KR101426119B1 - 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

METHODS FOR IDENTIFICATION AND DETECTION OF MYCOBACTERIUM USING REAL TIME POLYMERASE CHAIN REACTION AND MELTING CURVE ANALYSIS BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to a method for identification of mycobacteria using real-time PCR and melting curve analysis. More particularly, the present invention relates to a kit for identifying mycobacteria including a primer set for identifying mycobacteria, a primer set for identifying mycobacteria, a real-time PCR method using the kit, and a mycobacteria identification method using a melting curve analysis.

Nontuberculous mycobacteria refers to all mycobacteria other than Mycobacterium tuberculosis and Mycobacterium leprae. Acute respiratory tuberculosis was not only known shortly after the discovery of mycobacterium tuberculosis but recently it has become recognized as an important infectious disease.

Recently, the efforts of health authorities have reduced the prevalence of tuberculosis in Korea, but the incidence of TB infection is increasing. According to the report, 12% of the tuberculosis-positive and culture-positive specimens are separated from the acid-fast bacteria. In addition, the number of laboratories performing liquid tuberculosis culture test is increasing as the liquid tuberculosis culture method is applied in 2009, and when the tuberculosis is cultured by using the liquid medium, the detection of the acid-resistant tuberculosis bacteria increases more than the culture using the conventional solid medium.

In USA and Europe where the prevalence rate of M. tuberculosis is low, infections caused by acid-fast bacteria M. tuberculosis are increasing. According to the US report, the detection of acid-fast bacillus bacteria increased from 1/3 of the mycobacteria isolated in 1979 to 1980 to 3/4 of the isolated mycobacteria in 1992. In the United States, Canada, and Western Europe, it is known that 40 to 50% of aerobic bacteria isolated from sputum cause lung disease.

The diagnosis of infectious diseases caused by mycobacteria is based on the method of isolating bacteria through culture and identifying them by biochemical methods. However, this method is delayed in growth due to the bacterium characteristics of mycobacteria, and it takes a long time to cultivate and it is not possible to accurately identify various mycobacterial species by biochemical method alone. Recently, myco - bacterial identification methods using molecular biology techniques have been introduced into laboratories. Accuprobe, INNO-LiPA Mycobacteria, and GenoType Mycobacterium CM, AS. These test kits are short and sensitive to the inspection time, but they have the disadvantage of being contaminated in the process of hybridization of amplified products after the PCR reaction or limited identification of identifiable mycobacteria. Therefore, there is a need for a method for identification of mycobacteria that has many kinds of mycobacteria species that are simple, accurate, fast, cost-effective, and identifiable.

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

Another object of the present invention is to provide a kit for identifying mycobacteria comprising a primer set for identifying mycobacteria that can accurately detect and identify mycobacteria as a specific species.

It is another object of the present invention to provide a double-real-time polymerase chain reaction (PCR) using a kit for identification of mycobacteria comprising a primer set for identification of mycobacteria that can accurately detect and identify mycobacteria as a specific species ) And a method of identification of mycobacteria using a melting curve analysis.

In order to achieve the above object, a primer set specific to the internal transcribed spacer (ITS) gene of M.gastri comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2; A primer set specific for 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 for 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 for the ITS gene of M. fortuitum comprising the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8; A primer set specific for the ITS gene of M. fortuitum comprising a forward primer of SEQ ID NO: 7 and a set of reverse primers of SEQ ID NO: 9; A primer set specific to the IS6110 gene of the M. tuberculosis complex comprising the forward primer of SEQ ID NO: 10 and the reverse primer of SEQ ID NO: 11; A primer set specific for the 16S rRNA gene of the acidic non-Mycobacteria (Nontuberculous mycobacteria) comprising the forward primer of SEQ ID NO: 12 and the reverse primer of SEQ ID NO: 13; A primer set specific for 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 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 primer set specific for the 16S rRNA gene of M. intracellulare comprising the forward primer of SEQ ID NO: 18 and the reverse primer of SEQ ID NO: 19; A primer set specific for 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 for the ITS gene of M. terrae including the forward primer of SEQ ID NO: 22 and the reverse primer of SEQ ID NO: 23; 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 primer set specific for 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 for a 16S rRNA gene of M.kansasii or M. < RTI ID = 0.0 > gastri < / RTI > comprising a forward primer of SEQ ID NO: 28 and a set of reverse primers of SEQ ID NO: A primer set specific for 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; A primer set specific for 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 for the ITS gene of M. marinum or M. ulcerans comprising the forward primer of SEQ ID NO: 36 and the reverse primer of SEQ ID NO: 37; A primer set specific for the ITS gene of M. s. Mimae comprising the forward primer of SEQ ID NO: 38 and the reverse primer of SEQ ID NO: 39; A primer set specific for M.ulcerans HSP65 (Heat shock protein 65) comprising the forward primer of SEQ ID NO: 40 and the reverse primer of SEQ ID NO: 41; A primer set specific for 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 for the ITS gene of M. smegmatis including the forward primer of SEQ ID NO: 44 and the reverse primer of SEQ ID NO: 45; A primer set specific for the ITS gene of M. smegmatis comprising the forward primer of SEQ ID NO: 46 and the reverse primer of SEQ ID NO: 47; 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; A primer set specific for 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 for 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 for 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 for 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 for the ITS gene of M. abscessus comprising the forward primer of SEQ ID NO: 58 and the reverse primer of SEQ ID NO: 59; A primer set specific for the 16S rRNA gene of M. abscessus or M. chelonae comprising the forward primer of SEQ ID NO: 60 and the reverse primer of SEQ ID NO: 61; A primer set specific for 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 for 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 specific to the ITS gene of M. shimoidei comprising a forward primer of SEQ ID NO: 66 and an inverted primer of SEQ ID NO: 67. The primer set for identifying mycobacteria includes two or more sets of primers selected from the group consisting of:

The mycobacteria isolated from the primer sets, of the anti-Mycobacterium tuberculosis M.gastri acid rain, M.xenopi, M.fortuitum, M.abscessus, M.avium, M.intracellulare, M.szulgai, M.terrae, M.chelonae, M M. gordonae , M. marinum , M. siciae , M. ulcerans , M. septicum , M. smegmatis , M. scrofulaceum , M. haemophilum , M.celatum , M. peregrinum , M. mucogenicum , M. shimoidei Targets the 16S rRNA gene, the hsp65 (Heat Shock Protein 65) gene and the ITS (internal transcribed spacer) gene. It is known that the melting temperature (Tm) difference of the melting curve of the real- .

