Specific PCR primers and identification methods for Mycobacterium abscessus subsp.

Abstract

This invention belongs to the field of biodetection technology and discloses specific PCR primers and identification methods for the subspecies of Mycobacterium abscessus. The differentiation effect of this invention on the three subspecies of Mycobacterium abscessus is equivalent to whole-genome sequencing, while the established detection method does not rely on sequencing, is rapid and low-cost, and has low requirements for equipment and personnel, making it applicable to laboratories at all levels. The amplification method has low template requirements; it is applicable to raw samples such as sputum, bronchoalveolar lavage fluid, pleural and peritoneal fluid, cultures, and extracted nucleic acids, and is not culture-dependent. Identification of the three subspecies is based on product length rather than amplification negative / positive (presence or absence of bands), avoiding false negative results. The product length differences are significant (above 1000 bp), making results easy to interpret, reducing the impact of electrophoresis errors on results, and improving the accuracy and stability of detection. The detection reagents based on this invention can be widely used in clinical precision testing and epidemiological monitoring, among other related fields.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically, it relates to specific PCR primers and identification methods for Mycobacterium abscessus subspecies. Background Technology

[0002] Mycobacteroides abscessus (M. abscessus, also known as Mycobacterium abscessus) is an important pathogenic non-tuberculous mycobacterium (NTM), ranking first in isolation rate among common NTMs in clinical practice in my country (Lei Zhou et al., Frontiers in Public Health, 2020, 28;8:295). Currently, Mycobacteroides abscessus includes three subspecies: subsp. abscessus, subsp. bolletii, and subsp. massiliense. Mycobacterium abscessum can cause severe respiratory, skin, and mucosal infections in humans. It exhibits natural resistance to first- and second-line anti-tuberculosis drugs, and treatment primarily involves multidrug combination therapy centered on macrolides. However, because the abscess and bleae subspecies often carry the 23S rRNA methyltransferase gene erm(41), they exhibit induced resistance to macrolides, resulting in an overall cure rate of only 45.6%, making it considered one of the most difficult non-tuberculous mycobacteria to cure. Significant differences exist in antibiotic resistance and cure rates among the Mycobacterium abscessum subspecies. The cure rate for the abscess subspecies is only 33%, with the use of azithromycin, amikacin, and imipenem correlated with the cure rate. The masai subspecies, however, shows higher sensitivity to macrolides than the abscess subspecies, achieving a cure rate of 56.7%, but this is unrelated to the choice of the three antibiotics used (Kwak et al., European Respiratory Journal, 2019, 54: 1801991). Therefore, early and accurate subspecies identification is crucial for clinical treatment.

[0003] Methods for identifying mycobacterial species have been mainly classified into four categories during their development:

[0004] 1) Cell and colony morphology and biochemical phenotype;

[0005] 2) Single-gene sequencing or multi-gene combined sequencing of conserved genes such as 16S rRNA, rpoB, hsp65, ITS1 (intergenic region of 16S rRNA and 26S rRNA), and rpls, as well as detection methods such as probe method and melting curve method based on SNP sites of these genes.

[0006] 3) Based on the differences in sensitivity of abscesses and Maasai subspecies to macrolide drugs such as azithromycin, detection is based on the differences in erm(41) sequence in the drug resistance mechanism;

[0007] 4) Whole genome sequencing.

[0008] Among these methods, phenotypic analysis cannot distinguish between *Mycobacterium abscessus* subspecies. Previous analysis of erm(41) showed that among the 1259 *Mycobacterium abscessus* subspecies genomes published by NCBI, two genomes had a 246bp deletion of erm(41) instead of the complete 522bp; among the 617 *Mycobacterium masai* subspecies genomes, 581 had the 246bp deletion, and 11 genomes had the complete 522bp erm gene; all 130 *Mycobacterium bolyense* subspecies had the complete erm(41) gene. This indicates that the erm(41) gene cannot distinguish between *Mycobacterium abscessus* and *Mycobacterium bolyense* subspecies, and the defective erm(41) gene is not a common feature of the *Mycobacterium masaiense* subspecies, but rather accounts for a relatively high proportion. Among known single-gene identification methods, the rpoB gene has the highest resolution for mycobacteria. Although it has been used to identify the subspecies *Mycobacterium abscessum*, the results for species identification are inconsistent with those based on whole-genome sequencing (Tortoli et al., *International Journal of Systematic and Evolutionary Microbiology*, 2018, 68: 467-469). Therefore, accurate identification of the subspecies *Mycobacterium abscessum* currently relies on multi-gene sequencing or whole-genome sequencing, based on sequence similarity or the ANI value of the genome. However, sequencing methods are relatively expensive, especially whole-genome sequencing, which many laboratories cannot perform independently. Analyzing sequencing data and identifying subspecies also requires certain bioinformatics knowledge and techniques, and the overall time frame is long. Therefore, a new, highly specific, and universal marker for the subspecies is needed, and based on this, a simple, rapid detection method equivalent to whole-genome sequencing results should be established. Summary of the Invention

