Mycobacterium abscessus coding genes and uses thereof

Abstract

The present application relates to a kind of abscess mycobacteroides Mycobacteroides abscessus subsp abscessus ATCC 19977 sequence-specific coding gene MAB_3719c (-|3774251-3776008|), it can be used as abscess mycobacteroides complex group molecular identification molecular marker gene, for the molecular identification and clinical detection of abscess mycobacteroides complex group.

Description

Technical Field

[0001] This invention relates to the field of gene detection, specifically to the identification of pathogenic bacterial species. Background Technology

[0002] In recent years, non-tuberculous mycobacterial diseases (NTM) have shown a rapid growth trend, becoming a serious public health problem threatening human health globally, with both prevalence and incidence rates increasing year by year. A study in Japan showed that the incidence rate of NTM in Japan rose from 4.6 / 100,000 in 2001 to 10.1 / 100,000 in 2009, an increase of nearly 2.2 times. In South Korea, the incidence rate of NTM was 6.0 / 100,000 in 2008, but this figure rose to 19 / 100,000 by 2016. According to the "Guidelines for the Diagnosis and Treatment of Non-tuberculous Mycobacterial Diseases (2020 Edition)," although there is currently no large-scale epidemiological survey data on NTM in my country, relevant tuberculosis epidemiological survey data show that the isolation rate of NTM increased from 11.1% in 2000 to 22.9% in 2010, which also reflects a significant upward trend in NTM in my country.

[0003] NTM infection can cause damage to organs such as the lungs, lymph nodes, skin and soft tissues, and joints. As the most common form of NTM, approximately 75%-94% of patients with NTM present with lung lesions. Because the clinical manifestations and chest imaging of NTM lung disease closely resemble tuberculosis, and acid-fast bacilli can be found in sputum, NTM disease can be misdiagnosed as tuberculosis or bronchiectasis for a long time without bacterial identification.

[0004] Mycobacteroides abscessus complex (MABC) is a rapidly growing nontuberculous mycobacterium (RGM) that causes infectious diseases in humans and is an important NTM pathogen causing lung infections. It is further divided into three subspecies: *Mycobacterium abscessus* subsp. *abscessus*, *Mycobacterium abscessus* subsp. *massiliense*, and *Mycobacterium abscessus* subsp. *Bolletii*. In the United States, MABC accounts for 65%–80% of pathogenic RGM. In South Korea, lung diseases caused by MABC account for 70%–80% of lung diseases caused by RGM.

[0005] MABC (Multiple Organ Dysplastic Syndromes) are naturally resistant to first-line anti-tuberculosis drugs and are largely ineffective against common antibiotics. Treatment typically involves multi-drug regimens centered around macrolide antibiotics such as clarithromycin. Although many NTM (non-tumor inflammatory marker) strains exhibit strong biological and pathological similarities, different species show varying drug susceptibility, resulting in a low clinical cure rate. More seriously, MABC infections are often misdiagnosed as tuberculosis, and prolonged inappropriate drug use exacerbates drug resistance, leading to treatment failure. Therefore, MABC infection, due to its high drug resistance rate, numerous adverse drug reactions, long treatment duration, and poor prognosis, is one of the most difficult infections to treat. Early identification of the bacterial species and drug susceptibility testing are crucial for improving the cure rate of MABC lung disease and preventing drug resistance.

[0006] Currently, there are three main types of methods for identifying MABC: 1) Direct homologous gene sequence comparison: This method identifies pathogenic bacteria to the species level by analyzing differences in homologous DNA sequences. Commonly used sequences include the ITS region of 16S rDNA, 16S-23S rRNA, the β subunit of RNA polymerase (rpoB), and heat shock protein 65 (HSP65). However, its single-sequence identification ability is insufficient, and it is often necessary to use multiple homologous sequences in combination for species identification. 2) Indirect homologous gene sequence comparison: This method designs single nucleotide polymorphism (SNP) probes targeting specific homologous gene sequences and labels the probes on a solid matrix (chip). The species are identified by the binding of the probe to the target sequence. There are currently commercially available gene chip kits based on this method, but they are difficult to distinguish between MABC and Mycobacterium chelonae, and require a laser confocal scanner, making the operation relatively complex. 3) Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Due to the high similarity among MABC, Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium fortuitum complex, some NTM bacteria can only be distinguished at the complex level. Strains within a complex cannot be reliably identified by a single gene and require combined analysis of multiple gene loci.

