A targeted sequencing primer set, kit and application for mycobacterium identification and drug resistance detection

Abstract

The application belongs to the technical field of molecular biology detection, and particularly relates to a target sequencing primer group, a kit and application for mycobacterium identification and drug resistance detection. The detection method and the kit provide a primer group, a method, a kit and application for MTBC and its drug resistance mutation, NTM and other pathogen differential diagnosis. The primer group, the method, the kit and the application can perform differential diagnosis on 81 pathogens, including 3 MTB, 4 anti-tuberculosis first-line drugs, 38 clinically common NTM and 40 other pathogens, can realize tuberculosis identification and drug resistance detection, and can realize differentiation from the clinically common NTM and other pathogens. A touchdown PCR amplification method and a kit are used. The touchdown PCR can effectively improve the tuberculosis and mutation primer amplification efficiency and improve the tuberculosis detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a targeted sequencing primer set, reagent kit, and application for mycobacterial identification and drug resistance detection. Background Technology

[0002] Mycobacterium tuberculosis, commonly known as tuberculosis bacillus, is the pathogen that causes tuberculosis. The Mycobacterium tuberculosis complex mainly includes human-type Mycobacterium tuberculosis, bovine-type Mycobacterium tuberculosis, murine-type Mycobacterium tuberculosis, and African-type Mycobacterium tuberculosis. Among these, human-type, bovine-type, and African-type Mycobacterium tuberculosis are pathogenic to humans, while murine-type Mycobacterium tuberculosis does not cause tuberculosis in humans but mainly causes tuberculosis in rodents. Non-tuberculous mycobacteria (NTM) refer to a large group of mycobacteria excluding the Mycobacterium tuberculosis complex (including Mycobacterium tuberculosis, bovine, African-type, vole, goat, Pinnipedii, Suricattae, and Mungi) and Mycobacterium leprae. To date, more than 190 species and 14 subspecies of NTM have been discovered, most of which are parasitic, with only a small number being pathogenic to humans, belonging to the category of opportunistic pathogens. NTM disease refers to infection with NTM in humans, causing lesions in related tissues and organs. In recent years, NTM diseases have been increasing rapidly, becoming one of the important public health problems threatening human health.

[0003] High-throughput sequencing technology has significant advantages in the etiological diagnosis of infectious diseases and the diagnosis of drug resistance in common pathogenic microorganisms. It is also being used more and more widely in the field of mycobacterial diseases, providing evidence-based medicine for the diagnosis, differential diagnosis and drug resistance diagnosis of difficult mycobacterial diseases.

[0004] High-throughput sequencing methods are mainly divided into targeted sequencing, metagenomic sequencing, and whole-genome sequencing. Mycobacterial targeted sequencing technology first targets and captures mycobacterial-specific genes before performing high-throughput sequencing. It is primarily suitable for patients clinically suspected of having mycobacterial infection, aiding in the diagnosis of tuberculosis / non-tuberculous mycobacterial NTM disease and detecting drug resistance gene mutations, thus providing precise guidance for clinicians in medication use. The 2018 WHO Technical Guidance on the Detection of Drug-Resistant Tuberculosis-Related Gene Mutations Using High-Throughput Sequencing Technology emphasized the function of targeted sequencing technology in the diagnosis of drug-resistant tuberculosis.

[0005] The pathogen detection techniques for Mycobacterium tuberculosis are shown in Table 1 below.

[0006] Table 1. Pathogen detection techniques for Mycobacterium tuberculosis

[0007]

[0008] 1. Microscopic examination of smears

[0009] Similar to Mycobacterium tuberculosis, smear microscopy with fluorescent staining is recommended. Raney staining is still acceptable, but with a lower positive rate. Some NTMs, especially fast-growing mycobacteria, are less resistant to alcohol destaining compared to Mycobacterium tuberculosis, making false negatives more likely. Although some NTM species may exhibit distinctive acid-fast morphology, smear microscopy alone cannot differentiate between Mycobacterium tuberculosis and NTMs.

