Specific PCR (Polymerase Chain Reaction) primer and identification method of mycobacterium abscessus subspecies

Abstract

The invention belongs to the technical field of biological detection, and discloses a specific PCR (Polymerase Chain Reaction) primer and an identification method of mycobacterium abscessus subspecies. The distinguishing effect on the three subspecies of the mycobacterium abscessus is equal to that of whole genome sequencing, and the established detection method does not depend on sequencing, is rapid and low in cost, has low requirements on equipment and detection personnel, and is applicable to all levels of laboratories. The amplification method has low requirements on a template, is suitable for original samples such as sputum, alveolar lavage fluid, hydrothorax and ascites, culture and the like and extracted nucleic acid, and does not depend on culture. The identification of the three subspecies is realized according to the length of the product instead of amplification of negative / positive (existence or absence of bands), so that false negative results can be avoided. The product length difference is obvious (more than 1000bp), the result is easy to judge, the influence of electrophoresis error on the result is reduced, and the detection accuracy and stability are improved. The detection reagent based on the invention can be widely applied to the related fields of clinical precise detection, epidemiological monitoring and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to specific PCR primers and an identification method for Mycobacteroides abscessus subspecies. Background Art

[0002] Mycobacteroides abscessus (M. abscessus, formerly Mycobacterium abscessus) is an important pathogenic non-tuberculous mycobacterium (NTM). It ranks first in the isolation rate among clinically common NTMs in China (Lei Zhou et al., Frontiers in Public Health, 2020, 28;8:295). Currently, Mycobacteroides abscessus includes three subspecies, namely Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bolletii (formerly Mycobacterium bolletii), and Mycobacterium abscessus subsp. massiliense (formerly Mycobacterium massiliense). Mycobacteroides abscessus can cause severe respiratory, skin, and mucosal infections in humans. It is naturally resistant to first- and second-line anti-tuberculosis drugs. The treatment mainly uses multi-drug combination therapy centered on macrolide drugs. However, due to the fact that Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. bolletii often carry the 23S rRNA methylase gene erm(41), there is an induced drug resistance phenomenon to macrolide drugs. Therefore, the overall cure rate is only 45.6%, and it is regarded as the most difficult non-tuberculous mycobacterium to cure. There are significant differences in antibiotic resistance and cure rates among Mycobacteroides abscessus subspecies. The cure rate of Mycobacterium abscessus subsp. abscessus is only 33%. The use of azithromycin, amikacin, and imipenem is related to the cure rate. While Mycobacterium abscessus subsp. massiliense is more sensitive to macrolide drugs than Mycobacterium abscessus subsp. abscessus, and the cure rate can reach 56.7%, but it has nothing to do with the use selection of the three antibiotics (Kwak et al., European Respiratory Journal, 2019, 54: 1801991). Therefore, early and accurate subspecies identification is of great significance for clinical treatment.

[0003] The identification methods of mycobacterial species mainly fall into four categories during development: 1) Bacterial cells and colony morphology, biochemical phenotypes; 2) Single-gene sequencing or multi-gene combined sequencing of conserved genes such as 16S rRNA, rpoB, hsp65, ITS1 (the intergenic region between 16S rRNA and 26S rRNA), rpls, etc., as well as detection methods such as probe method and melting curve method based on SNP sites of these genes; 3) Detection based on the sequence difference of erm(41) in the drug resistance generation mechanism according to the sensitivity differences of abscess and Massilia subspecies to macrolide drugs such as azithromycin and clarithromycin; 4) Whole-genome sequencing.