[0009] The purpose of this invention is to provide specific molecular markers, PCR primers, and identification methods for the subspecies of Mycobacterium abscessus.

[0010] This invention, through analysis, provides two genomic regions that can distinguish the three subspecies of Mycobacterium abscessus, located at: 1) the coding genes of polysaccharide deacetylase family protein (MAB_RS06440) to CsbD family protein (MAB_RS06445); and 2) three consecutive coding gene regions (MAB_RS17785 to MAB_RS17800) of ABC1 kinase family protein.

[0011] To achieve the objectives of this invention, in a first aspect, this invention provides specific PCR primers for detecting *Mycobacteroides abscessus* subsp., including primer pair Mab-1 (SEQ ID NO: 1-2) and primer pair Mab-2 (SEQ ID NO: 3-4), with the following sequences:

[0012] Mab-1 forward primer (upstream primer): 5'-AGTTGACCGGCAAGTAGTTC-3',

[0013] Mab-1 reverse primer (downstream primer): 5'- CAGCTATGGCAGCAGAGAG-3';

[0014] Mab-2 forward primer (upstream primer): 5'-TGCCTCCCGTCTACCTGATG-3',

[0015] Mab-2 reverse primer (downstream primer): 5'-CCGCCTTCTCCAAGAGTTCG-3'.

[0016] Secondly, the present invention provides detection reagents or kits containing the primers.

[0017] Thirdly, the present invention provides the application of the primers or detection reagents or kits containing the primers in the identification of Mycobacterium abscessis subspecies.

[0018] Fourthly, the present invention provides a method for identifying subspecies of Mycobacterium abscessus (including for non-disease diagnostic purposes), comprising the following steps:

[0019] 1) Extract DNA from the sample to be tested;

[0020] 2) Using the extracted DNA or the original sample as a template, perform PCR amplification using primer pair Mab-1 and primer pair Mab-2, respectively;

[0021] 3) Analyze the PCR amplification products.

[0022] The samples to be tested can be raw samples such as sputum, bronchoalveolar lavage fluid, pleural effusion, peritoneal fluid, and cultures.

[0023] Preferably, the PCR amplification reaction conditions are: 98℃ for 2 min; 98℃ for 10 s, 58℃ for 10 s, 72℃ for 20 s, 30 cycles; 72℃ for 5 min.

[0024] The aforementioned method, step 3) includes:

[0025] ① Amplification results corresponding to primer pair Mab-1: The amplification products of abscess subspecies and masai subspecies are approximately 410bp in size, and the amplification product of bolai subspecies is approximately 2599bp in size;

[0026] ② Amplification results corresponding to primer pair Mab-2: The amplification product size of abscess subspecies is 1775bp, and the amplification product size of masai subspecies and bolai subspecies is about 386bp.

[0027] ③ Determine the type of Mycobacterium abscessus subspecies contained in the sample based on the size of the amplification product.

[0028] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0029] (i) The detection marker of the present invention has the same distinguishability for the three subspecies as whole genome sequencing.

[0030] (ii) This invention is based on PCR technology for detection, does not rely on sequencing, is low-cost and fast, with a detection time of 0.5-1.5 hours, is easy to operate, has low requirements for equipment and testing personnel, and has wider applicability in laboratories at all levels.

[0031] (iii) The amplification method has low requirements for templates and can be used for original samples such as sputum, bronchoalveolar lavage fluid, pleural effusion, ascites, and cultures, as well as extracted nucleic acids. It does not depend on culture and can shorten the clinical diagnosis time.

