordonae , M. marinum , M. simiae , M.ulcerans , M. septicum , M. smegmatis , M. scrofulaceum , M. celatum , M. peregrinum and M. mucogenicum It was confirmed that it can be recognized effectively.

In the case of Example 3, unlike Examples 1 and 2, M. haemophilum and M. shimoidei were not provided with a primer set for identification of mycobacteria capable of identification. In the case of M. haemophilum and M. shimoidei , it does not appear well in clinical specimens in Korea, so when including a primer set for identifying the strain, it is included in consideration of reduction in identification efficiency due to interactions between primer sets, etc. It is designed in a configuration that does not.

When performing the real-time polymerase chain reaction method and melting curve analysis according to the above embodiments, it can be confirmed that the mycobacteria can be identified as a specific strain with high reliability in a clinical sample efficiently, conveniently, accurately, quickly, and at low cost. there was.

<110> UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERATION <120> METHODS FOR IDENTIFICATION AND DETECTION OF MYCOBACTERIUM USING REAL TIME POLYMERASE CHAIN REACTION AND MELTING CURVE ANALYSIS <130> ADP-2013-0125 <160> 73 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.gastri <400> 1 gggcttgtct tggactcgt 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.gastri <400> 2 tggtgggaca acactcttgg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.gastri <400> 3 gcaacactcg ggcttgtctt 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.gastri <400> 4 atggtgggac aacactcttg g 21 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.xenopi <400> 5 gttgggcagc aggcagta 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.xenopi <400> 6 gttgcctcaa aacccaacag 20 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.fortuitum <400> 7 cccgagccgt gaggaac 17 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.fortuitum <400> 8 caatagtgtg tctggcagtc aaaa 24 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.fortuitum <400> 9 cgacacccgc aacaggat 18 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of IS6110 gene in M.tuberculosis complex <400> 10 cgaactcaag gagcacatca g 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of IS6110 gene in M.tuberculosis complex <400> 11 cagggttagc cacactttgc 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in Nontuberculous mycobacteria <400> 12 tktggtggaa agcttttgc 19 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in Nontuberculous mycobacteria <400> 13 cgtaggagtc tgggccgta 19 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.abscessus <400> 14 atgaactagg gaacataaag tatgca 26 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.abscessus <400> 15 aggatttaca aaacatattc accaagt 27 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.abscessus <400> 16 tcgaatgaac tagggaacat aaagta 26 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.abscessus <400> 17 gatttacaaa acatattcac caagtaga 28 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.intracellulare <400> 18 ggtctaatac cgrataggac cttta 25 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.intracellulare <400> 19 cgcaaaagct ttccaccwa 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.szulgai <400> 20 ggtcctgagg caacactca 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.szulgai <400> 21 ccaagatggt gggacaacag 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.terrae <400> 22 gattcccccg tacctcacat 20 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.terrae <400> 23 accaccgacc acccactac 19 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.avium <400> 24 gacctcaaga cgcatgtctt c 21 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.avium <400> 25 cgcaaaagct ttccaccag 19 <210> 26 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.chelonae <400> 26 tgtccacccc gtggata 17 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.chelonae <400> 27 gtgccagcgt ttcaattcta 20 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.kansasii or M.gastri <400> 28 gcaatctgcc ctgcacac 18 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.kansasii or M.gastri <400> 29 ccacaaggca tgctccaa 18 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.kansasii or M.gastri <400> 30 cggaaaggtc tcttcggaga c 21 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.kansasii or M.gastri <400> 31 tttcccaggc ttatcctggt 20 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.gordonae <400> 32 tgcaagcctt gagtggtca 19 <210> 33 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.gordonae <400> 33 ggggacagca ccagagg 17 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.gordonae <400> 34 taacacatgc aagtcgaaag gt 22 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.gordonae <400> 35 ctttccacca caggacatgt 20 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.marinum or M.ulcerans <400> 36 gggtcctgag gcaacatct 19 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.marinum or M.ulcerans <400> 37 caacatcccg aaaccaacag 20 <210> 38 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.simiae <400> 38 ctcggccgac ttcggtt 17 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.simiae <400> 39 agatggaggg acaccacttc a 21 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of HSP65(Heat shock protein 65) gene in M.ulcerans <400> 40 accgagaccc tgctcaaa 18 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of HSP65(Heat shock protein 65) gene in M.ulcerans <400> 41 gctccttggt ctcgacctct 20 <210> 42 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.septicum <400> 42 atggcctcgc acctgtag 18 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.septicum <400> 43 ccaatagtgt gtctggcagt tcta 24 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.smegmatis <400> 44 gagctggagc gctgtagtg 19 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.smegmatis <400> 45 gaaacagcgt ttcccacac 19 <210> 46 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.smegmatis <400> 46 gagccgtgag gagctgga 18 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.smegmatis <400> 47 accgcatctc aacgtttgc 19 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.scrofulaceum <400> 48 accatcgacg aaggctcac 19 <210> 49 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.scrofulaceum <400> 49 cacctaccgt caacccaca 19 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.scrofulaceum <400> 50 gcaacactcg gctcgttct 19 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.scrofulaceum <400> 51 agatggaggg acaccactca 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.haemophilum <400> 52 gcacaacagc aaatgaatcg 20 <210> 53 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.haemophilum <400> 53 acatgggaca agcctgagt 19 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.marinum or M.ulcerans <400> 54 gggtcctgag gcaacatc 18 <210> 55 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.marinum or M.ulcerans <400> 55 acagaacacg ccaccaaa 18 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.celatum <400> 56 cacgaaaaac actccgcatc 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.celatum <400> 57 gcgatttttc ccatttgttg 20 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.abscessus <400> 58 catttcccag tcgaatgaac ta 22 <210> 59 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.abscessus <400> 59 ggatttacaa aacatattca ccaagtagat ac 32 <210> 60 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.abscessus or M.chelonae <400> 60 ctaataccgg ataggaccac aca 23 <210> 61 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.abscessus or M.chelonae <400> 61 gcaaaagctt tgcaccact 19 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.peregrinum <400> 62 attcgttgga tggcctcac 19 <210> 63 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.peregrinum <400> 63 ccacgccaag tttgttgag 19 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.mucogenicum <400> 64 catttacatg ccctgatcca 20 <210> 65 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.mucogenicum <400> 65 cgacgacaat cccaacca 18 <210> 66 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.shimoidei <400> 66 gaagtcgagc cgtgagga 18 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of Internal transcribed spacer (ITS) gene in M.shimoidei <400> 67 accaccaaag atgaggcaac 20 <210> 68 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in Nontuberculous mycobacteria <400> 68 tgtggtggaa agcttttgc 19 <210> 69 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in Nontuberculous mycobacteria <400> 69 tttggtggaa agcttttgc 19 <210> 70 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.intracellulare <400> 70 ggtctaatac cggataggac cttta 25 <210> 71 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification specific fragments of 16S rRNA gene in M.intracellulare <400> 71 ggtctaatac cgaataggac cttta 25 <210> 72 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.intracellulare <400> 72 cgcaaaagct ttccaccaa 19 <210> 73 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification specific fragments of 16S rRNA gene in M.intracellulare <400> 73 cgcaaaagct ttccaccta 19