[0004] Among these methods, the phenotypic method cannot distinguish Mycobacterium abscessus subspecies. Previous analysis of erm(41) showed that among the 1259 abscess subspecies genomes published by NCBI, 2 genomes had erm(41) not as the complete 522 bp, but the deletion type of 246 bp; among the 617 Massilia subspecies genomes, 581 were of the deletion type of 246 bp, and 11 genomes had the complete 522 bp erm gene; all 130 Bolletii subspecies had the complete erm(41) gene. This indicates that the erm(41) gene cannot distinguish abscess and Bolletii subspecies, and the defective erm(41) gene is not a common feature of Massilia subspecies either, but only has a relatively high proportion. Among the known single-gene identification methods, the rpoB gene has the highest resolution for mycobacteria. Although it has been used for the identification of Mycobacterium abscessus subspecies, the species determination results of Mycobacterium abscessus subspecies are inconsistent with those based on the whole-genome sequence (Tortoli et al., International Journal of Systematic and Evolutionary Microbiology, 2018, 68: 467-469). Therefore, at present, the accurate identification of Mycobacterium abscessus subspecies relies on multi-gene sequencing or whole-genome sequencing, and is judged according to the sequence similarity or the ANI value of the genome. However, the cost of the sequencing method is relatively high, especially for whole-genome sequencing. Many laboratories cannot complete it independently in the laboratory. Analyzing the sequencing data and identifying subspecies also requires certain bioinformatics knowledge and technology, and the overall time cycle is relatively long. Therefore, a new universal identifier highly specific to subspecies is needed, and on this basis, a simple, rapid detection method equivalent to the whole-genome sequencing result is established. Summary of the Invention

[0005] The purpose of the present invention is to provide a specific molecular identifier, PCR primer and identification method for Mycobacterium abscessus subspecies.

[0006] Through analysis, the present invention provides two genomic regions that can distinguish three subspecies of Mycobacteroides abscessus, which are located in 1) the coding genes from polysaccharide deacetylase family protein (MAB_RS06440) to CsbD family protein (MAB_RS06445); 2) three consecutive coding gene regions of ABC1 kinase family protein (from MAB_RS17785 to MAB_RS17800).

[0007] To achieve the object of the present invention, in the first aspect, the present invention provides specific PCR primers for Mycobacteroides abscessus subsp., and the specific PCR primers for detecting M. abscessus subsp. abscessus, M. abscessus subsp. Bolletii, and M. abscessus subsp. Massiliense include primer pair Mab-1 (SEQ ID NO: 1-2) and primer pair Mab-2 (SEQ ID NO: 3-4), and the sequences are as follows: Mab-1 forward primer (upstream primer): 5’- AGTTGACCGGCAAGTAGTTC-3’, Mab-1 reverse primer (downstream primer): 5’- CAGCTATGGCAGCAGAGAG-3’; Mab-2 forward primer (upstream primer): 5’- TGCCTCCCGTCTACCTGATG-3’, Mab-2 reverse primer (downstream primer): 5’- CCGCCTTCTCCAAGAGTTCG-3’.

[0008] In the second aspect, the present invention provides a detection reagent or kit containing the above primers.

[0009] In the third aspect, the present invention provides the application of the above primers or a detection reagent or kit containing the above primers in the identification of Mycobacteroides abscessus subsp.

[0010] In the fourth aspect, the present invention provides a method for identifying Mycobacteroides abscessus subsp. (including for non-disease diagnosis purposes), which includes the following steps: 1) Extract the DNA of the sample to be tested; 2) Using the extracted DNA or the original sample as a template, perform PCR amplification using primer pairs Mab-1 and Mab-2 respectively; 3) Analyze the PCR amplification products.

[0011] The test sample can be an original sample such as sputum, bronchoalveolar lavage fluid, pleural effusion, ascites, culture, etc.

[0012] Preferably, the PCR amplification reaction conditions are: 98°C for 2 min; 98°C for 10 s, 58°C for 10 s, 72°C for 20 s, 30 cycles; 72°C for 5 min.

[0013] For the aforementioned method, step 3) includes: ① Amplification results corresponding to primer pair Mab-1: The amplified product sizes of the abscessus subspecies and the massiliense subspecies are approximately 410 bp, and the amplified product size of the bolletii subspecies is approximately 2599 bp; ② Amplification results corresponding to primer pair Mab-2: The amplified product size of the abscessus subspecies is 1775 bp, and the amplified product sizes of the massiliense subspecies and the bolletii subspecies are approximately 386 bp; ③ Determine the type of Mycobacterium abscessus subspecies contained in the test sample based on the amplified product size.

[0014] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: (1) The detection markers of the present invention have the same discrimination degree for the three subspecies as whole genome sequencing.