[0032] (iv) Identifying the three subspecies based on product length rather than amplification negative / positive (presence or absence of a band) can avoid false negative results. The product length difference is significant (above 1000 bp), making the results easier to interpret, reducing the impact of electrophoresis errors on the results, and improving the accuracy and stability of the detection. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the subspecies-specific fragment of the present invention. A and B represent the design regions of primers Mab-1 and Mab-2, respectively, and the same colored blocks indicate sequence homologous regions. Arrows indicate the positions of the primer pairs on the genome.

[0034] Figure 2 These are the simulated amplification results for the three subspecies of this invention.

[0035] Figure 3 The electrophoresis patterns of actual samples detected using two primer pairs are shown in the preferred embodiment of the present invention. The left image shows the amplification results of the Mab-1 forward / reverse primer pair; the right image shows the amplification results of the Mab-2 forward / reverse primer pair. The actual detected strains are: A1 is abscess subspecies strain ATCC19977; A2 is abscess subspecies strain FJ09038; M1 is a Marseille subspecies strain FJ05231; M2 is a Marseille subspecies strain FJ12077; B is a Boleyn subspecies strain DSM 45149. The products of Mab-1 are 410bp and 2599bp, respectively; the products of Mab-2 are 386bp and 1775bp, respectively. Detailed Implementation

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0037] Example 1: Analysis of differentially expressed regions in the genome of Mycobacterium abscessus subspecies

[0038] This invention utilizes over 2000 genomes of *Mycobacterium abscessus* from the NCBI database, employing Average Nucleotide Identity (ANI) and core genome phylogenetic analysis to obtain accurate subspecies classifications across the entire genome. Further genomic difference analysis between subspecies identified regions with significant differences between two different subspecies, suitable for subspecies differentiation. These sequence differences exist throughout the entire genome within a subspecies and are a shared characteristic. The differences between these two sequence segments are illustrated using the type strain genomes of three subspecies as examples. Figure 1The first differential region is located in the genomes of the Marseille and abscess subspecies at the coding genes of polysaccharide deacetylase family proteins and CsbD family proteins, separated by a 227 bp spacer. In the Bolei subspecies, these two genes are separated by the coding gene MASB_RS06435 of the penicillin acylase family protein. The penicillin acylase family protein gene is unique to the Bolei subspecies; no full-length homologous sequences are found in the genomes of the other two subspecies, but partial sequences of the 227 bp spacer region are highly similar to those in the other two subspecies. The second differential region is located at three consecutive coding genes of the ABC1 kinase family protein. In the genome of the abscess subspecies, two genes encoding GlcG / HbpS family heme-binding protein and SDR family oxidoreductase were inserted in reverse between genes 2 and 3. These two genes are specific to the abscess subspecies and there are no matching segments in the genomes of the Marseille and Boley subspecies.

[0039] Example 2 Development of specific PCR primers for Mycobacterium abscessus subspecies

[0040] Based on these two sequences, this invention designs specific PCR primers for Mycobacteroides abscessus subsp., which are used to detect M. abscessus subsp. abscessus, M. abscessus subsp. Bolletii, and M. abscessus subsp. Massiliense.

[0041] In primer design for Mab-1, forward primers were initially designed based on highly conserved regions of three subspecies: 5'-AACACTCCGTCGCCGTTG-3' and reverse primer 5'-AGTTGACCGGCAAGTAGTTC-3'. The products from different subspecies showed significant differences. Software-predicted TM values ​​were similar, with low complementarity and little or no secondary structure. However, the actual synthesized TM values ​​varied considerably. Non-specific bands appeared at lower annealing temperatures, while amplification efficiency was insufficient at higher temperatures. Subsequently, the position of the forward primer was changed, resulting in more consistent TM values. Although the product was longer, the amplification specificity and amplification efficiency were superior to the previous method. Mab-2 primers were designed with multiple primer pairs: forward primers 5'-CCTATGCGCGAATCATCGG-3' and 5'-TGCCTCCCGTCTACCTGATG-3', and reverse primers 5'-CGAGCACCTTGAACAGCTC-3', 5'-AGCACGCGATGCACCTTG-3', 5'-CCGCCTTCTCCAAGAGTTCG-3', and 5'-TTGCCAGTTTGGTCATTCGG-3', following the same design principles as Mab-1 primer pairs. PCR effects of different primers were tested under varying temperature conditions, and the primer pairs with the best amplification performance determined through comprehensive comparison were Mab-1 (SEQ ID NO:1-2) and primer pair Mab-2 (SEQ ID NO:3-4). The Mab-1 forward primer is located downstream of the MAB_RS06440 gene (positions 702 to 720) in the MAB_RS06445 reference genome ATCC 19977 (GCF_000069185.1), and the Mab-1 reverse primer is located at the 3' end of the MAB_RS06445 gene (positions 894-910, with an additional 3 bp in the non-coding region). The Mab-2 forward primer is located downstream of the MAB_RS17785 gene (positions 1187 to 1206), and the Mab-2 reverse primer is located upstream of the MAB_RS17800 gene (positions 151-132).

