Primer and probe combination for subspecies typing identification of mycobacterium abscessus as well as method and application of primer and probe combination

Abstract

The invention relates to the technical field of typing identification, in particular to a primer and probe combination for typing identification of mycobacterium abscessus subspecies and a method and application of the primer and probe combination. The primer and probe combination comprises three subspecies specific amplification primer pairs and three subspecies specific TaqMan fluorescent probes; the sequences of the three pairs of subspecies specific amplification primers are SEQ ID NO. 1 to SEQ ID NO. 6; wherein the TaqMan fluorescent probe comprises three TaqMan fluorescent probes corresponding to mycobacterium abscessus subtypes, two ends of each TaqMan fluorescent probe are respectively provided with a fluorophore and a quenching group, and each sequence is SEQ ID NO.7 to SEQ ID NO.9. The invention further discloses a kit with the PCR reaction liquid, and by means of the PCR reaction liquid, the mycobacterium abscessus can be rapidly and accurately detected and identified, and subspecies typing identification can be conducted on the mycobacterium abscessus.

Description

Technical Field

[0001] The present invention relates to the technical field of genotyping identification, and particularly relates to a primer and probe combination for genotyping identification of Mycobacterium abscessus subspecies, and a method and application thereof. Background Art

[0002] Mycobacterium abscessus (MAB) is a rapidly growing non-tuberculous mycobacterium (NTM), which is associated with pulmonary infections, especially in patients with cystic fibrosis, and has become one of the most common NTM flora isolated from pulmonary samples around the world. In addition, MAB can also cause severe extrapulmonary diseases, including keratitis, endocarditis, meningitis, and even disseminated diseases, MAB bacteremia, etc. MAB contains three subspecies, namely Mycobacterium abscessus subsp. abscessus (MABS), Mycobacterium abscessus subsp. massiliense (MMAS), and Mycobacterium abscessus subsp. bolletii (MBOL).

[0003] MAB was previously thought to be independently acquired from the environment by susceptible individuals. However, recent studies have shown that more than 70% of MAB infections in CF patients are acquired through person-to-person transmission (possibly through contaminants and aerosols), and it is evolving from an environmental saprophyte to an obligate pulmonary pathogen.

[0004] MAB has been subdivided into three independent subspecies, which differ in treatment methods and potential clinical outcomes. Macrolide antibiotics may be ineffective in treating MAB infections. This is because there is an inducible functional erythromycin resistance methylase (erm41) gene, which produces macrolide resistance in both the M. bolletii and M. abscessus subgroups, but is non-functional and non-inducible in M. massiliense. Therefore, the correct identification of MABC subspecies has high clinical significance.

[0005] Traditional Mycobacterium identification methods are based on the phenotypic characteristics of isolates after cultivation, including medium selectivity, colony morphology, growth rate, pigmentation, and biochemical tests. The results of these tests can take up to two weeks. Although these are generally considered the gold standard, they rely on bacterial growth and usually take a long time, especially for slow-growing Mycobacterium species (more than 7 days). The three subspecies of Mycobacterium abscessus are generally considered phenotypically indistinguishable, so alternative methods are needed.

[0006] Molecular techniques and mass spectrometry methods have largely replaced biochemical techniques for the identification of MAB isolates. Multilocus sequence typing (MLST) has been used, including changes in genes such as 16S rRNA, rpoB, hsp65, and secA1, which have been used to distinguish Mycobacterium abscessus subspecies with relatively high accuracy. Usually, sequencing of a single 16S rRNA or rpoB cannot distinguish MAB subspecies because these loci are almost identical. The diagnostic challenge of developing a rapid and simple detection method to distinguish MAB subspecies lies in the need to sequence various subspecies gene targets. But in each case, the detection method requires PCR (Polymerase Chain Reaction) amplification followed by DNA sequencing. However, these research techniques are labor-intensive, subjective in result interpretation, and not suitable for routine clinical microbiology laboratories. The advancement of whole-genome sequencing (WGS) technology enables MAB clinical isolates to be classified into three subspecies even at the whole-genome level, but this method is time-consuming and expensive, while mass spectrometry-based methods such as matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) require the use of expensive equipment and trained personnel. Additionally, linear probe assays (GenoType NTM-DR) have been used to identify MAB at the subspecies level. But this method also requires post-PCR lid-opening operations and has a risk of contamination.