[0015] (2) The present invention performs detection based on PCR technology, does not rely on sequencing, has low cost, is fast, the detection time is 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.

[0016] (3) The amplification method has low requirements for templates, is applicable to original samples such as sputum, bronchoalveolar lavage fluid, pleural effusion, ascites, culture, etc. and the extracted nucleic acids, does not rely on culture, and can shorten the clinical diagnosis time.

[0017] (4) Identification of the three subspecies is achieved based on the product length rather than the amplification negative / positive (presence or absence of bands), which can avoid the occurrence of false negative results. The product length differences are significant (more than 1000 bp), the results are easy to judge, reducing the influence of electrophoresis errors on the results, and improving the accuracy and stability of detection. Description of the Drawings

[0018] Figure 1 It is a schematic diagram of the subspecies-specific fragment of the present invention. A and B respectively indicate the designed regions of primers Mab-1 and Mab-2, and the same color blocks represent sequence homologous regions. The arrows mark the positions of the primer pairs on the genome.

[0019] Figure 2 These are the simulated amplification results of the three subspecies of the present invention.

[0020] Figure 3 This is the electrophoresis pattern of the actual sample detection using two pairs of primers in a preferred embodiment of the present invention; among them, the left figure is the amplification result of the Mab-1 forward / reverse primer pair; the right figure is the amplification result of the Mab-2 forward / reverse primer pair; the actual detected strains are: A1 is the abscess subspecies strain ATCC19977; A2 is the abscess subspecies strain FJ09038; M1 is the massiliensis subspecies strain FJ05231; M2 is the massiliensis subspecies strain FJ12077; B is the bolletii 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 manners

[0021] The following examples are used to illustrate the present invention, but not to limit the scope of the present 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.

[0022] Example 1 Analysis of the genomic differential regions of Mycobacterium abscessus subsp. abscessus In the present invention, more than 2,000 genomes of Mycobacterium abscessus in the NCBI database were subjected to average nucleotide identity (ANI) and core genome phylogenetic analysis to obtain accurate subspecies classification of all genomes. Through further genomic difference analysis between subspecies, regions with obvious differences between two different subspecies were identified, which can be used for subspecies differentiation and identification. This sequence difference exists in all genomes within the subspecies and is a common feature within the subspecies. Taking the genomic sequences of the type strains of the three subspecies as an example, the differences between these two sequences among the subspecies are shown ( Figure 1). The first differential region is located at the coding genes of polysaccharide deacetylase family protein and CsbD family protein on the genomes of M. massiliense and M. abscessus subsp. abscessus, with a 227-bp spacer region between the two genes. In M. abscessus subsp. Bolletii, these two genes are separated by the coding gene MASB_RS06435 of penicillin acylase family protein. The penicillin acylase family protein gene is specific to M. abscessus subsp. Bolletii, and there is no full-length homologous sequence in the genomes of the other two subspecies, but it has a relatively high sequence identity with partial sequences of the 227-bp gene spacer region in the other two subspecies. The second differential region is located at three consecutive coding genes of ABC1 kinase family protein. On the genome of M. abscessus subsp. abscessus, two coding genes of GlcG / HbpS family heme-binding protein and SDR family oxidoreductase are inserted in reverse between the second and third genes. These two genes are specific to M. abscessus subsp. abscessus and there are no matching fragments in the genomes of M. massiliense and M. abscessus subsp. Bolletii.

[0023] Example 2 Development of specific PCR primers for Mycobacteroides abscessus subsp. Based on these two sequences, the present invention designs specific PCR primers for Mycobacteroides abscessus subsp. for detecting M. abscessus subsp. abscessus, M. abscessus subsp. Bolletii, and M. abscessus subsp. Massiliense.