M.kansasii or 16S rRNA gene specific primer sets on the M.gastri including a set of a reverse primer of SEQ ID NO: 28 and a reverse primer of SEQ ID NO: 29 is specifically to the strain of the M.kansasii or M.gastri, real-time The polymerase chain reaction and melting curve analysis were designed to distinguish the two strains from each other, although the two strains can not be distinguished from each other because the nucleotide sequences of the two strains are the same.

In SEQ ID NO: specific for the strains of the specific primer set or M.kansasii M.gastri M.kansasii or the 16S rRNA gene of M.gastri containing 30 forward primer and a reverse primer of SEQ ID NO: 31 small, real-time polymerase chain In the analysis of the reaction and melting curve, the two strains have the same nucleotide sequence so that they can not be distinguished from each other, but they are designed to be distinguishable from other mycobacterial strains.

A primer set specific for the ITS gene of M. marinum or M. ulcerans comprising the forward primer of SEQ ID NO: 36 and the reverse primer of SEQ ID NO: 37 is specific for strains of M. marinum or M. ulcerans , In the analysis of the reaction and melting curve, the two strains have the same nucleotide sequence so that they can not be distinguished from each other, but they are designed to be distinguishable from other mycobacterial strains.

A primer set specific for 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, is specific for strains of M. marinum or M. ulcerans , In the analysis of the reaction and melting curve, the two strains have the same nucleotide sequence so that they can not be distinguished from each other, but they are designed to be distinguishable from other mycobacterial strains.

A primer set specific for the 16S rRNA gene of M.abscessus or M.chelonae comprising the forward primer of SEQ ID NO: 60 and the reverse primer of SEQ ID NO: 61 specifically binds to M. abscessus or M.chelonae strains, In the analysis of the chain reaction and the melting curve, the two strains have the same nucleotide sequence so that they can not be distinguished from each other, but they are designed to be distinguishable from other mycobacterial strains.

In the primer set for identifying mycobacteria, the Mycobacterium tuberculosis and the acid-fast TB mycobacteria are specific for the IS6110 gene of Mycobacterium tuberculosis and the 16S rRNA of the anti-acidoblast Mycobacterium tuberculosis, respectively, and designed so that the melting temperature (Tm) difference between the fusion curves of the real- will be.

The primer sets are 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 (5'-tktggtggaaagcttttgc-3 ') of SEQ ID NO: 12 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) at a ratio of 1: 1.

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

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

In order to accomplish this another object, there is provided a primer set specific to an internal transcribed spacer (ITS) gene of M.gastri comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A primer set specific for the internal transcribed spacer (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 for 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 for the ITS gene of M. fortuitum comprising the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8; A primer set specific for the ITS gene of M. fortuitum comprising a forward primer of SEQ ID NO: 7 and a set of reverse primers of SEQ ID NO: 9; A primer set specific to the IS6110 gene of the M. tuberculosis complex comprising the forward primer of SEQ ID NO: 10 and the reverse primer of SEQ ID NO: 11; A primer set specific for the 16S rRNA gene of the acidic non-Mycobacteria (Nontuberculous mycobacteria) comprising the forward primer of SEQ ID NO: 12 and the reverse primer of SEQ ID NO: 13; A primer set specific for 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 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 primer set specific for the 16S rRNA gene of M. intracellulare comprising the forward primer of SEQ ID NO: 18 and the reverse primer of SEQ ID NO: 19; A primer set specific for 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 for the ITS gene of M. terrae including the forward primer of SEQ ID NO: 22 and the reverse primer of SEQ ID NO: 23; 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 primer set specific for 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 for a 16S rRNA gene of M.kansasii or M. < RTI ID = 0.0 > gastri < / RTI > comprising a forward primer of SEQ ID NO: 28 and a set of reverse primers of SEQ ID NO: A primer set specific for 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; A primer set specific for 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 for the ITS gene of M. marinum or M. ulcerans comprising the forward primer of SEQ ID NO: 36 and the reverse primer of SEQ ID NO: 37; A primer set specific for the ITS gene of M. s. Mimae comprising the forward primer of SEQ ID NO: 38 and the reverse primer of SEQ ID NO: 39; A primer set specific for M.ulcerans HSP65 (Heat shock protein 65) comprising the forward primer of SEQ ID NO: 40 and the reverse primer of SEQ ID NO: 41; A primer set specific for 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 for the ITS gene of M. smegmatis including the forward primer of SEQ ID NO: 44 and the reverse primer of SEQ ID NO: 45; A primer set specific for the ITS gene of M. smegmatis comprising the forward primer of SEQ ID NO: 46 and the reverse primer of SEQ ID NO: 47; 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; A primer set specific for 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 for 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 for 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 for 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 for the ITS gene of M. abscessus comprising the forward primer of SEQ ID NO: 58 and the reverse primer of SEQ ID NO: 59; A primer set specific for the 16S rRNA gene of M. abscessus or M. chelonae comprising the forward primer of SEQ ID NO: 60 and the reverse primer of SEQ ID NO: 61; A primer set specific for 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 for 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 of two or more primers selected from the group consisting of a forward primer of SEQ ID NO: 66 and a reverse primer of SEQ ID NO: 67 and a primer set specific to the ITS gene of M. shimoidei , and a set of primers for identifying mycobacteria A kit for identifying bacteria is provided.