[0007] There is an urgent clinical need for a rapid, sensitive, and simple method to directly distinguish MAB subtypes from clinical samples or culture broths to guide antibiotic use. Summary of the Invention

[0008] The purpose of the present invention is to provide a primer and probe combination, method, and application for the subspecies typing and identification of Mycobacterium abscessus, which can rapidly and accurately detect and identify Mycobacterium abscessus and perform subspecies typing and identification on it.

[0009] To achieve the above technical purposes and reach the above technical effects, the present invention is realized through the following technical solutions: A primer and probe combination for the subspecies typing and identification of Mycobacterium abscessus, comprising 3 pairs of subspecies-specific amplification primer pairs and 3 subspecies-specific TaqMan fluorescent probes; The primer pairs include: Forward primer MABS-F, whose sequence is shown in SEQ ID NO.1, and reverse primer MABS-R, whose sequence is shown in SEQ ID NO.2; Forward primer MMAS-F, whose sequence is shown in SEQ ID NO.3, and reverse primer MMAS-R, whose sequence is shown in SEQ ID NO.4; Forward primer MBOL-F, whose sequence is shown in SEQ ID NO.5, and reverse primer MBOL-R, whose sequence is shown in SEQ ID NO.6; The TaqMan fluorescent probes include: MABS-Probe, whose sequence is shown in SEQ ID NO.7; MMAS-Probe, whose sequence is shown in SEQ ID NO.8; MBOL-Probe, whose sequence is shown in SEQ ID NO.9.

[0010] Furthermore, the fluorescent groups of SEQ ID NOs. 7-9 are ROX, HEX, and CY5 respectively, and the corresponding quenching groups are BHQ2, BHQ1, and BHQ3 respectively.

[0011] On the other hand, the present invention provides a fluorescent PCR reaction solution for typing and identifying Mycobacterium abscessus, which comprises the above-mentioned primer pairs and probe combinations, as well as a qPCR premix; Wherein, the qPCR premix is 2×Animal Detection U+Probe qPCR Super PreMix, and contains a FAM fluorescently labeled probe and primers for detecting Mycobacterium abscessus species.

[0012] Furthermore, for the TaqMan fluorescent probe and primers with the quenching group BHQ1, as long as it belongs to the abscess species, a FAM signal will be generated in the detection system.

[0013] Furthermore, in the 3 pairs of subspecies-specific amplification primer pairs, the molar usage ratio of each upstream and downstream primer is 1:1; The difference in Tm values of the upstream and downstream primers of the 3 subspecies-specific primers does not exceed 2°C, and the Tm value of each probe is 8-10°C higher than the Tm value of any primer in the corresponding primer pair; The molar usage ratio of the 3 subspecies-specific TaqMan fluorescent probes is 1:1:1.

[0014] On the other hand, the present invention provides a method for typing and identifying subspecies of Mycobacterium abscessus, which comprises the following steps: S1: Extract the bacterial genomic DNA of the sample to be tested and store it at -80 °C; S2: Using the genomic DNA as a template, perform PCR amplification with the above-mentioned fluorescence PCR reaction solution. The reaction conditions include: Stage 1: Incubate at 37 °C for 2 min; Stage 2: Pre-denature at 95 °C for 30 s; Stage 3: Denature at 95 °C for 10 s, anneal / extend at 60 °C for 30 s, and repeat for 35 cycles; S3: According to the results of fluorescence signal interpretation, the rules are as follows: If the Ct value of the FAM signal ≤ 35 and the ROX signal is positive, it is identified as the abscess subspecies; If the Ct value of the FAM signal ≤ 35 and the HEX signal is positive, it is identified as the Marseille subspecies; If the Ct value of the FAM signal ≤ 35 and the CY5 signal is positive, it is identified as the Bolley subspecies; If there is no FAM signal, it is determined to be negative, indicating that no Mycobacterium abscessus is detected.