[0010] 2. Isolation and Culture

[0011] Some NTMs require special culture media, specific incubation temperatures, or longer incubation times. For example, *Haemophilus haemolyticus* can only grow on iron-containing media, while *Mycobacterium ulcerans* is easier to culture with the addition of egg yolk. The optimal growth temperature for *Mycobacterium ulcerans* is 25–33 °C, for *Mycobacterium marinum* it is 28–32 °C, and for *Mycobacterium bufo* it is 45 °C. For skin tissue specimens, it is recommended to conduct parallel incubation at two temperature gradients: 28–30 °C and 35–37 °C, to improve the positive rate. Slow-growing mycobacteria generally require 2–3 weeks or even longer to form visible colonies on solid media, therefore isolation and culture are usually time-consuming. Preliminary species identification can be achieved, but it is only used for preliminary differentiation between MTB complexes and NTMs. Commonly used methods include the p-nitrobenzoic acid selective medium method: after culturing the mycobacterial strain, the strain is inoculated into p-nitrobenzoic acid medium; most NTMs can grow, while MTBs cannot. MBP64 antigen detection method: MPB64 is one of the main proteins secreted by the MTB complex during growth in liquid culture medium, while NTMs mostly do not secrete this protein. Therefore, it can be used for preliminary identification of cultured mycobacteria, but this method cannot identify NTM species.

[0012] 3. Molecular detection technology

[0013] (1) Quantitative Real-Time PCR: PCR technology has become the "gold standard" for identifying mycobacterial species. Commonly used sequences for mycobacterial species identification include the 16S RNA coding gene (16S DNA), the 16S-23S rRNA intergenic region (ITS), the β subunit of RNA polymerase (rpoB), and the heat shock protein 65 (HSP65) coding gene. It has high detection sensitivity and can detect pathogens and drug resistance genes, but the number of detection targets is limited.

[0014] (2) Metagenomic next-generation sequencing technology: It helps in early identification of bacterial species and rapid diagnosis. The test results are less affected by antibiotic treatment and can also be used to track the spread of NTM in specific populations. With the increasing popularity and lower cost of metagenomic next-generation sequencing technology, it will play an increasingly important role in the diagnosis of skin NTM diseases.

[0015] (3) Whole genome sequencing technology: Sequencing the entire genome of an organism can obtain complete genomic information, which is a method for further identification of NTM species and subspecies. However, it is expensive and not suitable for routine diagnostic screening.

[0016] (4) Targeted sequencing: target fragments are captured by multiplex PCR or probes, and the target fragments are sequenced. Combined with bioinformatics comparison analysis, pathogen identification and drug resistance gene detection can be achieved. This method has high sensitivity and more detection targets than PCR technology, and has been widely used in clinical practice.

[0017] Existing solutions:

[0018] Based on authoritative guidelines, consensus statements, and literature, the testing scope is determined to cover Mycobacterium tuberculosis identification and drug resistance, common clinical NTMs, and co-infectious or differentially diagnosed pathogens. Specific primers are designed for each pathogen, and multiplex amplification targeted sequencing technology is used to differentiate between 3 MTBs, 4 first-line anti-tuberculosis drugs, 38 common clinical NTMs, and 40 other pathogens. It can identify MTBs, indicate tuberculosis drug resistance, differentiate common clinical NTMs from other pathogens, and distinguish mixed infections.

[0019] The main drawback is:

[0020] Mycobacterium tuberculosis and non-tuberculous mycobacteria are difficult to distinguish morphologically and through acid-fast staining of images, leading to significant differences in their clinical treatment. Many non-tuberculous mycobacteria exhibit natural resistance to anti-tuberculosis drugs, resulting in poor response to conventional chemotherapy. Therefore, the identification of mycobacteria is currently of great importance for the diagnosis, differential diagnosis, and effective treatment of tuberculosis. Studies show that retreated tuberculosis requires a longer diagnostic time compared to newly diagnosed tuberculosis (73 days vs. 35 days); imaging examinations and molecular biological detection methods have low specificity for the diagnosis of retreated tuberculosis, posing a significant challenge to its diagnosis. The clinical and imaging manifestations of retreated pulmonary tuberculosis often lack specificity. In the absence of positive etiological evidence, a comprehensive judgment needs to be made by combining clinical symptoms, treatment history, and comparison with previous imaging data. Furthermore, it is necessary to differentiate it from other infectious and non-infectious pulmonary diseases. Accurate and rapid differentiation of mycobacteria from other infectious and non-infectious diseases can avoid misdiagnosis, mistreatment, missed diagnosis, and overdiagnosis, enabling individualized treatment and contributing to the control of the spread of tuberculosis and NTM (non-tuberculous tuberculosis).