[0007] In summary, current strategies for identifying Mycobacterium abscessus complex (MABC) suffer from problems such as complex operation, cumbersome procedures, low specificity, and high cost. Therefore, establishing a rapid, accurate, and inexpensive method for detecting Mycobacterium abscessus complex and effectively identifying the infecting bacteria in MABC is the prerequisite and foundation for the diagnosis and treatment of MABC-related lung diseases, and also a new task that clinical laboratories urgently need to accomplish in detecting Mycobacterium abscessus. Summary of the Invention

[0008] One object of the present invention is to provide an isolated nucleotide that is a sequence-specific coding gene for Mycobacterium abscessus (ATCC 19977), the gene being MAB_3719c(-|3774251-3776008|), whose sequence information can be used as a barcode molecular marker for Mycobacterium abscessus complex to identify Mycobacterium abscessus complex, and its sequence is shown in SEQ ID NO.1.

[0009] Other objects of the present invention include providing specific PCR primers for amplifying the above-mentioned coding genes and providing a detection method for detecting or identifying the presence of Mycobacterium abscessis complex in a sample; the present invention also provides detection kits related to the above-mentioned coding genes and applications of the above-mentioned genes.

[0010] According to one aspect of the present invention, comparative genomics analysis revealed a protein-coding sequence with significant sequence differences within ATCC 19977, which effectively distinguishes MABC from other NTM species. This gene is a Mycobacterium abscessus ATCC 19977MAB_3719c(-|3774251-3776008|), and NCBI-BLASTP analysis showed that this gene is a presumptive functional protein with an unclear specific function, exhibiting low sequence homology in GenBank. Comparative genomics studies have demonstrated that this gene sequence can differentiate Mycobacterium abscessus complex (MABC) strains from other NTM species.

[0011] Specifically, primers capable of specifically amplifying the MAB_3719c(-|3774251-3776008|) gene of MABC were designed, which are the primers proposed in this invention, and the primer sequences are as follows:

[0012] F:5'-ACGTGGGGGGATTGGCCCGGATCTA-3';

[0013] R:5'-TGTAAAGCCCGAAGCAGATACCGAC-3'.

[0014] MABCs can be quickly and accurately identified based on the presence or absence of the PCR product of the gene's DNA sequence in the sample being tested, or by differences in the DNA sequence.

[0015] According to another aspect of the present invention, based on the novel standard coding gene of Mycobacterium abscessus ATCC 19977, the present invention specifically establishes a method for detecting or identifying Mycobacterium abscessus complexes, the steps of which are as follows:

[0016] (1) Isolate and extract genomic DNA from the sample to be tested;

[0017] (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the following primers:

[0018] F: 5'-ACGTGGGGGGATTGGCCCGGATCTA-3' (SEQ ID NO. 3);

[0019] R: 5'-TGTAAAGCCCGAAGCAGATACCGAC-3' (SEQ ID NO. 4).

[0020] (3) The DNA product amplified in step (2) was analyzed by gel electrophoresis. The size was consistent with the theoretical size.

[0021] (4) Sequencing analysis was performed on the DNA product amplified in step (2), and it was compared with the barcode gene MAB_3719c(-|3774251-3776008|). If the homology was greater than 90%, the sample to be tested was determined to contain Mycobacterium abscessus complex.

[0022] Furthermore, based on the DNA barcoding principle, the above detection method initially performs electrophoretic analysis on the PCR products. If the test strain does not have the target band, it indicates that the strain is not MABC; if there is a band, further sequencing verification can be performed. The sequenced sequence is compared and aligned with the standard sequence of MAB_3719c(-|3774251-3776008|) to obtain the similarity between the sequences. If the sequence homology is greater than 99%, the strain can be determined to be possibly MABC. Based on the clustering of the DNA barcoding sequence of the test strain with the standard sequence, MABC can be distinguished from Mycobacterium tuberculosis H37Rv, other NTM bacteria, and common respiratory pathogens.