The kit for identifying mycobacteria may include a reagent for amplifying DNA by a real-time PCR. Reagents for amplifying the DNA by real-time PCR include DNA polymerase, dNTPs, PCR buffer, and the like. The kit for identifying mycobacteria may include a primer designed to target a gene specific to a specific strain and designed to have a high melting temperature difference so as to reliably distinguish mycobacteria from each other . The primer sets are 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 detection kit of Mycobacterium tuberculosis and acid-fast bacterium according to one embodiment of the present invention, a fluorescent dye such as SYBR Green lowers the polymerase chain reaction at a high concentration, and a high-resolution melting curve high-resolution melting curve analysis, and therefore may include fluorescent pigments such as SYTO 9, EvaGreen, and LCGreen. In other words, the reaction solution for the real-time PCR and the fusion curve analysis may include fluorescent dyes such as SYTO 9, EvaGreen and LCGreen. For example, EvaGreen fluorescent dye may be included in the reaction solution for polymerase chain reaction and melting curve analysis. In one embodiment of the present invention, real-time PCR 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 identifying mycobacteria comprises a primer set specific to the ITS gene of M.gastri comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, the forward primer of SEQ ID NO: And a primer set specific to the ITS gene of M. xenopi comprising the reverse primer of SEQ ID NO: 6, a forward primer of SEQ ID NO: 7, and a primer set specific to the ITS gene of M. fortuitum comprising the reverse primer of SEQ ID NO: A primer set specific for the IS6110 gene of the Mycobacterium tuberculosis including the forward primer of SEQ ID NO: 10 and the reverse primer of SEQ ID NO: 11, a forward primer of SEQ ID NO: 12, and a 16S rRNA gene of the anti-acidic mycobacteria including the reverse primer of SEQ ID NO: Identification of a first mycobacteria comprising a primer set specific for Primer set; A primer set specific for the ITS gene of M. abscessus comprising the forward primer of SEQ ID NO: 14 and the reverse primer of SEQ ID NO: 15, the forward primer of SEQ ID NO: 18, and the 16S rRNA of M. intracellulare containing the reverse primer of SEQ ID NO: 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 containing the reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: A set of primers for identifying a second mycobacteria comprising a primer set specific to the ITS gene of M. terrae ; Of SEQ ID NO: M.chelonae containing specific primer sets, the forward primer of SEQ ID NO: 26 and a reverse primer of SEQ ID NO: 27 in the 16S rRNA gene in M.avium containing 24 of a forward primer and a reverse primer of SEQ ID NO: 25 ITS A primer set specific for 16S rRNA gene of M. kansasii or M. gastri comprising a forward primer of SEQ ID NO: 28 and a reverse primer of SEQ ID NO: 29, a forward primer of SEQ ID NO: 32 and a primer set of SEQ ID NO: A primer set for identification of a third mycobacteria comprising a primer set specific to the ITS gene of M. gordonae comprising the reverse primer of SEQ ID 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 in the ITS of the gene or M.marinum M.ulcerans comprising a reverse primer of SEQ ID NO: 36 and a reverse primer of SEQ ID NO: 37 a primer set specific for the ITS gene of simiae , a forward primer of SEQ ID NO: 40 and a primer set specific for HSP65 of M. ulcerans comprising the reverse primer of SEQ ID NO: 41, a forward primer of SEQ ID NO: 42 and a reverse primer of SEQ ID NO: fourth mycobacterial identification comprising a set of primers specific to the gene of the ITS M.smegmatis comprising a specific primer set and the forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45 in the ITS of the gene containing the M.septicum Primer set; 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, a forward primer of SEQ ID NO: 50, and an ITS of M. scrofulaceum containing the reverse primer of SEQ ID NO: A primer set specific for the gene, a forward primer of SEQ ID NO: 52 and a reverse primer of SEQ ID NO: 53, a primer set specific for the ITS gene of M. haemophilum including the reverse primer of SEQ ID NO: 53, a forward primer of SEQ ID NO: 56 and a reverse primer of SEQ ID NO: 57 A set of primers for identifying a fifth mycobacteria comprising a primer set specific to the ITS gene of M.celatum ; A primer set specific for the ITS gene of M. abscessus comprising the forward primer of SEQ ID NO: 58 and the reverse primer of SEQ ID NO: 59, the forward primer of SEQ ID NO: 62 and the reverse primer of SEQ ID NO: 63, and the ITS of M. peregrinum A primer set specific for the gene, 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 including the reverse primer of SEQ ID NO: 66 and a reverse primer of SEQ ID NO: 67 And a set of primers for identifying a sixth mycobacteria comprising a primer set specific for the ITS gene of M. shimoidei .