[0021] Most current methods for identifying mycobacteria are based on multiplex PCR technology, including real-time quantitative PCR, isothermal amplification, probe-reverse hybridization, and probe-melting curve techniques. These methods are convenient and fast, but they are mostly based on a specific SNP site for species identification, limiting the number of species that can be identified and making it difficult to identify subspecies. First-generation sequencing methods for species identification are mostly based on the 16S rRNA region, but the 16S rRNA region has insufficient resolution, and some closely related species cannot be accurately identified.

[0022] Therefore, developing an efficient and accurate methodology and primer set for mycobacterial identification is crucial for identifying Mycobacterium tuberculosis and its drug-resistant mutations, covering first-line tuberculosis drugs such as rifampin, isoniazid, ethambutol, and pyrazinamide; and for differential diagnosis of common clinical NTM and other pathogens. This has significant application value in the detection of tuberculosis / drug-resistant tuberculosis / NTM disease. Summary of the Invention

[0023] The purpose of this invention is to address existing problems by providing a targeted sequencing primer set, kit, and application for mycobacterial identification and drug resistance detection.

[0024] This invention is achieved through the following technical solution:

[0025] A targeted sequencing primer set for mycobacterial identification and drug resistance detection, the primer set comprising primers with nucleotide sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 206.

[0026] Furthermore, the 5' end of each primer also contains a universal sequence that can bind to the sequencing primers of the sequencing platform;

[0027] The universal 5' sequence of the upstream primer in the primer set is CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT, and the universal 5' sequence of the downstream primer in the primer set is TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT.

[0028] A kit for mycobacterial identification and drug resistance detection, comprising the aforementioned targeted sequencing primer set.

[0029] Furthermore, it also includes reverse transcriptase, DNA polymerase, DNA purification magnetic beads, nuclease-free water, anhydrous ethanol, and Qubit quantitative reagent.

[0030] Furthermore, the kit is used to detect MTBC, NTM, and Staphylococcus aureus, Klebsiella pneumoniae, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, and Aspergillus flavus.

[0031] The application of the targeted sequencing primer set or the kit described herein in the preparation of products for the identification of mycobacteria, detection of drug-resistant mutations in Mycobacterium tuberculosis, differential diagnosis of nontuberculous mycobacteria and other pathogens, or differentiation of mixed infections.

[0032] A targeted sequencing method for mycobacterial identification and drug resistance detection includes the following steps:

[0033] (1) Extract total nucleic acids, including DNA and RNA, from the sample to be tested;

[0034] (2) Reverse transcribe the RNA obtained in step (1) into cDNA;

[0035] (3) Using the targeted sequencing primer set described above, perform multiplex PCR amplification on the templates in steps (1) and (2);

[0036] (4) The reaction product obtained in step (3) is purified and amplified twice to obtain a sequencing library;

[0037] (5) Perform high-throughput sequencing on the sequencing library obtained in step (4) to obtain sequencing sequences;

[0038] (6) Perform bioinformatics analysis on the sequencing sequence in step (5), compare it with the pathogen sequences in the database, and determine the type of pathogen and drug resistance gene mutation in the sample to be tested.

[0039] Furthermore, the high-throughput sequencing uses the BGI sequencing platform or the Illumina sequencing platform.

[0040] Furthermore, when using the MGI sequencing platform, the sequencing mode was SE50, and the data volume was 2M.

[0041] The present invention has the following advantages over the prior art:

[0042] 1. The present invention provides a primer set for the differential diagnosis of MTBC (Mycobacterium tuberculosis complex) and its drug-resistant mutations, NTM (non-tuberculous Mycobacteria), and other pathogens. The method, kit, and application can differentiate and diagnose 81 pathogens, including 3 MTBs (Mycobacterium tuberculosis), 4 first-line anti-tuberculosis drugs, 38 clinically common NTMs, and 40 other pathogens. It can achieve tuberculosis identification and drug resistance detection, as well as differentiation from clinically common NTMs and other pathogens. A landing PCR amplification method and kit are also provided. Using landing PCR can effectively improve the amplification efficiency of primers for tuberculosis and its mutations, thereby increasing the sensitivity of tuberculosis detection.

[0043] 2. This invention can detect MTB and drug resistance mutations, differentiate and diagnose common clinical NTMs and other pathogens, and distinguish mixed infections. Attached Figure Description

[0044] Figure 1 This is the result of the landing PCR detection. Detailed Implementation

[0045] To further explain the present invention, the following specific embodiments are described.