[0015] On the other hand, the present invention provides a fluorescence PCR kit for typing and identification of Mycobacterium abscessus, comprising: (a) The above-mentioned fluorescence PCR reaction solution; (b) Bacterial genomic DNA extraction reagent.

[0016] On the other hand, the present invention provides the application of the above-mentioned primer and probe combination, fluorescence PCR reaction solution, and kit in the preparation of products for subspecies typing detection of Mycobacterium abscessus.

[0017] Furthermore, the detection product is used for rapid typing and identification of clinical samples, including Mycobacterium abscessus in clinical cultures or samples such as sputum and lavage fluid.

[0018] Advantages of the present invention: The present invention realizes typing and identification with high specificity and accuracy, and can accurately identify the specific subspecies of 3 MABs in whole-genome sequencing data, clinical cultures, and clinical samples. The proposed 3 pairs of subspecies-specific amplification primers and the corresponding 3 TaqMan fluorescence probe combinations ensure that each pair of primers and probes can accurately identify specific subspecies (abscess subspecies MABS, Marseille subspecies MMAS, Bolley subspecies MBOL). The unique sequences labeled by the fluorescence probes ensure that only the genomic DNA of the target subspecies binds to them, avoiding cross-reaction with other mycobacteria or common bacteria. Through detailed analysis and comparison of the target gene sequences, the accuracy of subspecies identification during the detection process is guaranteed.

[0019] The present invention does not require traditional culturing steps and can directly extract DNA from clinical samples such as sputum and lavage fluid of patients for detection, completely skipping the mycobacterium culturing step that takes several weeks, significantly shortening the diagnostic cycle. This improvement not only avoids the result deviation caused by culturing failure but also greatly reduces the complexity of laboratory operations, making it particularly suitable for popularization and application in routine clinical microbiology laboratories.

[0020] The present invention provides a rapid and efficient detection process. Traditional methods rely on culturing and phenotypic identification, usually requiring long-time sample processing and analysis. In contrast, the present invention adopts qPCR technology. Through real-time monitoring of fluorescence signals, the detection time is significantly shortened. The PCR amplification process only takes less than one hour, including pre-incubation, denaturation, renaturation, and extension stages. The automated signal interpretation criteria (Ct value ≤ 35 and positive specific fluorescence signal) ensure rapid and accurate result determination, making it suitable for immediate screening and diagnosis of clinical samples.

[0021] The present invention realizes multi-target detection in one tube through multiplex fluorescence labeling. TaqMan probes labeled with three different fluorescent groups (ROX, HEX, CY5) are used for specific recognition of three subspecies, enabling the simultaneous detection of multiple targets in the same reaction system. The selection of fluorescent groups and the optimization of their combination with quenching groups ensure clear identification and non-interference of multi-color fluorescence in the unified reaction system, greatly improving the detection throughput and information content. This allows for the completion of multi-target detection in a single reaction, avoiding the consumable and time costs required for multiple reactions and enhancing the economy and operational simplicity of the detection.