[0023] This detection method can be used for species identification studies of the Mycobacterium abscessus complex, as well as for rapid clinical testing. The sample to be tested can be MABC strains, H37Rv strains, other NTM strains, and other common respiratory pathogens.

[0024] Based on the above method, the present invention also provides a detection kit. The kit container contains reagents for detecting the ATCC 19977 standard coding gene of Mycobacterium abscessus, and may also include manufacturing, use, and sales information of the drug or biological product approved by a drug regulatory authority. For example, reagents for directly detecting the MAB_3719c(-|3774251-3776008|) gene in a sample after PCR amplification may contain one or more of the following: amplification primers, dNTPs, DNA polymerase for PCR reaction and its buffer, reagents required for enzyme digestion and / or sequencing reactions, etc. Those skilled in the art will know that the above components are merely illustrative; for example, the primers may be the specific PCR primers described above, and the DNA polymerase for PCR reaction may be an enzyme capable of PCR amplification. The detection of the coding gene of the present invention can also be provided in an integrated manner, such as a gene chip.

[0025] Beneficial Effects: This invention provides a standard gene and molecular identification method for the molecular identification of Mycobacterium abscessus complex (MABC). This gene can effectively distinguish MABC from Mycobacterium tuberculosis H37Rv and other NTM bacteria. The identification method using this gene overcomes the shortcomings of existing Mycobacterium abscessus identification processes, such as primer design multiplicity and poor result reproducibility. It has the characteristics of being universal, easy to amplify, and easy to compare, and can accurately identify this group from other closely related NTM bacteria or other respiratory tract infection bacteria. It provides a powerful technical means and research tool for tuberculosis epidemiological surveys and rapid diagnosis and identification of clinical tuberculosis patients. Attached Figure Description

[0026] Figure 1 DNA sequence alignment of MAB_3719c (-|3774251-3776008|) with H37Rv; DNA sequence alignment (Coverage: 98%, Identity: 77.29%). The upper sequence is Mycobacterium abscessus ATCC 19977, and the lower sequence is Mycobacterium tuberculosis H37Rv.

[0027] Figure 2 : Correspondence diagram of the DNA sequence encoding protein sequence of MAB_3719c;

[0028] Figure 3Amino acid sequence alignment of MAB_3719c (-|3774251-3776008|) with H37Rv; AA sequence alignment (Coverage: 97%, Identity: 79.44%). The upper sequence is Mycobacterium abscessus ATCC 19977, and the lower sequence is Mycobacterium tuberculosis H37Rv.

[0029] Figure 4 Comparison of gene sequence homology between MAB_3719c(-|3774251-3776008|) and other NTM standard strains;

[0030] Figure 5 The PCR amplification product of the MAB_3719c(-|3774251-3776008|) specific primer can distinguish Mycobacterium abscessus from Mycobacterium tuberculosis and other NTM strains;

[0031] Figure 6 The PCR amplification products of the MAB_3719c(-|3774251-3776008|) specific primers can distinguish Mycobacterium abscessus from 16 other respiratory tract infection pathogens; the specific information of each lane sample is shown in Table 1.

[0032] Figure 7 The PCR amplification product of the MAB_3719c(-|3774251-3776008|) specific primer can identify clinically isolated Mycobacterium abscesses.

[0033] Figure 8 Comparison of PCR amplification and sequencing results of the MAB_3719c(-|3774251-3776008|) gene with standard sequences;

[0034] Figure 9 The positional relationship between the primers and the MAB_3719c(-|3774251-3776008|) gene is shown in single underline, with the MAB_3719c gene sequence represented by a gray background. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, but this does not limit the scope of the claims. All reagents used in this invention are commercially available.

[0036] Example 1: Identifying a specific segment of the ATCC 19977 MAB_3719c gene

[0037] 1.1 Comparative genomics reveals MABC sequence-specific genes

[0038] First, the genome sequences of *M. abscessus subsp. abscessus ATCC 19977* (GCF_004028015.1_ASM402801v1_genomic) and *M. tuberculosis H37Rv* (GCF_000195955.2_ASM19595v2_genomic) were downloaded from NCBI. Comparative genomic analysis revealed significant differences between the two genome sequences in the MAB_3719c(-|3774251-3776008|) region, with a sequence coverage of 98% and a sequence similarity of 77.29%. The DNA sequence alignment results of *M. abscessus subsp. abscessus ATCC 19977* and *M. tuberculosis H37Rv* in the MAB_3719c(-|3774251-3776008|) region are as follows: Figure 1 As shown.