[0046] 1. This invention provides a list of pathogens and primer sets for identifying Mycobacterium tuberculosis, non-tuberculous mycobacteria, and other pathogens or mixed infections. The pathogen list is as follows:

[0047] Mycobacterium tuberculosis complex, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium abscessus, Mycobacterium abscessus subspecies, Mycobacterium abscessus masai subspecies, Mycobacterium abscessus borei subspecies, Mycobacterium avium complex, Mycobacterium avium, Intracellular Mycobacterium, Mycobacterium turcica, Mycobacterium smegmatis, Mycobacterium aureum, Mycobacterium neo-aureum, Mycobacterium non-chromogenic, Mycobacterium azoospermum, Mycobacterium bufota, Mycobacterium Gordon, Mycobacterium ulcerans, Mycobacterium marineum, Mycobacterium surugani, Mycobacterium simianum, Mycobacterium commune, Mycobacterium marmosetum, Mycobacterium scrofula, Mycobacterium haemophilus, Mycobacterium kansasii, Mycobacterium chimera, Mycobacterium stoloniferum, Mycobacterium myxogenerum, Mycobacterium asiaticum, Mycobacterium lataniae, Mycobacterium nevarii, Mycobacterium schrenckii, Mycobacterium auricularis, Mycobacterium canaliculata Mycobacterium leprae, Mycobacterium thermosepticum, Mycobacterium triplex, Acinetobacter baumannii, Burkholderia melioides, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Legionella pneumophila, Nocardia dermatophytes, Nocardia gaillesii, Nocardia abscessii, Nocardia asteroides, Nocardia guinea pig otitis media, Nocardia brassicae, Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis Fungi, Candida parapsilosis, Candida guinea, Cryptococcus neoformans, Cryptococcus gutella, Pneumocystis jirovecii, Blue styloides marneffei, Histoplasma capsulatum, Mucor, Rhizopus, Rhizopus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Human herpesvirus type 5 (CMV), Balamchia amoebae, Trichomonas vaginalis, Leishmania, Plasmodium;