[0042] Primer alignment analysis was performed on the core NT libraries of five genera of mycobacteria (Mycobacterium, Mycobacterium pseudomycobacterium, Mycobacterium spp., Mycobacterium spp., and Mycobacterium spp.) using primer blast to detect primer specificity. The results showed that, except for *Mycobacterium abscessus*, all genomes, including *Mycobacterium turcica* within the *Mycobacterium* genus, failed to produce amplification products, indicating that the primers had high specificity.

[0043] The specific sequence is as follows:

[0044] Mab-1 forward primer (upstream primer): 5'-AGTTGACCGGCAAGTAGTTC-3',

[0045] Mab-1 reverse primer (downstream primer): 5'- CAGCTATGGCAGCAGAGAG-3';

[0046] Mab-2 forward primer (upstream primer): 5'-TGCCTCCCGTCTACCTGATG-3',

[0047] Mab-2 reverse primer (downstream primer): 5'-CCGCCTTCTCCAAGAGTTCG-3'.

[0048] A schematic diagram of the subspecies-specific fragment of this invention is shown below. Figure 1 A and B represent the design regions for primers Mab-1 and Mab-2, respectively, with identical colored blocks indicating sequence homologous regions. Arrows indicate the positions of the primer pairs on the genome. Simulated amplification results for the three subspecies are shown below. Figure 2 .

[0049] Example 3: Identification of bacterial species from plate isolates

[0050] 1) The abscess subspecies ATCC19977, abscess subspecies FJ09038, Marseille subspecies FJ05231, Marseille subspecies FJ12077 and Boley subspecies DSM 45149 were streaked onto 7H10-OADC plates and incubated at 37°C for 3 days.

[0051] 2) Pick a single colony with a pipette tip, grind it into 100 μL of water, and prepare an amplification template.

[0052] 3) Prepare a 20μL reaction system using TSINGKE TSE101 Gold Mix (green) (Beijing Qingke Biotechnology Co., Ltd.), as follows:

[0053]

[0054] 4) Amplification on the instrument, reaction conditions: 98℃ for 2 min, (98℃ for 10 s, 58℃ for 10 s, 72℃ for 20 s) for 30 cycles, 72℃ for 5 min, 4℃ to infinity. Total time: 27 min.

[0055] 5) 1% agarose gel electrophoresis, load 3 μL of sample, and read the results using a Bio-rad gel reader.

[0056] 6) Identification of bacterial species, such as Figure 3 As shown.

[0057] 7) The detection sensitivity is 100%. PCR detection technology has a sensitivity of up to 3 RFU (plaque-forming units), and the minimum detection limit in bacteriology is 3 bacteria. Using plasmids as a reference, the detection limit of conventional PCR is approximately 1.0 × 10⁻⁶.4 Copy / μl.

[0058] Example 4 Detection in sputum samples

[0059] 1) Simulated sputum samples of *Abscessus* subspecies ATCC19977, *Abscessus* subspecies FJ09038, *Abscessus masei* subspecies FJ05231, *Abscessus masei* subspecies FJ12077, and *Abscessus bleae* subspecies DSM 45149. Depending on the viscosity of the sputum sample, add 1–2 times the volume of 4% NaOH solution, tighten the screw cap, and vortex for 30 seconds to homogenize the sample. Incubate at room temperature until fully liquefied (no obvious solid matter and no stringing upon aspiration), no more than 15 minutes. Centrifuge at 12000 rpm for 5 minutes. Rinse the precipitate with sterile physiological saline and resuspend in 200 μL of physiological saline.