[0039] Therefore, the sequence of the MAB_3719c(-|3774251-3776008|) region of the above gene was extracted, and the following sequence was obtained:

[0040] ATGTCATCACCGGCAGCAGTCCCTGTAAAGCCCGAAGCAGATACCGACTTT GACGTACTGATCGTCGGATCCGGATTCGGCGGAAGCGTCACCGCCATGCGCCTGACCGAGAAGGGTTATCGCGTAGGTGTCCTGGAGGCGGGCCGACGGT TCGCGGACGAGGAATTCGCCGAGACCTCATGGGATCTGCGCAAGTTCCTAT GGGCGCCGATGTTCAAGTGCTTCGGCATCCAGCGGATCCACCTGCTGAGCAACTGCATGATCCTGGCCGGTGCCGGGGTGGGTGGAGGCTCGCTGAACTA CGCCAACACCCTCTACGTGCCACCGGACCCGTTCTTCAACGATCCGCAGTGGAAGGGCATCACCGACTGGCGCGCCGAACTCTCACCCCACTATGAGCAG GCCCAGCGGATGCTCGGTGTGGTGAAGAACCCCACCTTCACCGACGCCGACCGGCTCATCAAGGAGGTTGCCGACGACATGGGTGCCGGCGACACCTTTG TCGCGACACCAGTGGGTGTGTACTTCGGGCCCGACGGCACCAAGACCCCT GGCGTCAAAGTTCCCGATCCATACTTCGGCGGGGCCGGACCCGACCGGGTGGGCTGCACCGAGTGTGGATCCTGCATGACCGGCTGCCGGGTCGGCGCCA AGAACACTCTGGTCAAGAACTACCTGGGACTGGCTGAATCCAATGGTGCCCAGGTCATTCCGCTCACCACCGTGACCGCGGTGCAGCAGGGCGCGGACGG TGTCTGGCGCGTCTCCACCAAGTCCACCGGCCGTTGGGTGCGTAAGCAGC GCAAGACCTACACGGCCAAGTACGTGGTGTTGGCCGCCGGCACCTGGGGCACGCAGAACCTGCTGTTCAAGATGAAGGACAAGGGGTTGCTGCCCAAGCTGTCACAGCGGCTGGGCGTGCTGACCCGCACAAACTCCGAATCCATCGTCGGCGCGGGCCGGTTGGAGTACAAGGACGACCTGGACCTCACCCACGGTGT GGCCATCACGTCTTCCTTCCACCCGACCAGCGACACCCATATCGAGCCGGT CCGTTACGGCAAGGGCTCCAACGCGATGGGGCTGCTGCAGACCCTGATGACCGACGGCGATGGGCCGAAGTCGAGGTGGCGCCAGCTTATTGAGAACGCC CGTGCCGACTGGCGGGGCACGTTCCGGATGTTCAACGTGACGCAGTGGAGCGAGCGCACCGTTATCGCGCTGGTCATGCAGCACCTGGACAACTCGATCA CCACCTTCACCAAGAAGACGTTGTTCTGGCGGCGTCTGGACAGCAAGCAA GGCCACGGGGAGCCCAACCCCACCTGGATTCCCGCGGGTAACGAGGCCACGCGGCGCCTGGCCGCCAAGATCGACGGCGTTGCCGGCGGCACCTGGGGC GAGCTGTTCAACATTCCGCTGACCGCGCACTTCCTGGGCGGTGCCGTGATCGGTACCAGCCCGGAGAACGGCGTCATCGACCCCTACCACCGGGTGTACGG CTACCCGACCATGTACGTGGTGGACGGAGCGTCGATCTCGGCCAACCTGG GCGTCAACCCGTCACTGAGCATTACCGCACAGGCCGAGCGTGCGGCGTCGTTGTGGCCCAACAAGGGTGAGACGGATCTGCGTCCGGAGCAGGGCCTGCC GTATCAGCGGATGGCCCCGGTGCCCCCGGTGCACCCGGTGGTCCCCGCCG ATGCCCCGGGGGCGCTGCGGCGTCTGCCGATCGAACCGGTGAGCTCGGCCAGTTAG(SEQ ID NO.1)

[0041] The correspondence between the DNA sequence and the encoded amino acid sequence is shown in Figure 2 below.