[0022] Of course, it is not necessary for any product implementing the present invention to simultaneously achieve all the above-mentioned advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 Flow chart for obtaining primers and probes for MAB subspecies typing through pan-genomics; Figure 2 Venn diagram of the number of core genes of three subspecies of Mycobacterium abscessus; Figure 3Single, double, and triple fluorescence curves of Mycobacterium abscessus subspecies; a Fluorescence curves of Mycobacterium abscessus abscessus (MABS) (FAM and ROX); b Fluorescence curves of Mycobacterium abscessus massiliense (MMAS) (FAM and HEX); c Fluorescence curves of Mycobacterium abscessus bolletii (MBOL) (FAM and CY5); d Fluorescence curves of MABS+MMAS (FAM, ROX, and HEX); e Fluorescence curves of MABS+MBOL (FAM, ROX, and CY5); f MMAS+MBOL (FAM, HEX, and CY5); g Fluorescence curves of MABS+MMAS+MBOL (FAM, ROX, HEX, and CY5). Detailed implementation mode

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0026] Example 1 Based on the pan-genomics method, the present invention screens specific target genes of three subspecies of Mycobacterium abscessus through the following steps.

[0027] (1) Download and selection of Mycobacterium abscessus genomes Download the publicly available Mycobacterium abscessus genomes from the Genome database of NCBI, and a total of 1959 assembled chromosomal genomes are obtained. Based on the reference genome provided by NCBI, use the fastANI software to confirm the Mycobacterium abscessus genomes. If the ANI of the downloaded genome and the reference genome is not less than 95%, the strain belongs to this species. After determining the species, select 348 genomes with good assembly results for pan-genomics analysis, including the genomes of 136 MABS, 202 MMAS, and 10 MBOL strains.

[0028] (2) Screening of candidate target genes by pan-genomics analysis Use the Prokka software to annotate the genomes of the above 348 strains, and then use the Roray software for pan-genomics analysis to obtain the core genes of each subspecies ( Figure 2 ). Screen the core genes specific to each subspecies, and use the medium-length members among them as candidate target genes (Table 1).

[0029] Table 1 Number of candidate target genes of each subspecies of Mycobacterium abscessus Example 2 Use the Mafft software to perform multiple sequence alignment on the candidate target genes to determine the regions conserved within the species for each target gene. Select the conserved regions as long as possible for primer and probe design. In the present invention, a forward primer, a reverse primer, and a TaqMan probe are designed within the conserved regions obtained from the pan-genome analysis. The distance between the forward and reverse primers is within the range of 50 - 300 bp, and the TaqMan probe is located between the forward and reverse primers. Use Beacon Designer to design the primers and probes. The probe should meet the requirements such as having a length between 20 - 30 bp, a GC content between 40% - 60%, a melting temperature differing from the PCR primers by 8 - 10 °C, and not forming a secondary structure by itself. Details of the primers and probes screened from the Mycobacterium abscessus subspecies sequences are shown in Table 3 and Table 4.

[0030] Among them, the 5' end of the probe sequence is labeled with a fluorophore, and the 3' end is labeled with a corresponding quencher group. The fluorophore and quencher group are not limited to the above specific types, and the positions of the fluorophore and quencher group can also be interchanged.

[0031] Example 3 Blast the above-downloaded 1959 Mycobacterium abscessus genomes with the above primers and probes, and the reverse complementary sequences corresponding to the primers and probes, and screen the results with 100% sequence identity and length. If a strain has the forward primer, reverse primer, and probe of a species, it is considered that this strain belongs to this subspecies. The results show that the sensitivities of the above primers and probes for identifying MABS, MMAS, and MBOL are 99.75%, 99.84%, and 100% respectively, and the specificities are all 100% (Table 2).

[0032] Table 2 Detection results of subspecies primers and probes based on the MAB genome Example 4 A primer and probe combination for typing and identification of Mycobacterium abscessus subspecies described in this example includes 3 pairs of subspecies-specific amplification primer pairs and 3 subspecies-specific TaqMan fluorescence probes; The subspecies-specific amplification primer pairs include: Mycobacterium abscessus abscessus (MABS) primer pair: SEQ ID NO.1 (forward primer) and SEQ ID NO.2 (reverse primer); Mycobacterium abscessus massiliense (MMAS) primer pair: SEQ ID NO.3 (forward primer) and SEQ ID NO.4 (reverse primer); Mycobacterium abscessus bolletii (MBOL) primer pair: SEQ ID NO.5 (forward primer) and SEQ ID NO.6 (reverse primer); Three subspecies-specific TaqMan fluorescent probes, the nucleotide sequences of which are respectively: MABS probe: SEQ ID NO.7, labeled with ROX fluorescent group and BHQ2 quenching group at both ends respectively; MMAS probe: SEQ ID NO.8, labeled with HEX fluorescent group and BHQ1 quenching group at both ends respectively; MBOL probe: SEQ ID NO.9, labeled with CY5 fluorescent group and BHQ3 quenching group at both ends respectively.