According to an embodiment of the present invention, the kit for identifying mycobacteria comprises a primer set specific to the ITS gene of M.gastri including a forward primer of SEQ ID NO: 3 and an reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: And a primer set specific to the ITS gene of M. xenopi comprising the reverse primer of SEQ ID NO: 6, a forward primer of SEQ ID NO: 7, and a primer set specific to the ITS gene of M. fortuitum comprising the reverse primer of SEQ ID NO: A primer set specific for the IS6110 gene of the Mycobacterium tuberculosis including the forward primer of SEQ ID NO: 10 and the reverse primer of SEQ ID NO: 11, a forward primer of SEQ ID NO: 12, and a 16S rRNA gene of the anti-acidic mycobacteria including the reverse primer of SEQ ID NO: Identification of a first mycobacteria comprising a primer set specific for Primer set; 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, the forward primer of SEQ ID NO: 18 and the 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 containing the reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: A set of primers for identifying a second mycobacteria comprising a primer set specific to the ITS gene of M. terrae ; Of SEQ ID NO: M.chelonae containing specific primer sets, the forward primer of SEQ ID NO: 26 and a reverse primer of SEQ ID NO: 27 in the 16S rRNA gene in M.avium containing 24 of a forward primer and a reverse primer of SEQ ID NO: 25 ITS A primer set specific for 16S rRNA gene of M. kansasii or M. gastri comprising a forward primer of SEQ ID NO: 28 and a reverse primer of SEQ ID NO: 29, a forward primer of SEQ ID NO: 32 and a primer set of SEQ ID NO: A primer set for identification of a third mycobacteria comprising a primer set specific to the ITS gene of M. gordonae comprising the reverse primer of SEQ ID NO: 33; The HSP65 of M.ulcerans containing the reverse primer of the primer set specific for SEQ ID NO: 40 of the forward primer and SEQ ID NO: 41 in the ITS of M.simiae gene containing a forward primer of SEQ ID NO: 38 and SEQ ID NO: 39 reverse primer 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 a set of primers for identifying a fourth mycobacteria comprising a primer set specific to the ITS gene of .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 ITS gene of M. marinum or M. ulcerans comprising 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 for identifying a fifth mycobacteria comprising a primer set specific for the ITS gene of M.celatum comprising a forward primer of SEQ ID NO: 56 and an inverted primer of SEQ ID NO: 57; And a primer set specific to the 16S rRNA gene of M. abscessus or M. chelonae comprising the forward primer of SEQ ID NO: 60 and the reverse primer of SEQ ID NO: 61, the forward primer of SEQ ID NO: 62 and the reverse primer of SEQ ID NO: A primer set specific for the ITS gene of M. peregrinum , 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 and a forward primer of SEQ ID NO: 66 and a primer set of SEQ ID NO: And a primer set specific for the ITS gene of M. shimoidei comprising the reverse primer of M. shimoidei .

According to an embodiment of the present invention, the kit for identifying mycobacteria comprises a primer set specific to the ITS gene of M. xenopi comprising a forward primer of SEQ ID NO: 5 and an inverted primer of SEQ ID NO: 6, a forward primer of SEQ ID NO: A primer set specific for the ITS gene of M. peregrinum including the reverse primer of SEQ ID NO: 63, a forward primer of SEQ ID NO: 10, and a primer set specific for IS6110 gene of Mycobacterium tuberculosis including the reverse primer of SEQ ID NO: A primer set for identifying a first mycobacteria comprising a primer set specific for a 16S rRNA gene of an acid-resistant Mycobacterium tuberculosis comprising a forward primer of 12; and a reverse primer of SEQ ID NO: 13; 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, the forward primer of SEQ ID NO: 18 and the 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 containing the reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: A set of primers for identifying a second mycobacteria comprising a primer set specific to the ITS gene of M. terrae ; Of SEQ ID NO: M.chelonae containing specific primer sets, the forward primer of SEQ ID NO: 26 and a reverse primer of SEQ ID NO: 27 in the 16S rRNA gene in M.avium containing 24 of a forward primer and a reverse primer of SEQ ID NO: 25 ITS A primer set specific for the gene, a forward primer of SEQ ID NO: 30 and a reverse primer of SEQ ID NO: 31, a primer set specific for the 16S rRNA gene of M. kansasii or M.gastri , a forward primer of SEQ ID NO: 7 and a primer set of SEQ ID NO: A set of primers for identification of a third mycobacteria comprising a set of primers specific to the ITS gene of M. fortuitum comprising a set of reverse primers of SEQ ID NO : The HSP65 of M.ulcerans containing the reverse primer of the primer set specific for SEQ ID NO: 40 of the forward primer and SEQ ID NO: 41 in the ITS of M.simiae gene containing a forward primer of SEQ ID NO: 38 and SEQ ID NO: 39 reverse primer A primer set specific for 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 forward primer of SEQ ID NO: 34 and an M a set of primers for identifying a fourth mycobacteria comprising a set of primers specific to the 16S rRNA gene of .gordonae ; 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: 54 and the reverse primer of SEQ ID NO: 55, M. marinum or M a primer set specific for the ITS gene of E. coli, a forward primer of SEQ ID NO: 46, and a primer set specific for the ITS gene of M. smegmatis including the reverse primer of SEQ ID NO: 47; And SEQ ID NO: 3 of the forward primer and SEQ ID NO: M.mucogenicum containing specific primer sets, the 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 ITS 4 A primer set specific for the gene, and a set of primers for identifying a sixth mycobacteria comprising a primer set specific for the ITS gene of M.celatum including the forward primer of SEQ ID NO: 56 and the reverse primer of SEQ ID NO: 57.