[0042] Further analysis revealed that its DNA sequence (SEQ ID NO.1) is 1,758 bp in length, encoding 585 amino acids, with a theoretical molecular weight of 63.45 kDa, which is the MAB_3719c(-|3774251-3776008|) gene. The amino acid sequence of the theoretically encoded product of this gene is shown in SEQ ID NO.2:

[0043] MSSPAAVPVKPEADTDFDVLIVGSGFGGSVTAMRLTEKGYRVGVLEAGRRFA DEEFAETSWDLRKFLWAPMFKCFGIQRIHLLSNCMILAGAGVGGGSLNYANTLYVPPDPFFNDPQWKGITDWRAELSPHYEQAQRMLGVVKNPTFTDADRLIKE VADDMGAGDTFVATPVGVYFGPDGTKTPGVKVPDPYFGGAGPDRVGCTECGSCMTGCRVGAKNTLVKNYLGLAESNGAQVIPLTTVTAVQQGADGVWRVSTK STGRWVRKQRKTYTAKYVVLAAGTWGTQNLLFKMKDKGLLPKLSQRLGVL TRTNSESIVGAGRLEYKDDLDLTHGVAITSSFHPTSDTHIEPVRYGKGSNAMGLLQTLMTDGPKSRWRQLIENARADWRGTFRMFNVTQWSERTVIALVMQ HLDNSITTFTKKTLFWRRLDSKQGHGEPNPTWIPAGNEATRRLAAKIDGVAGGTWGELFNIPLTAHFLGGAVIGTSPENGVIDPYHRVYGYPTMYVVDGASISAN LGVNPSLSITAQAERAASLWPNKGETDLRPEQGLPYQRMAPVPPVHPVVPAD APGALRRLPIEPVSSAS*(SEQ IDNO.2)

[0044] The amino acid sequence of the theoretical gene-encoded product shown in SEQ ID NO.2 was compared with that of H37Rv, and the sequence matched the protein Rv3409c in H37Rv. Rv3409c is cholesterol oxidase ChoD. The sequence coverage of the two proteins was 97%, and the similarity was also low at 79.44% (see [link to original text]). Figure 3 This indicates that the gene MAB_3719c(-|3774251-3776008|) also differs significantly from H37Rv at the protein level.

[0045] We performed a comparative genome-wide BLAST analysis of the DNA sequence of the MAB_3719c(-|3774251-3776008|) gene, such as... Figure 4 As shown, the results indicate that the MAB_3719c gene sequence is significantly different from other NTM bacteria, with low sequence similarity, which can distinguish MABC from H37Rv and other NTM bacteria.

[0046] Example 2: Establishing a method for identifying MABC complex groups

[0047] (1) Primer design:

[0048] Based on the CDS sequence of the MAB_3719c(-|3774251-3776008|) gene as shown in SEQ ID NO.1, PCR primers were designed using Oligo 7.0. The primer sequences are as follows:

[0049] F: 5'-ACGTGGGGGGATTGGCCCGGATCTA-3' (SEQ ID NO. 3);

[0050] R: 5'-TGTAAAGCCCGAAGCAGATACCGAC-3' (SEQ ID NO. 4).

[0051] The positional relationship between the above primers and the MAB_3719c(-|3774251-3776008|) gene is as follows: Figure 9 As shown, the subscripts for the primer positions are underlined, and the MAB_3719c gene sequence is represented by a gray background.

[0052] (2) Total DNA was extracted from the test strains, including M. abscessus subsp. abscessus ATCC 19977. All strains underwent 16S rRNA gene sequencing, alignment, and NCBI sequence submission. The test strains are shown in Table 1.

[0053] Table 1. Selected relevant strains

[0054]

[0055]

[0056]

[0057] (3) Amplify the DNA fragment and perform polymerase chain reaction (PCR) using the above-mentioned F / R primers.