[0024] When designing primers for Mab-1, initially, forward primers: 5’-AACACTCCGTCGCCGTTG-3’ and reverse primer 5’-AGTTGACCGGCAAGTAGTTC-3’ were designed based on three highly conserved regions of different subspecies. The products of different subspecies were significantly different. The software predicted that the TM values were close, the self-complementarity was low, and there was no or weak secondary structure. However, after actual synthesis, the TM values differed greatly. There were non-specific bands at lower annealing temperatures and insufficient amplification efficiency at higher temperatures. Subsequently, the position of the forward primer was changed, and the actual TM values were closer. Although the product was longer, the amplification specificity and amplification effect were better than the former. When designing Mab-2, multiple primer pairs were designed simultaneously, namely 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’. The design principle was the same as that of the Mab-1 primer pair. The PCR effects of different primers were tested under different temperature conditions, and the primer pairs with the best amplification effect determined by comprehensive comparison were Mab-1 (SEQ ID NO:1-2) and primer pair Mab-2 (SEQ ID NO:3-4). Among them, the Mab-1 forward primer was located downstream of the MAB_RS06440 gene (positions 702 to 720) of Mycobacterium abscessus reference genome ATCC 19977 (GCF_000069185.1), and the Mab-1 reverse primer was located at the 3' end of the MAB_RS06445 gene (894-910, and there were also 3 bp in the non-coding region). The Mab-2 forward primer was located downstream of the MAB_RS17785 gene (positions 1187 to 1206), and the Mab-2 reverse primer was located upstream of MAB_RS17800 (positions 151-132).

[0025] The primer blast was used to perform primer alignment analysis on the core nt library of 5 genera related to Mycobacterium (Mycobacterium, Mycobacteroides, Mycobacteracella, Mycobacteroides, Mycobacteriella) to detect the specificity of the primers. The results showed that except for Mycobacterium abscessus, all genomes including Mycobacteroides chelonae in the genus Mycobacteroides could not produce amplification products, indicating that the primers had high specificity.

[0026] The specific sequences are as follows: Mab-1 forward primer (upstream primer): 5’- AGTTGACCGGCAAGTAGTTC-3’, Mab-1 reverse primer (downstream primer): 5’- CAGCTATGGCAGCAGAGAG-3’; Mab-2 forward primer (upstream primer): 5’- TGCCTCCCGTCTACCTGATG-3’, Mab-2 reverse primer (downstream primer): 5’- CCGCCTTCTCCAAGAGTTCG-3’.

[0027] The schematic diagram of the subspecies-specific fragment of the present invention is shown in Figure 1 , where A and B respectively indicate the designed regions of primers Mab-1 and Mab-2, and the same color blocks represent the sequence homologous regions. The arrows mark the positions of the primer pairs on the genome. The simulated amplification results of the three subspecies are shown in Figure 2 .

[0028] Example 3 Identification of Bacterial Strains Isolated from Colonies on Plates 1) Streak inoculate the strains of Mycobacterium abscessus subsp. abscessus ATCC19977, Mycobacterium abscessus subsp. abscessus FJ09038, Mycobacterium massiliense subsp. massiliense FJ05231, Mycobacterium massiliense subsp. massiliense FJ12077 and Mycobacterium bolletii DSM 45149 on a 7H10-OADC plate by the three-phase line method and culture at 37°C for 3 days.

[0029] 2) Pick a single colony with a pipette tip, grind it into 100 μL of water to prepare an amplification template.

[0030] 3) Prepare a 20 μL reaction system using TSINGKE TSE101 Gold Mix (green) (Beijing Tsingke Biotechnology Co., Ltd.) as follows:

[0031] 4) Amplify on the machine with the reaction conditions: 98°C for 2 min, (98°C for 10 s, 58°C for 10 s, 72°C for 20 s) for 30 cycles, 72°C for 5 min, 4°C ∞. The whole process takes 27 min.

[0032] 5) Perform 1% agarose gel electrophoresis, load 3 μL of the sample, and read the results with a Bio-rad gel reader.

[0033] 6) The strain judgment is as shown in Figure 3 .

[0034] 7) The detection sensitivity is 100%. The sensitivity of the PCR detection technology can reach 3 RFU (plaque-forming units), and the minimum detection rate in bacteriology is 3 bacteria. When using a plasmid as a reference, the detection limit of ordinary PCR is about 1.0×10 4 copies / μl.