According to an embodiment of the present invention, the kit for identifying mycobacteria comprises a primer set specific to the ITS gene of M. xenopi comprising a forward primer of SEQ ID NO: 5 and an inverted primer of SEQ ID NO: 6, a forward primer of SEQ ID NO: A primer set specific for the ITS gene of M. peregrinum including the reverse primer of SEQ ID NO: 63, a forward primer of SEQ ID NO: 10, and a primer set specific for IS6110 gene of Mycobacterium tuberculosis including the reverse primer of SEQ ID NO: A primer set for identifying a first mycobacteria comprising a primer set specific for a 16S rRNA gene of an acid-resistant Mycobacterium tuberculosis comprising a forward primer of 12; and a reverse primer of SEQ ID NO: 13; 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, the forward primer of SEQ ID NO: 18 and the 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 containing the reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: A set of primers for identifying a second mycobacteria comprising a primer set specific to the ITS gene of M. terrae ; Of SEQ ID NO: M.chelonae containing specific primer sets, the forward primer of SEQ ID NO: 26 and a reverse primer of SEQ ID NO: 27 in the 16S rRNA gene in M.avium containing 24 of a forward primer and a reverse primer of SEQ ID NO: 25 ITS A primer set specific for the gene, a forward primer of SEQ ID NO: 30 and a reverse primer of SEQ ID NO: 31, a primer set specific for the 16S rRNA gene of M. kansasii or M.gastri , a forward primer of SEQ ID NO: 7 and a primer set of SEQ ID NO: A set of primers for identification of a third mycobacteria comprising a set of primers specific to the ITS gene of M. fortuitum comprising a set of reverse primers of SEQ ID NO : The HSP65 of M.ulcerans containing the reverse primer of the primer set specific for SEQ ID NO: 40 of the forward primer and SEQ ID NO: 41 in the ITS of M.simiae gene containing a forward primer of SEQ ID NO: 38 and SEQ ID NO: 39 reverse primer A primer set specific for 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 forward primer of SEQ ID NO: 34 and an M a set of primers for identifying a fourth mycobacteria comprising a set of primers specific to the 16S rRNA gene of .gordonae ; 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: 54 and the reverse primer of SEQ ID NO: 55, M. marinum or M a primer set specific for the ITS gene of E. coli, a forward primer of SEQ ID NO: 46, and a primer set specific for the ITS gene of M. smegmatis including the reverse primer of SEQ ID NO: 47; And SEQ ID NO: 3 of the forward primer and SEQ ID NO: M.mucogenicum containing specific primer sets, the 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 ITS 4 A primer set specific for the gene, and a set of primers for identifying a sixth mycobacteria comprising a primer set specific for the ITS gene of M.celatum including the forward primer of SEQ ID NO: 56 and the reverse primer of SEQ ID NO: 57.

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

According to another aspect of the present invention, there is provided a method of separating DNA from a specimen, Subjecting the DNA to a real-time polymerase chain reaction using the kit for identifying mycobacteria; Melting the amplification product amplified by the real-time PCR reaction while changing the temperature to obtain a melting curve; And confirming the melting temperature of the melting curve.

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

The present invention relates to a primer set for identifying mycobacteria which can accurately detect and identify mycobacteria as a specific species, a kit for identifying mycobacteria comprising the same, a real-time PCR method using the same, and a mycobacterial identification method 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.

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

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but it should be understood that the present invention is not limited thereto.

Example: Identification of Mycobacteria Using Double Real-Time Polymerase Chain Reaction and Melting Curve Analysis

1. Target site and primer design

end. Target gene

Mycobacteria of the 16S rRNA gene, hsp65 (Heat Shock Protein 65) gene, ITS (internal transcribed spacer) gene the sequence data was analyzed with Sequencher 4.10 M.gastri, M.xenopi, M.fortuitum, M.abscessus, M. M. avium , M. intracellulare , M. szulgai , M. terrae , M. chelonae , M. kansasii , M. gordonae , M. marinum , M. siciae , M. ulcerans , M. septicum , M. smegmatis , M. scrofulaceum , unusual primer M.haemophilum, M.celatum, M.peregrinum, M.mucogenicum, M.shimoidei was designed using techniques can primer3 programs.

The nucleotide sequence data were obtained from the NCBI database, and the nucleotide sequence data used in the design of 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 (A F156238.1, 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

Target gene: 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 DEG C

2) M.gastri 2

Target gene: 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 캜

3) M.xenopi

Target gene: 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 ° C

4) M. fortuitum 1

Target gene: 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 ° C

5) M. fortuitum 2

Target gene: 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 DEG C

6) M.tuberculosis complex (Mycobacterium tuberculosis)

Target 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 DEG C

7) Nontuberculous mycobacteria (anti-acid bacteria)

Target 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 DEG C

8) M.abscessus 1

Target gene: 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 ° C

9) M.abscessus 2

Target gene: 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 ° C

10) M.intracellulare

Target 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 캜 or 76.90 캜

11) M.Szulgai

Target gene: 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 ° C

12) M. terrae

Target gene: ITS (Internal transcribed spacer)

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

Reverse primer: 5'-accaccgaccaccactac-3 '(SEQ ID NO: 23)

Amplification Product Size: 80bp

Amplification product melting temperature (Tm): 82.86 ° C

13) M.avium

Target 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 DEG C

14) M.chelonae

Target gene: 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 DEG C

15) M.kansasii / M.gastri 1

Target 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 DEG C

16) M.kansasii / M.gastri 2

Target 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 ° C

17) M.gordonae 1

Target gene: 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 占 폚

18) M.gordonae 2

Target 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 DEG C

19) M. marinum / M.ulcerans 1

Target gene: 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 ° C

20) M.simiae

Target gene: 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 ° C

21) M.ulcerans

Target gene: 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 ° C

22) M. septicum

Target gene: 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 ° C

23) M. smegmatis 1

Target gene: 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 ° C

24) M. smegmatis 2

Target gene: 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 ° C

25) M. scrofulaceum 1

Target 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 DEG C

26) M. scrofulaceum 2

Target gene: 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 DEG C

27) M. haemophilum

Target gene: 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 ° C

28) M. marinum / M.ulcerans 2

Target gene: 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 DEG C

29) M.celatum

Target gene: 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 DEG C

30) M.abscessus 3

Target gene: 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 ° C

31) M.abscessus / M.chelonae

Target 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 ° C

32) M. peregrinum

Target gene: 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 ° C

33) M. mucogenicum

Target gene: 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 DEG C

34) M. shimoidei

Target gene: 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 ° C

2. Real-time polymerase chain reaction and melting curve analysis

end. Isolation of DNA

100 isolates from clinical specimens and 68 isolates from Mycobacterium 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 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, and M. xenopi KMRC 42001.

100 strains isolated from clinical specimens were cultured in liquid medium (MGIT mycobacteria medium) or solid medium (Ogawa medium). The ATCC and KCTC strains were cultured in liquid medium and KMRC were cultured in solid medium.

The DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the cultured MGIT mycobacterial culture tubes well, 500 μl of the liquid medium was 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 sterilized distilled water was added to the remaining portion of the needle, and the mixture was heated in boiling water for 10 minutes. After heating in a hot water bath, the supernatant was centrifuged at 14,000 rpm for 5 minutes, and the supernatant was used as template DNA for the polymerase chain reaction.

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

I. Primers mix

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

1) Tube I

 a. Tube I-A

M.gastri 1 primer 10 pmole / l

9 pmole / l of M. xenopi primer

M.fortuitum 1 primer 6 pmole / l

M.tuberculosis complex primer 14 pmole / l

Nontuberculous mycobacteria primer 8 pmole / l

 b. Tube I-B

M.gastri 2 primer 8 pmole / l

9 pmole / l of M. xenopi primer

M.fortuitum 1 primer 6 pmole / l

M.tuberculosis complex primer 14 pmole / l

Nontuberculous mycobacteria primer 8 pmole / l

 c. Tube I-C

9 pmole / l of M. xenopi primer

M. peregrinum primer 12 pmole / l

M.tuberculosis complex primer 14 pmole / l

Nontuberculous mycobacteria primer 8 pmole / l

2) Tube II

 a. Tube II-A

M.abscessus 1 primer 10 pmole / l

10 pmole / l of M. intracellulare primer

M.szulgai primer 10 pmole / l

10 pmole / l of M. terrae primer

 b. Tube II-B

M.abscessus 2 primer 12 pmole / l

10 pmole / l of M. intracellulare primer

M.szulgai primer 10 pmole / l

10 pmole / l of M. terrae primer

3) Tube III

 a. Tube III-A

10 pmole / l of M. avium primer

M.chelonae primer 14 pmole / l

M. kanasii / M.gastri 1 primer 8 pmole / l

M.gordonae 1 primer 10 pmole / l

 b. Tube III-B

10 pmole / l of M. avium primer

M.chelonae primer 14 pmole / l

M. kanasii / M.gastri 2 primer 8 pmole / l

M. fortuitum 2 primer 10 pmole / l

4) Tube IV

 a. Tube IV-A

10 pmole / l of M. marinum / M.ulcerans 1 primer

M.simiae primer 10 pmole / l

12 ul / l of M.ulcerans primer

10 pmole / l M.septicum primer

Msmegmatis 1 primer 8 pmole / l

 b. Tube IV-B

M.simiae primer 10 pmole / l

12 ul / l of M.ulcerans primer

10 pmole / l M.septicum primer

Msmegmatis 1 primer 8 pmole / l

 c. Tube IV-C

M.simiae primer 10 pmole / l

12 ul / l of M.ulcerans primer

10 pmole / l M.septicum primer

M.gordonae 2 primer 10 pmole / l

5) Tube V

 a. Tube V-A

M. scrofulaceum 1 primer 12 pmole / l

M. scrofulaceum 2 primer 12 pmole / l

10 pmole / l of M. haemophilum primer

8 pmole / l of M.celatum primer

 b. Tube V-B

M. scrofulaceum 1 primer 12 pmole / l

10 pmole / l of M. haemophilum primer

10 pmole / l of M. marinum / M.ulcerans 2 primer

8 pmole / l of M.celatum primer

 c. Tube V-C

M. scrofulaceum 1 primer 12 pmole / l

10 pmole / l of M. marinum / M.ulcerans 2 primer

M.smegmatis 2 primer 10 pmole / l

6) Tube VI

 a. Tube VI-A

M.abscessus 3 primer 10 pmole / l

10 pmole / l of M. peregrinum primer

Mmucogenicum primer 10 pmole / l

4 pmole / l of M. shimoidei primer

 b. Tube VI-B

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

10 pmole / l of M. peregrinum primer

Mmucogenicum primer 10 pmole / l

4 pmole / l of M. shimoidei primer

 c. Tube VI-C

M.gastri 2 primer 10 pmole / l

Mmucogenicum primer 10 pmole / l

8 pmole / l of M.celatum primer

All. Double Real-Time Polymerase Chain Reaction and Melting Curve Analysis

Real-time PCR 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 with one cycle of denaturation at about 95 ° C for about 5 minutes, denaturation at about 95 ° C for about 10 seconds, annealing at about 60 ° C for about 15 seconds, and extension at one cycle. After the final polymerase chain reaction, the fluorescence wavelength of 510 nm was measured while increasing the temperature from about 68 ° C to about 93 ° C at a rate of about 0.2 ° C per second, and the melting curve was analyzed. At this time, the composition of the reactant to perform double real-time PCR is shown in Table 1 below.

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

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

≪ Example 1 >

A, Tube III-A, Tube IV-A, Tube V-A and Tube VI-A are used to identify mycobacteria. Mycobacteria were identified according to the real-time PCR and melting curve analysis described above.

≪ Example 2 >

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 real-time PCR and melting curve analysis described above.

≪ Example 3 >

This is a method for identifying mycobacteria using the primer mixes 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 real-time PCR and melting curve analysis described above.

la. Real-time polymerase chain reaction and melting curve analysis

≪ Example 1 >

FIGS. 1 to 5 show amplification products obtained by real-time PCR of M. fortuitum , M. gastri , Nontuberculous mycobacteria, M. tuberculosis complex, and M. xenopi using a primer mix of Tube I- ). ≪ / RTI >

6 to 9 are respectively using the primer mix of the tube Ⅱ-A M.abscessus, M.intracellulare, M. szulgai and the melting curve of the amplified product (amplicon) is obtained by performing a real-time polymerase chain reaction of M.terrae The results of the analysis are shown.

10 to 13 show the melting curves of the amplicons obtained by real-time PCR of M. avium , M. chelonae , M. gordonae and M.kansasii using the primer mix of Tube III-A, respectively The results of the analysis are shown.