[0058] The PCR system (25 μL) consisted of dd H2O (9.5 μL), 2×Taq PCR MasterMix (TIANGEN, 12.5 μL), primer F (10 μM, 1 μL), primer R (10 μM, 1 μL), and DNA template (1 μL).

[0059] Amplification program: 95℃ pre-denaturation for 4 min, 95℃ denaturation for 1 min, 60℃ annealing for 1 min, 72℃ extension for 90 s, for a total of 35 cycles, followed by 72℃ extension for 10 min.

[0060] (4) Electrophoretic detection of amplification products: Electrophoresis was performed on a 1.5% agarose gel in 1×TBE buffer. Results are as follows: Figure 5 As shown, MABC showed an amplified band at 1,253 bp, and the amplification result was consistent with expectations. However, other NTM strains did not amplify bands at the expected position.

[0061] (5) Figure 6 As shown, MABC and the positive control (collected clinical isolates of Mycobacterium abscessus) showed an amplification band at 1,253 bp, and the amplification results were consistent with expectations. However, no specific target-size amplification band was observed in the 16 respiratory tract infection pathogens.

[0062] (6) Figure 7 As shown, MABC ATCC 19977 and 24 collected clinical isolates of Mycobacterium abscessus showed an amplification band at 1,253 bp, and the amplification results were consistent with expectations.

[0063] (7) To further verify the sequence of the amplified DNA, we sequenced the amplified sequence and compared it with the missing annotated sequence, such as... Figure 8 As shown, the results are exactly as expected, and the sequence is correct.