[0060] 2) Prepare a 20 μL reaction system using 2×EasyTaq® PCR SuperMix (TransGen Biotech). The components are as follows:

[0061]

[0062] 3) Amplification on the instrument, reaction conditions: 95℃ for 5 min, (95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min) 30 cycles, 72℃ for 5 min, 4℃ ∞. Total time: 70 min.

[0063] 4) 1% agarose gel electrophoresis, load 3 μL of sample, and read the results using a Bio-rad gel reader.

[0064] 5) Identification of bacterial species, such as Figure 3 As shown.

[0065] Example 5: Identification of bacterial strains in cultures

[0066] 1) Fresh Löwensch-Nutritive culture media of *Abscessus* subspecies ATCC19977, *Abscessus* subspecies FJ09038, *Massai* subspecies FJ05231, *Massai* subspecies FJ12077, and *Bole* subspecies DSM 45149. A certain amount of culture was scraped from Löwensch-Nutritive medium using an inoculation loop and added to 400 μL of physiological saline to prepare a bacterial suspension.

[0067] 2) Extract genomic DNA using the CTAB method or heat in a 100°C water bath for 5 minutes, centrifuge at 12000 rpm for 5 minutes, and take the supernatant to prepare a boil-in template.

[0068] 3) Using Premix Taq ™ (TaKaRa Taq ™ Prepare a 20 μL reaction system using (Version 2.0) (TAKARA Corporation), as follows:

[0069]

[0070] 4) Amplification on the instrument, reaction conditions: 95℃ for 5 min, (95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min) 30 cycles, 72℃ for 5 min, 4℃ ∞. Total time: 70 min.

[0071] 5) 1% agarose gel electrophoresis, load 3μL of sample, and observe the results using a blue light gel cutter.

[0072] 6) Identification of bacterial species, such as Figure 3 As shown.

[0073] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Mycobacterium abscessus subsp. ( Mycobacteroides abscessus A specific PCR primer set for subsp., characterized in that, Used to detect abscess subtypes ( M. abscessus subsp. abscessus ), Boley subspecies ( M. abscessus subsp. Bolletii) and Masai subspecies ( M. abscessus subsp. Massiliense The specific PCR primers include primer pair Mab-1 and primer pair Mab-2, with the following sequences: Mab-1 forward primer: 5'-AGTTTGACCGGCAAGTAGTTC-3', Mab-1 reverse primer: 5'-CAGCTATGGCAGCAGAGAG-3'; Mab-2 forward primer: 5'-TGCCTCCCGTCTACCTGATG-3', Mab-2 reverse primer: 5'-CCGCCTTCTCCAAGAGTTCG-3'.

2. A detection reagent or kit containing the primer set of claim 1.

3. The application of the primer set of claim 1 or the detection reagent or kit of claim 2 in the identification of subspecies Mycobacterium abscessus; The application is for non-disease diagnosis purposes.

4. A method for identifying the subspecies of Mycobacterium abscessus, characterized in that, Includes the following steps: 1) Extract DNA from the sample to be tested; 2) Using the extracted DNA or the original sample as a template, perform PCR amplification using the primer pair Mab-1 and primer pair Mab-2 described in claim 1, respectively; 3) Analyze the PCR amplification products; Step 3) includes: ① Amplification results corresponding to primer pair Mab-1: The amplification product size of abscess subspecies and masai subspecies is 410bp, and the amplification product size of bolai subspecies is 2599bp; ② Amplification results corresponding to primer pair Mab-2: The amplification product size of abscess subspecies is 1775bp, and the amplification product size of masai subspecies and bolai subspecies is 386bp; ③ Determine the type of Mycobacterium abscessus subspecies contained in the sample based on the size of the amplification product; The method described is not for disease diagnosis purposes.

5. The method according to claim 4, characterized in that, PCR amplification reaction conditions: 98℃ for 2 min; 98℃ for 10 s, 58℃ for 10 s, 72℃ for 20 s, 30 cycles; 72℃ for 5 min.

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