[0048] Table 2 Primer set

[0049] Number Forward primer Reverse primer 1 GTGATCCTTCGAAACGACCAG GATGAACGTCGAGAGCAGC 2 ACCTGAAACCGTGTGCCTAC GGTCCAGAACACGCCACTAT 3 AGTCTGTTGATGAGGCGGTC CTCATCGGTGACCCCGTC 4 GATGGGATCGATGGTGAACG TTGCCCGACATCAATATTCCG 5 GAGGGACTTGCGGCTATTC CAGGACCTGAGAATGAACGC 6 GTCACGAACATGAAAATCCGTT CGGAAAACTCCTTGATCCTGG 7 GCCGGTATGTCGTTCGTC TGTCGTCAAAGATGGACCAGCT 8 TTGTTGGTACCGATATTGCCG GCGATCTCAATACCGCTATCG 9 ACGGATCGTTCGTCAAGCG CGACGTCGACGAGATAACGG 10 GACCGCATCGAATGGCTTAG ACCATGCCCTGCCCAAATC 11 AACCTGTGCGCCAATATCTTC ACTTCTACATCGCCTACCGG 12 ACAACTGAGTCGTCTGATGGG TCCGAATCCGTCACTATCCAG 13 TTGGCGATACCGAATTGGATG CGTAATCCAAGATGTGCTCGG 14 CATGACAACGATCTAACTCCGG TGCATGTCCCCTTGTTGAATG 15 TGTGAATTCATACAAGCCGTAGTC CCCGTAGCGTTACTGAGAAATTGC 16 TGCGACTGAACACAATGTCAG CGTCGGGTGCAATATTGGTAG 17 TTCGAAATCATCAACCGCCAG CACAAAACAAACACCACACCC 18 AAAGAACCACGAGCATGGAAC CTCATCCTTGGCAAATCGACC 19 CTTCTCAGTGACCAGCAGTTC GTAAGTACTCCGCCTGTTTGG 20 TATCCAAATGCTCTTGGCGAC CTGGTCGAACCGGGAGTAATC 21 CCGTTGATGCTCATCGATGTC TTCGTCCGTACTTCTCTACCG 22 ATCCACACTCGCTACTCATCG TATATCCACCAGTACCGCACC 23 CTGACCGATGATCAGCATGG GTCATGTATGCCAGCACCATC 24 CTTCGCCGATTCAAATTTGGC CCATTGGGTTTCTCTTCCAGC 25 ATCGCCTCGAAATGGTTTCAG TCATTTCTGGGAAATCACCGC 26 AGATGAACAGGTTGGACCCC CTATTACATCGACGTGGCACC 27 GGCATTTCGTCGTACAGTTGG TCGAGGTCGAGTTACCAGATG 28 GAATTGATCACCGACCGCTTC CATGCACATCCAGCAGTAGTC 29 CTTGGCCGATAACGACTTAGC TCCTTTGCTGCGCATAGATTG 30 CCATAACCCGAGTCATGTTGC CATGGTAGCCACGGTTCTTAC 31 GTAACCGAACACGACCACAC TCTCGACGTACCCGGATTG 32 CTGGTCGACTTCGTCAAAGAC CTTTGAGGACGTCGTTCATGG 33 TGGGATGGACAAACGGC GTCACCCGTTCAGTCTCCAC 34 CGAACCATTGTCCACTTGGG TGGAAACCGAAATCCTTTGGG 35 ACGTCGTAGTACTCGGACTTC CGATATCGTGACGACATGGAAG 36 ATCTACGAGAGGGTGAGGAGG TTCTTGTCCGGATGCAAATCG 37 CTTGCCAGAACTCTACGAACG GGATGGATCGTGTAATGCAGC 38 CCGCAGATGGCCAGATAGTAG GTTGCGAATCGTGTTGACAC 39 GCCCTGTGCCATGATTTGT GCTCGACGTCGATTGGGA 40 GTCGACAGGGGAGTGAGTATC CTTTCTCAACGGCCAGTACAC 41 GTTCATGGACTCAGTCTGTGC GGAATCCCGATGCACTACTTC 42 ACTATCACCACAAAATCGCCG TGGATCAAAACCGTTGGTGC 43 ACGATCAGATCCGGTTTGAGG ATGACCTGGGACGGATACTTC 44 GATTCGACGCGTTTGTTCCAC CAATCACCCTGCTCTGTGAAG 45 GCGCAAGTCCGAACTGTATG CGTCAACAACAGCGTGGAAA 46 AATGTGGAGAAAGCGCGCAT CGTCAACAACAGCGTGGAAA 47 CCGAGACCATGGGCAACTAC CTCGTCGATTTCCCTCAGCA 48 CGCTGTCGGGGTTGACC GCGGTCAGGTACACGATCTC 49 CCCAGCGCCGACAGTC TCGGCGAGCTGATCCAAAAC 50 CCGATCGAAACCCCTGAGG GCGGTCAGGTACACGATCTC 51 ATGGTCTCGTCGAAGTACAC CTGACCCTCGTTTCGACGAT 52 CGATCAAGGAGTTCTTCGGC GCGGTCAGGTACACGATCTC 53 CGCTCGTGGACATACCGATT CATTCGACGCCAAACAGCC 54 GCTGGATGAGCGGATTCAG GTCAGTGGCCCATACCCATG 55 TCACAGCCCGATAACACCAA CTCCGCGGCATAGTCGAAC 56 GAGCTCGTATGGCACCGG TCCGCTCATAGATCGGATCC 57 CGGCATCGAGGTCGTATGG TCCGCTCATAGATCGGATCC 58 CCGTCATCCTGACCGTGG TCAGCAAGACCATGGCCG 59 CATGTCATCGGCGCGAATTC TCAGCAAGACCATGGCCG 60 TGTCCAACTATTTCCGCTGGT TCAGCAAGACCATGGCCG 61 CAGCGACGCCAGTCTGTG TCAGCAAGACCATGGCCG 62 GTTCAACAACGGCCTGCG GATGACGGCGCACCAAGATC 63 ATCCTCCGGGCTGCCG AGCTCCTCCTCAGGCCG 64 