[0033] According to the embodiments of the present invention, the fluorescent PCR reaction solution and kit for typing and identification of Mycobacterium abscessus will be used to identify three types of Mycobacterium abscessus, namely: Mycobacterium abscessus subsp. abscessus (MABS), Mycobacterium abscessus subsp. massiliense (MMAS), and Mycobacterium abscessus subsp. bolletii (MBOL).

[0034] In this embodiment, the conserved gene fragment of the corresponding Mycobacterium abscessus in the sputum sample (or cultured bacterial liquid sample) of the subject to be tested is amplified by PCR. The sequences of three pairs of amplification primers are preferably as shown in Table 3 below: Table 3 Meanwhile, the following three TaqMan fluorescent probes of the corresponding Mycobacterium abscessus subtypes are used. Fluorescent groups and quenching groups are labeled at both ends of the probes respectively, and they are added to the PCR reaction solution together. The probe sequences are as shown in Table 4 below: Table 4 Among them, ROX, HEX, and CY5 are fluorescent groups, and BHQ2, BHQ1, and BHQ3 are quenching groups.

[0035] The fluorescent groups and quenching groups corresponding to each sequence are not limited to the above specific types, and the positions of the fluorescent groups and quenching groups can also be interchanged.

[0036] In some embodiments, in addition to the above-mentioned primers and probes, the PCR reaction solution may further include DNA polymerase, 10× Buffer, dNTP, etc., for formulating the PCR reaction solution and performing PCR amplification. The amplification program may include high-temperature denaturation, low-temperature renaturation, and extension. In this embodiment, the multiplex TaqMan probe-based real-time fluorescence quantitative PCR technology is a highly specific detection method based on the 5'→3' exonuclease activity of Taq DNA polymerase. Multiple sets of specific primers and probes are introduced into the same reaction system to achieve synchronous detection of multiple targets. Its core principle is as follows: After the probe (5'-end reporter group, 3'-end quenching group) binds to the template, it is enzymatically cleaved and hydrolyzed during the PCR extension stage, releasing a fluorescence signal. The number of cleaved fluorescent molecules is proportional to the number of PCR products. Therefore, by detecting the fluorescence intensity in the PCR reaction system, the purpose of detecting the amplification amount of PCR products can be achieved; different fluorescent-labeled probes (such as FAM, ROX, HEX) are used to match independent detection channels to distinguish multiple targets. In this embodiment, different Mycobacterium abscessus subtypes can be distinguished through the discrimination of fluorescence signals.

[0037] In addition to the above-mentioned primers and probes, the PCR reaction solution further includes 5 μL of 2× Animal Detection U+ProbeqPCR Super PreMix. This premix contains DNA polymerase (Taq enzyme), related buffers, Mg2+, dNTP, etc., for formulating the PCR reaction solution and performing PCR amplification. The amplification program may include denaturation, renaturation, and extension. When the target bacterial species to be tested is added to the amplification system (including dNTP, Taq enzyme, Mg2+ and related buffers, 4 forward primers, 4 reverse primers, and 4 TaqMan probes), the amplification reaction is initiated, and the 5'-3' exonuclease activity of Taq enzyme cleaves and degrades the probe, and the fluorescence signal is monitored by the system. In this experiment, different forward primers, reverse primers, and probes are designed according to different subspecies to detect the accuracy of this design system. Only when a fluorescence signal and an amplification curve appear when the nucleic acid template of the target bacterial species matching the designed subspecies is added to the system, the detection system design is correct and has subspecies specificity. The sample addition to the system is operated according to Table 5. Figure 3 Single, double, and triple fluorescence curve graphs for Mycobacterium abscessus subspecies.