Claims (4)

A primer set specific for the ITS gene of M.gastri including the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: 5, and an ITS gene of M. xenopi including the reverse primer of SEQ ID NO: A first primer set for identifying a first mycobacteria comprising a primer set specific for the ITS gene of M. fortuitum comprising a forward primer of SEQ ID NO: 7 and an inverted primer of SEQ ID NO: 8;
A primer set specific for the ITS gene of M. abscessus comprising the forward primer of SEQ ID NO: 16 and the reverse primer of SEQ ID NO: 17, a forward primer of SEQ ID NO: 18, and a reverse primer of SEQ ID NO: 19, 16S rRNA of M. intracellulare A primer set specific for the gene, a forward primer of SEQ ID NO: 20 and a reverse primer of SEQ ID NO: 21, a primer set specific for the ITS gene of M. szulgai , and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: A set of primers for identification of a second mycobacteria comprising a primer set specific to the ITS gene of M. terrae ;
A primer set specific for the 16S rRNA gene of M. avium comprising the forward primer of SEQ ID NO: 24 and the reverse primer of SEQ ID NO: 25, a forward primer of SEQ ID NO: 26, and an ITS of M. chelonae comprising the reverse primer of SEQ ID NO: A primer set specific for the 16S rRNA gene of M. kansasii or M. gastri comprising a set of primers specific to the gene, a forward primer of SEQ ID NO: 28 and a reverse primer of SEQ ID NO: 29, a forward primer and sequence of SEQ ID NO: A set of primers for identifying a third mycobacteria comprising a primer set specific to the ITS gene of M. gordonae comprising the reverse primer of SEQ ID NO: 33;
A primer set specific for the ITS gene of M. simiae comprising the forward primer of SEQ ID NO: 38 and the reverse primer of SEQ ID NO: 39, the forward primer of SEQ ID NO: 40, and the HSP65 of M. ulcerans containing the reverse primer of SEQ ID NO: M including the specific primers, SEQ ID NO: 42 of the forward primer and the specific primer set and the forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45 to the ITS gene of M. septicum comprising a reverse primer of SEQ ID NO: 43 the fourth mycobacteria identifying primer set comprising a set of primers specific to the gene of the ITS smegmatis;
A primer set specific for the 16S rRNA gene of M. scrofulaceum comprising the forward primer of SEQ ID NO: 48 and the reverse primer of SEQ ID NO: 49, the forward primer of SEQ ID NO: 52 and the reverse primer of SEQ ID NO: 53, and the ITS of M. haemophilum A primer set specific for the gene, a forward primer of SEQ ID NO: 54 and a reverse primer of SEQ ID NO: 55, a primer set specific for the ITS gene of M. marinum or M. ulcerans , a forward primer of SEQ ID NO: 56 and a primer set of SEQ ID NO: 57 A set of primers for identification of a fifth mycobacteria comprising a primer set specific to the ITS gene of M. celatum including a reverse primer; And
Specific primer sets on M. abscessus or 16S rRNA gene of M. chelonae comprising a forward primer SEQ ID NO: 60 and SEQ ID NO: 61 of the reverse primer, M comprising a reverse primer of SEQ ID NO: 62 and a reverse primer of SEQ ID NO: 63 a primer set specific for the ITS gene of P. ceregrinum , a forward primer of SEQ ID NO: 64 and a reverse primer of SEQ ID NO: 65, a primer set specific for the ITS gene of M. mucogenicum , a forward primer of SEQ ID NO: 66, Characterized in that it comprises a primer set for identification of a sixth mycobacteria comprising a primer set specific to an ITS gene of M. shimoidei including a reverse primer. delete The kit for identification of mycobacteria according to claim 1, wherein the primer set for identifying the first mycobacteria to the sixth mycobacteria is separated by a separate storage means. Obtaining DNA from the separated sample;
Subjecting the DNA to a real-time polymerase chain reaction using the kit for identifying mycobacteria according to claim 1;
Melting the amplification product amplified by the real-time PCR reaction while changing the temperature to obtain a melting curve; And
And confirming the melting temperature of the melting curve.

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