14 to 18 are each tube Ⅳ-A using a primer mix of M.marinum, M.septicum, M.simiae, to perform a real-time polymerase chain reaction of M.smegmatis and M.ulcerans obtained amplification product (amplicon ). ≪ / RTI >

19 to 21 show the results of melting curve analysis of an amplicon obtained by real-time PCR of M.celatum , M. haemophilum and M. scrofulaceum using a primer mix of Tube V-A, respectively .

22 to 25 show the melting curves of amplicons obtained by real-time PCR of M.abscessus , M. mucogenicum , M. peregrinum and M. shimoidei using the primer mix of Tube VI-A, The results of the analysis are shown.

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

Since Figure 1 and to Figure 2, using a primer mix of the tube-A Ⅰ perform real-time polymerase chain reaction (PCR), each with a peak (about 87.54 ℃) for the acid rain wherein Mycobacterium tuberculosis (Nontuberculous mycobacteria) M. amplification product melting temperature (Tm) peak corresponding to fortuitum (about 81.70 ° C) and M.gastri (about 75.06 ° C). In the case of M.xenopi , however, only the amplification product melting temperature (Tm) peak (about 80.86 ° C) corresponding to M. acidophilus mycobacteria was observed (see FIG. 5).

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

21, since real-time PCR is performed using a primer mix of Tube V-A (including a primer set of M. scrofulaceum 1 and M. scrofulaceum 2), in the case of a strain of M. scrofulaceum , (Tm) peak (about 77.50 占 폚), and the amplification product melting temperature (Tm) peak corresponding to the primer set of M. scrofulaceum 1 and M. scrofulaceum 2 all corresponded to about 77.50 占 폚, .

In Example 1, a primer set capable of detecting M. abscessus by detecting M. abscessus in Tube II-A ( M.abscessus 1) and Tube VI-A ( M.abscessus 3) was provided . it is possible to identify abscessus . In addition, it was confirmed that M. scrofulaceum 1 and M. scrofulaceum 2 were contained in Tube V-A, and M. scrofulaceum could be identified in a clinical sample.

Through the first embodiment, whether Mycobacterium tuberculosis Mycobacterium tuberculosis that the anti-acid rain, acid rain, wherein Mycobacterium tuberculosis Among 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 , respectively.

≪ Example 2 >

Figures 26 to 30 show the amplification products obtained by real-time polymerase chain reaction of M. fortuitum , M. gastri , Nontuberculous mycobacteria, M. tuberculosis complex and M. xenopi using primer mix of Tube I-B, respectively ). ≪ / RTI >

31 to 34 show the melting curves of amplicons obtained by real-time PCR of M.abscessus , M. intracellulare , M. szulgai and M. terrae using primer mixes of tubes II-B, respectively. The results of the analysis are shown.

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

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

Figure 43 to 47 is each tube Ⅴ-B by using a primer mix of the obtained by performing a real-time polymerase chain reaction of M.celatum, M.haemophilum, M.scrofulaceum, M.marinum and M.ulcerans amplification product (amplicon ). ≪ / RTI >

Figure 48 to Figure 52, each using a primer mix of the tube-B Ⅵ M. abscessus, M.chelonae, M.mucogenicum, M.peregrinum and amplification products obtained by performing a real-time polymerase chain reaction of M.shimoidei (amplicon ). ≪ / RTI >

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

Referring to FIG. 26 and FIG. 27, since the real-time PCR reaction is performed using the primer mix of Tube I-B, the PCR reaction is carried out with a peak corresponding to Nontuberculous mycobacteria (about 87.54 ° C.) . amplification product melting temperature (Tm) peak corresponding to fortuitum (about 81.70 ° C) and M.gastri (about 78.10 ° C). 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. 30).

46 and 47, a real-time PCR was carried out using a primer mix of Tube V-B, and the amplification product melting temperature (Tm) peak corresponding to M. marinum / M.ulcerans 2 (about 83.56 ° C), M. marinum or M. ulcerans showed amplification product melting temperature (Tm) peaks corresponding to about 83.56 ° C. In this case, polymerase chain reaction was performed using the primer mix of Tube VI-B, and compared with the results of the melting curve analysis, the amplification product melting temperature (Tm) peak corresponding to M. ulcerans 79.10 < 0 > C), the strain can be identified with M. ulcerans , and when the peak does not appear, the strain can be identified with M. marinum . In addition, real-time PCR was performed using a primer mix of Tube V-B to reveal an amplification product melting temperature (Tm) peak (about 83.56 ° C) corresponding to M. marinum / M.ulcerans 2, using a mix of primers B performing the polymerase chain reaction, if carried out by the melting curve analysis of the amplification products receive the melting temperature (Tm) peak (about 79.10 ℃) for the M.ulcerans, M.marinum in the sample and It can be identified as having M. ulcerans .

While having the above-described primer set can each be distinguished by detecting the M.abscessus, M.chelonae in Example 2-B Ⅱ tube (M.abscessus 2) and the tube Ⅲ-A (M.chelonae) in the tube Ⅵ- to B it can not distinguish M.abscessus M.chelonae and is not provided with a high specificity M.abscessus / M.chelonae for M.abscessus or M.chelonae, effectively identifying the M.abscessus or M.chelonae from clinical specimens I can confirm that I can do it.

Conducted through a second example, if M. tuberculosis M. tuberculosis that the anti-acid rain, acid rain from wherein Mycobacterium tuberculosis is 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 , respectively.

≪ Example 3 >

53 to 56 show the melting curves of amplicons obtained by real-time PCR of M. xenopi , M. peregrinum , Nontuberculous mycobacteria and M. tuberculosis complex using primer mix of Tube I-C, respectively The results of the analysis are shown.

Figures 57 to 60 show the melting curves of amplicons obtained by real-time PCR of M. abscessus , M. intracellulare , M. szulgai and M. terrae using primer mixes of tubes II-B, respectively The results of the analysis are shown.