[0038] Table 5 Figure 3Single, double, and triple fluorescence curves of Mycobacterium abscessus subspecies; a Fluorescence curves of Mycobacterium abscessus subsp. abscessus (MABS) (FAM and ROX); b Fluorescence curves of Mycobacterium abscessus subsp. massiliense (MMAS) (FAM and HEX); c Fluorescence curves of Mycobacterium abscessus subsp. bolletii (MBOL) (FAM and CY5); d Fluorescence curves of MABS + MMAS (FAM, ROX, and HEX); e Fluorescence curves of MABS + MBOL (FAM, ROX, and CY5); f MMAS + MBOL (FAM, HEX, and CY5); g Fluorescence curves of MABS + MMAS + MBOL (FAM, ROX, HEX, and CY5).

[0039] Example 5 Specificity verification. Multiplex TaqMan probe-based real-time fluorescence quantitative PCR amplification was performed on standard strains and clinical isolates (1 ng / μL). These strains included the standard strain MTB H37Rv (ATCC 25293) and Mycobacterium bovis BCG.In addition, it also includes 33 NTM strains accurately identified by whole-genome sequencing (WGS), including Mycobacterium avium subsp. avium, Mycobacterium avium subsp. hominissuis, Mycobacterium massiliense, Mycobacterium intracellulare, Mycobacterium intracellulare subsp. yongonense, Mycobacterium paraintracellulare, Mycobacterium intracellulare subsp. chimaera, Mycobacterium colombiense, Mycobacterium chelonae, Mycobacterium abscessus subsp. abscessus (MABS), Mycobacterium abscessus subsp. massiliense (MMAS), Mycobacterium abscessus subsp. bolletii (MBOL), Mycobacterium kansasii, Mycobacterium gordonae, Mycobacterium paragordonae, Mycobacterium scrofulaceum, Mycobacterium parascrofulaceum, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium marinum, Mycobacterium simiae, Mycobacterium triplex, Mycobacterium lentiflavum, Mycobacterium brisbanense, Mycobacterium iranicum, Mycobacterium mageritense, Mycobacterium mucogenicum, Mycobacterium neoaurum, Mycobacterium novocastrense, Mycobacterium obuense, Mycobacterium peregrinum, Mycobacterium seoulense, Mycobacterium shimoidei.And 6 clinical strains other than Mycobacterium identified by mass spectrometry: Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Cryptococcus neoformans, and Aspergillus fumigatus. According to the detection process in Example 4, among the 41 strains, only Mycobacterium abscessus subsp. Abscessus (MABS), Mycobacterium abscessus subsp. massiliense (MMAS), and Mycobacterium abscessus subsp. Bolletii (MBOL) had corresponding positive signals, and the others were all negative (Table 6).

[0040] Table 6 Example 6 To verify the accuracy of the present invention, clinical strains and samples were detected using the method involved in the present invention. Clinical strains: 72 clinical isolates of Mycobacterium abscessus differentiated by WGS sequencing were collected from the tuberculosis laboratory of Hangzhou Red Cross Hospital. These strains included 51 strains of Mycobacterium abscessus subsp. (MABS), 20 strains of Mycobacterium massiliense subsp. (MMAS), and 1 strain of Mycobacterium bolletii subsp. (MBOL). All results could correctly distinguish the 3 subspecies.