[0064] This demonstrates that the method for identifying MABC complexes based on the DNA sequence of the MAB_3719c(-|3774251-3776008|) gene is authentic and reliable. SEQUENCE LISTING <110> Military Medical Research Institute of the Academy of Military Sciences of the Chinese People's Liberation Army <120> Mycobacterium abscessus encoding genes and their applications <130> BJ1936-21P150581 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1758 <212> DNA <213> M. abscessus subsp. abscessus <400> 1 atgtcatcac cggcagcagt ccctgtaaag cccgaagcag ataccgactt tgacgtactg 60 atcgtcggat ccggattcgg cggaagcgtc accgccatgc gcctgaccga gaagggttat 120 cgcgtaggtg tcctggaggc gggccgacgg ttcgcggacg aggaattcgc cgagacctca 180 tgggatctgc gcaagttcct atgggcgccg atgttcaagt gcttcggcat ccagcggatc 240 cacctgctga gcaactgcat gatcctggcc ggtgccgggg tgggtggagg ctcgctgaac 300 tacgccaaca ccctctacgt gccaccggac ccgttcttca acgatccgca gtggaagggc 360 atcaccgact ggcgcgccga actctcaccc cactatgagc aggcccagcg gatgctcggt 420 gtggtgaaga accccacctt caccgacgcc gaccggctca tcaaggaggt tgccgacgac 480 atgggtgccg gcgacacctt tgtcgcgaca ccagtgggtg tgtacttcgg gcccgacggc 540 accaagaccc ctggcgtcaa agttcccgat ccatacttcg gcggggccgg acccgaccgg 600 gtgggctgca ccgagtgtgg atcctgcatg accggctgcc gggtcggcgc caagaacact 660 ctggtcaaga actacctggg actggctgaa tccaatggtg cccaggtcat tccgctcacc 720 accgtgaccg cggtgcagca gggcgcggac ggtgtctggc gcgtctccac caagtccacc 780 ggccgttggg tgcgtaagca gcgcaagacc tacacggcca agtacgtggt gttggccgcc 840 ggcacctggg gcacgcagaa cctgctgttc aagatgaagg acaaggggtt gctgcccaag 900 ctgtcacagc ggctgggcgt gctgacccgc acaaactccg aatccatcgt cggcgcgggc 960 cggttggagt acaaggacga cctggacctc acccacggtg tggccatcac gtcttccttc 1020 cacccgacca gcgacaccca tatcgagccg gtccgttacg gcaagggctc caacgcgatg 1080 gggctgctgc agaccctgat gaccgacggc gatgggccga agtcgaggtg gcgccagctt 1140 attgagaacg cccgtgccga ctggcggggc acgttccgga tgttcaacgt gacgcagtgg 1200 agcgagcgca ccgttatcgc gctggtcatg cagcacctgg acaactcgat caccaccttc 1260 accaagaaga cgttgttctg gcggcgtctg gacagcaagc aaggccacgg ggagcccaac 1320 cccacctgga ttcccgcggg taacgaggcc acgcggcgcc tggccgccaa gatcgacggc 1380 gttgccggcg gcacctgggg cgagctgttc aacattccgc tgaccgcgca cttcctgggc 1440 ggtgccgtga tcggtaccag cccggagaac ggcgtcatcg acccctacca ccgggtgtac 1500 ggctacccga ccatgtacgt ggtggacgga gcgtcgatct cggccaacct gggcgtcaac 1560 ccgtcactga gcattaccgc acaggccgag cgtgcggcgt cgttgtggcc caacaagggt 1620 gagacggatc tgcgtccgga gcagggcctg ccgtatcagc ggatggcccc ggtgccccg 1680 gtgcacccgg tggtccccgc cgatgccccg ggggcgctgc ggcgtctgcc gatcgaaccg 1740 gtgagctcgg ccagttag 1758 <210> 2 <211> 585 <212> PRT <213> M. abscessus subsp. abscessus <400> 2 Put Sir Sir Pro Ala Ala Val Pro Val Lys Pro Glu Ala Asp Thr Asp 1 5 10 15 Phe Asp Val Leu Ile Val Gly Ser Gly Phe Gly Gly Ser Val Thr Ala 20 25 30 Met Arg Leu Thr Glu Lys Gly Tyr Arg Val Gly Val Leu Glu Ala Gly 35 40 45 Arg Arg Phe Ala Asp Glu Glu Phe Ala Glu Thr Ser Trp Asp Leu Arg 50 55 60 Lys Phe Leu Trp Ala Pro Met Phe Lys Cys Phe Gly Ile Gln Arg Ile 65 70 75 80 His Leu Leu Ser Asn Cys Met Ile Leu Ala Gly Ala Gly Val Gly Gly 85 90 95 Gly Ser Leu Asn Tyr Ala Asn Thr Leu Tyr Val Pro Pro Asp Pro Phe 100 105 110 Phe Asn Asp Pro Gln Trp Lys Gly Ile Thr Asp Trp Arg Ala Glu Leu 115 120 125 Ser Pro His Tyr Glu Gln Ala Gln Arg Met Leu Gly Val Val Lys Asn 130 135 140 Pro Thr Phe Thr Asp Ala Asp Arg Leu Ile Lys Glu Val Ala Asp Asp 145 150 155 160 Met Gly Ala Gly Asp Thr Phe Val Ala Thr Pro Val Gly Val Tyr Phe 165 170 175 Gly Pro Asp Gly Thr Lys Thr Pro Gly Val Lys Val Pro Asp Pro Tyr 180 185 190 Phe Gly Gly Ala Gly Pro Asp Arg Val Gly Cys Thr Glu Cys Gly Ser 195 200 205 Cys Met Thr Gly Cys Arg Val Gly Ala Lys Asn Thr Leu Val Lys Asn 210 215 220 Tyr Leu Gly Leu Ala Glu Ser Asn Gly Ala Gln Val Ile Pro Leu Thr 225 230 235 240 Thr Val Thr Ala Val Gln Gln Gly Ala Asp Gly Val Trp Arg Val Ser 245 250 255 Thr Lys Ser Thr Gly Arg Trp Val Arg Lys Gln Arg Lys Thr Tyr Thr 260 265 270 Ala Lys Tyr Val Val Leu Ala Ala Gly Thr Trp Gly Thr Gln Asn Leu 275 280 285 Leu Phe Lys Met Lys Asp Lys Gly Leu Leu Pro Lys Leu Ser Gln Arg 290 295 300 Leu Gly Val Leu Thr Arg Thr Asn Ser Glu Ser Ile Val Gly Ala Gly 305 310 315 320 Arg Leu Glu Tyr Lys Asp Asp Leu Asp Leu Thr His Gly Val Ala Ile 325 330 335 Thr Ser Ser Phe His Pro Thr Ser Asp Thr His Ile Glu Pro Val Arg 340 345 350 Tyr Gly Lys Gly Ser Asn Ala Met Gly Leu Leu Gln Thr Leu Met Thr 355 360 365 Asp Gly Asp Gly Pro Lys Ser Arg Trp Arg Gln Leu Ile Glu Asn Ala 370 375 380 Arg Ala Asp Trp Arg Gly Thr Phe Arg Met Phe Asn Val Thr Gln Trp 385 390 395 400 Ser Glu Arg Thr Val Ile Ala Leu Val Met Gln His Leu Asp Asn Ser 405 410 415 Ile Thr Thr Phe Thr Lys Lys Thr Leu Phe Trp Arg Arg Leu Asp Ser 420 425 430 Lys Gln Gly His Gly Glu Pro Asn Pro Thr Trp Ile Pro Ala Gly Asn 435 440 445 Glu Ala Thr Arg Arg Leu Ala Ala Lys Ile Asp Gly Val Ala Gly Gly 450 455 460 Thr Trp Gly Glu Leu Phe Asn Ile Pro Leu Thr Ala His Phe Leu Gly 465 470 475 480 Gly Ala Val Ile Gly Thr Ser Pro Glu Asn Gly Val Ile Asp Pro Tyr 485 490 495 His Arg Val Tyr Gly Tyr Pro Thr Met Tyr Val Val Asp Gly Ala Ser 500 505 510 Ile Ser Ala Asn Leu Gly Val Asn Pro Ser Leu Ser Ile Thr Ala Gln 515 520 525 Ala Glu Arg Ala Ala Ser Leu Trp Pro Asn Lys Gly Glu Thr Asp Leu 530 535 540 Arg Pro Glu Gln Gly Leu Pro Tyr Gln Arg Met Ala Pro Val Pro Pro 545 550 555 560 Val His Pro Val Val Pro Ala Asp Ala Pro Gly Ala Leu Arg Arg Leu 565 570 575 Pro Ile Glu Pro Val Ser Ser Ala Ser 580 585 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 acgtgggggg attggcccgg atcta 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 tgtaaagccc gaagcagata ccgac 25