61 to 64 show the melting curves of amplicons obtained by real-time PCR of M. avium , M. chelonae , M. fortuitum and M.kansasii using primer mix of Tube III-B, respectively The results of the analysis are shown.

65 to 68 show the melting curves of amplicons obtained by real-time polymerase chain reaction of M. septicum , M. s.miae , M. gordonae and M. ulcerans using primer mix of Tube IV-C, respectively The results of the analysis are shown.

69 to 71 show the results of melting curve analysis of an amplicon obtained by real-time PCR of M. scrofulaceum , M. marinum and Ms. megmatis using a primer mix of Tube V-B, respectively .

72 to 74 show the results of the melting curve analysis of amplicons obtained by real-time PCR of M. gastri , M. mucogenicum and M.celatum using primer mix of Tube VI-B, respectively .

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

54, since a real-time PCR reaction is performed using a primer mix of Tube I-C, a peak corresponding to Nontuberculous mycobacteria (about 87.54 ° C) and M. peregrinum (about 81.70 ° C (Tm) peak corresponding to the amplification product melting temperature (Tm). 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. 53).

Carried through Examples 3, wherein that the acid rain that the M. tuberculosis M. tuberculosis, M. tuberculosis are among the anti-acid rain 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. s . Mimae , M. ulcerans , M. septicum , M. smegmatis , M. scrofulaceum , M.celatum , M. peregrinum and M. mucogenicum It can be confirmed that it can be identified effectively.

In the case of Example 3, unlike Examples 1 and 2, it has a constitution which does not include a set of primers for identifying mycobacteria that can be identified for M. haemophilum and M. shimoidei . Since M. haemophilum and M. shimoidei are not well expressed in clinical specimens in Korea, they are included in consideration of reduction in identification efficiency due to interaction between primer sets when a primer set for identifying the strain is included It is designed with no configuration.

According to the above-described embodiments, when real-time PCR and melting curve analysis are performed, it is confirmed that the mycobacteria can be identified as a specific strain with high reliability in a clinical sample in a simple, accurate, quick, and inexpensive manner 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> DP-2011-0063 <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 accaccac 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 gene (ITS) 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 gene (ITS) 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          ITS (Internal transcribed spacer) 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          ITS (Internal transcribed spacer) 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          ITS (Internal transcribed spacer) 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          ITS (Internal transcribed spacer) 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 (7)

delete delete A primer set specific to the ITS gene of M.gastri including the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, a forward primer of SEQ ID NO: 5, and an ITS gene of M. xenopi comprising the reverse primer of SEQ ID NO: A 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: 14 and the reverse primer of SEQ ID NO: 15, the forward primer of SEQ ID NO: 18, and the 16S rRNA of M. intracellulare containing the reverse primer of SEQ ID NO: 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 containing the reverse primer of SEQ ID NO: 21, and a forward primer of SEQ ID NO: 22 and a reverse primer of SEQ ID NO: A set of primers for identifying a second mycobacteria comprising a primer set specific to the ITS gene of M. terrae ;
Of SEQ ID NO: M.chelonae containing specific primer sets, the forward primer of SEQ ID NO: 26 and a reverse primer of SEQ ID NO: 27 in the 16S rRNA gene in M.avium containing 24 of a forward primer and a reverse primer of SEQ ID NO: 25 ITS A primer set specific for 16S rRNA gene of M. kansasii or M. gastri comprising a forward primer of SEQ ID NO: 28 and a reverse primer of SEQ ID NO: 29, a forward primer of SEQ ID NO: 32 and a primer set of SEQ ID NO: A primer set for identification of a third mycobacteria comprising a primer set specific to the ITS gene of M. gordonae comprising the reverse primer of SEQ ID 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 in the ITS of the gene or M.marinum M.ulcerans comprising a reverse primer of SEQ ID NO: 36 and a reverse primer of SEQ ID NO: 37 a primer set specific for the ITS gene of simiae , a forward primer of SEQ ID NO: 40 and a primer set specific for HSP65 of M. ulcerans comprising the reverse primer of SEQ ID NO: 41, a forward primer of SEQ ID NO: 42 and a reverse primer of SEQ ID NO: fourth mycobacterial identification comprising a set of primers specific to the gene of the ITS M.smegmatis comprising a specific primer set and the forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45 in the ITS of the gene containing the M.septicum Primer set;
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, a forward primer of SEQ ID NO: 50, and an ITS of M. scrofulaceum containing the reverse primer of SEQ ID NO: A primer set specific for the gene, a forward primer of SEQ ID NO: 52 and a reverse primer of SEQ ID NO: 53, a primer set specific for the ITS gene of M. haemophilum including the reverse primer of SEQ ID NO: 53, a forward primer of SEQ ID NO: 56 and a reverse primer of SEQ ID NO: 57 A set of primers for identifying a fifth mycobacteria comprising a primer set specific to the ITS gene of M.celatum ; And
A primer set specific for the ITS gene of M. abscessus comprising the forward primer of SEQ ID NO: 58 and the reverse primer of SEQ ID NO: 59, the forward primer of SEQ ID NO: 62, and the ITS gene of M. peregrinum including the reverse primer of SEQ ID NO: A forward primer of SEQ ID NO: 64, a reverse primer of SEQ ID NO: 66, a primer set specific to the ITS gene of M. mucogenicum comprising the reverse primer of SEQ ID NO: 65, a forward primer of SEQ ID NO: 66 and a reverse primer of SEQ ID NO: 67 Characterized in that it comprises a primer set for identification of a sixth mycobacteria comprising a primer set specific to the ITS gene of M. shimoidei . delete delete [Claim 4] The kit for identifying mycobacteria according to claim 3, 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 a kit for identifying mycobacteria according to claim 3;
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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