[0035] Example 4 Detection in Sputum Samples 1) Simulated sputum samples of strains of subspecies abscessus ATCC19977, subspecies abscessus FJ09038, subspecies massiliense FJ05231, subspecies massiliense FJ12077, and subspecies bolletii DSM 45149. Depending on the viscosity of the sputum specimen, add 1 - 2 volumes of 4% NaOH solution, tighten the screw cap, and vortex for 30 s to homogenize the specimen; let it stand at room temperature until fully liquefied (no obvious solid matter and no filament-detaching phenomenon when aspirating), for no more than 15 minutes, then centrifuge at 12,000 rpm for 5 minutes; rinse the precipitate with sterilized normal saline and resuspend it in 200 μL of normal saline.

[0036] 2) Prepare a 20 μL reaction system using 2×EasyTaq® PCR SuperMix (TransGen Biotech), with the following components:

[0037] 3) Perform amplification on the machine, with the reaction conditions: 95°C for 5 min, (95°C for 30 s, 58°C for 30 s, 72°C for 1 min) for 30 cycles, 72°C for 5 min, 4°C ∞. The whole process takes 70 min.

[0038] 4) Perform 1% agarose gel electrophoresis, load 3 μL, and read the results using a Bio-rad gel reader.

[0039] 5) Bacterial species determination is as Figure 3 shown.

[0040] Example 5 Identification of Bacterial Species in Cultures 1) Fresh Lowenstein-Jensen medium cultures of strains of subspecies abscessus ATCC19977, subspecies abscessus FJ09038, subspecies massiliense FJ05231, subspecies massiliense FJ12077, and subspecies bolletii DSM 45149. Use an inoculation loop to scrape a certain amount of the culture from the Lowenstein-Jensen medium and make a bacterial suspension with 400 μL of normal saline.

[0041] 2) Extract genomic DNA by the CTAB method or heat in a 100°C water bath for 5 minutes, centrifuge at 12,000 rpm for 5 min, and take the supernatant to prepare a boiled template.

[0042] 3) Use Premix Taq ™ (TaKaRa Taq ™ , Version 2.0) (TAKARA) to prepare a 20 μL reaction system, with the system as follows:

[0043] 4) Perform amplification on the machine, with the reaction conditions: 95°C for 5 min, (95°C for 30 s, 58°C for 30 s, 72°C for 1 min) for 30 cycles, 72°C for 5 min, 4°C ∞. The whole process takes 70 min.

[0044] 5) Perform 1% agarose gel electrophoresis, load 3 μL of the sample, and observe the results using a blue light gel cutting instrument.

[0045] 6) The strain identification is as Figure 3 shown.

[0046] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. Specific PCR primers for Mycobacteroides abscessus subsp., characterized in that: Specific PCR primers for detecting M. abscessus subsp. abscessus, M. abscessus subsp. Bolletii, and M. abscessus subsp. Massiliense include primer pair Mab-1 and primer pair Mab-2, the sequences of which are as follows: Mab-1 forward primer: 5'-AGTTGACCGGCAAGTAGTTC-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 primers according to claim 1.

3. Use of the primers according to claim 1 or the detection reagent or kit according to claim 2 in the identification of Mycobacterium abscessus subspecies.

4. A method for identifying subspecies of Mycobacterium abscessus, characterized in that: The following steps are involved: 1) Extract DNA from the sample to be tested; 2) Using the extracted DNA or original sample as a template, perform PCR amplification using primer pair Mab-1 and primer pair Mab-2 respectively; 3) Analyze PCR amplification products; The method is not for disease diagnosis purposes.

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

6. The method according to claim 4 or 5, characterized in that: Step 3) includes: ① The amplification results corresponding to the primer pair Mab-1: the amplification product size of the abscess subspecies and the massi subspecies is 410 bp, and the amplification product size of the bolai subspecies is 2599 bp; ② The amplification results corresponding to the primer pair Mab-2: the amplification product size of the abscess subspecies was 1775 bp, and the amplification product size of the Masai and Bolai subspecies was 386 bp; ③ Determine the type of Mycobacterium abscessus subspecies contained in the sample to be tested based on the size of the amplified product.

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