Claims

1. A method for differentiating Mycobacterium abscessus subsp. from other mycobacteria, comprising the following steps: (1) Isolate and extract genomic DNA from the sample to be tested; (2) Using the DNA obtained in step (1) as a template, add amplification primers and perform polymerase chain reaction; (3) The DNA product obtained from step (2) was analyzed by gel electrophoresis, and its size was consistent with the theoretical size; (4) Sequencing analysis was performed on the DNA product amplified in step (2), and the nucleotides were compared with those of the standard detection gene. Based on their homology, it was determined whether Mycobacterium abscessus was present in the sample to be tested. The nucleotides used as the standard detection gene are Mycobacterium abscessus. M. abscessus subsp abscessus The ATCC 19977 encoding gene MAB_3719c (-|3774251-3776008|) has the nucleotide sequence shown in SEQ ID NO.1; The identification method described herein is not used for the diagnosis and treatment of diseases.

2. The identification method according to claim 1, wherein the amplification primer sequence in step (2) is: F: 5'-ACGTGGGGGGATTGGCCCGGATCTA-3'; R: 5'-TGTAAGCCCGAAGCAGATACCGAC-3'.

3. The application of a reagent for detecting the encoding gene MAB_3719c (-|3774251-3776008|) of Mycobacterium abscessus ATCC 19977 in epidemiological surveys of Mycobacterium abscessus subspecies and / or in the preparation of rapid diagnostic and differential diagnostic reagents for clinical patients infected with Mycobacterium abscessus subspecies, wherein the nucleotide sequence of the encoding gene MAB_3719c (-|3774251-3776008|) of Mycobacterium abscessus ATCC 19977 is shown in SEQ ID NO.1, and the detection reagent is an amplification primer, the sequence of which is: F: 5'-ACGTGGGGGGATTGGCCCGGATCTA-3'; R: 5'-TGTAAGCCCGAAGCAGATACCGAC-3'.

Images