[0041] Clinical samples: 202 respiratory specimens from the sample bank of the tuberculosis laboratory of Hangzhou Red Cross Hospital collected previously. These specimens were clinically diagnosed as Mycobacterium infection during the period from March 2021 to March 2023 and the pathogens were determined by tNGS technology. Among them, for the 30 abscess samples (specifically: 27 cases of MABS and 3 cases of MMAS), the detection situation was as follows: the sensitivity of positive samples was 96.7% (29 / 30); the remaining 172 cases were non-abscess samples, and the specificity of negative samples was 99.4% (171 / 172).

[0042] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A primer and probe combination for the subtyping identification of Mycobacterium abscessus, characterized in that, It includes 3 pairs of subspecies-specific amplification primer pairs and 3 subspecies-specific TaqMan fluorescent probes; The primer pairs include: Forward primer MABS-F, whose sequence is shown in SEQ ID NO.1, and reverse primer MABS-R, whose sequence is shown in SEQ ID NO.2; Forward primer MMAS-F, whose sequence is shown in SEQ ID NO.3, and reverse primer MMAS-R, whose sequence is shown in SEQ ID NO.4; Forward primer MBOL-F, whose sequence is shown in SEQ ID NO.5, and reverse primer MBOL-R, whose sequence is shown in SEQ ID NO.6; The TaqMan fluorescent probes include: MABS-Probe, whose sequence is shown in SEQ ID NO.7; MMAS-Probe, whose sequence is shown in SEQ ID NO.8; MBOL-Probe, whose sequence is shown in SEQ ID NO.

9.

2. A fluorescence PCR reaction solution for typing and identification of Mycobacterium abscessus, characterized in that, It includes the primer and probe combination as described in claim 1, and a qPCR premix; wherein, the qPCR premix is 2×Animal Detection U+Probe qPCR Super PreMix, and contains a FAM fluorescently labeled probe and primers for detecting Mycobacterium abscessus species.

3. The fluorescent PCR reaction solution according to claim 2, wherein: In the 3 pairs of subspecies-specific amplification primer pairs, the molar dosage ratio of each upstream and downstream primer is 1:

1.

4. The fluorescent PCR reaction solution according to claim 2, wherein: The Tm values of the 3 pairs of subspecies-specific upstream and downstream primers differ by no more than 2°C, and the Tm value of each probe is 8 - 10°C higher than the Tm value of any primer in the corresponding primer pair.

5. The fluorescent PCR reaction solution according to claim 2, wherein: The molar dosage ratio of the 3 subspecies-specific TaqMan fluorescent probes is 1:1:

1.

6. A method for typing and identifying Mycobacterium abscessus subspecies, characterized in that, It includes the following steps: S1: Extract the bacterial genomic DNA of the sample to be tested; S2: Using the genomic DNA as a template, perform amplification with the fluorescent PCR reaction solution as described in any one of claims 2 - 5, and the reaction conditions include: Stage 1: Incubate at 37°C for 2 min; Stage 2: Pre-denature at 95°C for 30 s; Stage 3: Denature at 95°C for 10 s, anneal / extend at 60°C for 30 s, and repeat for 35 cycles; S3: According to the interpretation result of the fluorescent signal, the rules are as follows: If the Ct value of the FAM signal ≤ 35 and the ROX signal is positive, it is identified as the abscess subspecies; If the Ct value of the FAM signal ≤ 35 and the HEX signal is positive, it is identified as the massiliense subspecies; If the Ct value of the FAM signal ≤ 35 and the CY5 signal is positive, it is identified as the bolletii subspecies; If there is no FAM signal, it is determined to be negative.

7. A fluorescence PCR kit for typing and identification of Mycobacterium abscessus, characterized in that, It contains: (a) The fluorescent PCR reaction solution as described in any one of claims 2 - 5; (b) Reagents for extracting bacterial genomic DNA.

8. The application of the primer and probe combination as described in claim 1 in the preparation of a Mycobacterium abscessus subspecies typing detection product.

9. The application according to claim 8, wherein The detection product is used for rapid typing identification of clinical samples, including Mycobacterium abscessus in sputum, lavage fluid or culture broth.

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