AACCGCGGAAGAAAGAAATTG TCGGAAGCGCCTAGAAAAAAG 65 CGTTCTGCGAAAACCGAAA ACCAACCCCCAGTTTCCCG 66 CCGTGATGCCCTTTAGATGC TCCTGGTTCATGACGCTGAT 67 GCGTGGTGAGATTGAAGTCAG TAGTCAACTCCAGCGATCCTG 68 TTACCAGATATTTGCGCTGCC ATGAGTTGGCCAACATCGATC 69 CCCATCCACAGGCACAGAT ATTGACACCGCTCGTGCG 70 TATCAAGCAAAACGAGAGCGG AGGATGCTTGAAGAAACGTCG 71 GTCGAGAGCTTTCCGTAATCG ACTCTGTCTACGCTCCATCAG 72 CCATGTTGGGTCCAAGTGATG GTTTGCCGAGTTTGGTCATTC 73 GTTAAACGTTCTGCTTGCCAC ACAGTGGCCATTTGAAGCAAC 74 AACTGAGTCTCTGTCACCTGG TTGGAATCCGTTTGGTCAAGG 75 CTCAGCGGGGTTTTAGGTTTC CGCAGTCCCTCCAATTGAATC 76 GTGGCCGAGAGAGAAATTGAC ATAACAGAGATGGGGTCGTCG 77 ACAATGGTACCAAACAGCAGG TTCTCCGACATTGCTGACATG 78 ACGGTTCACCACAGGAACAT AGTTGACCCAGAGGGTAGCA 79 TGTGGATATGGAGGAATGGGAG CGGATACCATGTGTGTGTGTG 80 GCCACAGCTAGCAACCGT TGGCTCAGTCCAGGTAAGAG 81 TCGCTATTCGTAATTTGGCCG CCACCTGCTTGAATATCAGCC 82 TTTCATCCATCACATCGCTCC AAGCGACTTCAACCCATCAAC 83 TACAGGATGGAGTGTCGCTAC AGCATTGATCCGTCTTTTCCG 84 CCTTGTTCACGATGGTCTTCG CATCGTGAGCGTCAAGGTC 85 GTGATCCGGTTGTACCACTTG TTGCAGCCATTGTGGAAGATG 86 AGTCGGACAACGCTGAACATG AACCACCAGCACACTCTACG 87 TGATAGAGCACGAGCATCTCC TGTTCCTCACGGTCGTCATC 88 GAACTCCTCGCCATCATGC GAACTGTTGCTACTCGCCG 89 TCGATCCCAACAAGCCCTG CACGATCAGCTCAACAACTCC 90 CGCCTCTGGTCAAGTGGATC GTCACACCGACTATTGGAGC 91 TAAGTGAGTGGCGTGAAGAGG ATTCACGGACAACATTCGGAC 92 AGCAATAAAATACCGGCTGCC CGTGAGGGATTATGCGTTTCC 93 CACTGTTATTCGCTCCTCTGC AGAAGGTCTTGCTGTAGGTGG 94 CATCCTAGATTCCCCCCTCTC AAGAGCGATTGCAGAGAAGAG 95 CTGTTGATCGAGCATTTTCGC TTACAACGCGCCTCTATTGAG 96 GGGATCAAACTCCCGATAACG GTGTTTTAGCATTGCAACCCG 97 GCAGGATTATCCGCAGCTATG CATTTCCAAGACAGGAGGCTG 98 GTCGCGATGGTCCAAAATTTC ATGGACGTGGACTCTTCTCTC 99 CTGTGGCGGAATGTCTAACAG CATGATCAGCAGTGAGTCACG 100 CAAGTCAGATTATCCCGCCAC TGCAATGCTGAGCGATAAGAC 101 TTCTTCCAGTCATCACCCCTC TACCATGCCAGCAACTGAATC 102 CATCCGATACGCACCATTGG CAGCAGATTCTCGACCTCACC 103 TGGTTCAGCCGTATCTTATGG AGGCGTCTTCAGCAGAAACAT

[0050] The 5' end of the primer contains a universal sequence that can be combined with sequencing primers for the MGI platform, Illumina, or other domestic sequencing platforms. For example, when used with the MGI sequencing platform, the universal sequence at the 5' end of the upstream primer is CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT, and the universal sequence at the 5' end of the downstream primer is TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT.

[0051] 2. The present invention provides a detection kit comprising a multiplex PCR specific primer mixture, reverse transcriptase, DNA polymerase, DNA purification magnetic beads, nuclease-free water, anhydrous ethanol, and Qubit quantitative reagent.

[0052] 3. This invention provides a detection method and its application, comprising the following steps:

[0053] Step 1: Extract total nucleic acids, including DNA and RNA, from the sample to be tested;

[0054] Step 2: Reverse transcribe the RNA obtained in Step 1 into cDNA;

[0055] Step 3: Perform ultramultiplex PCR amplification on the templates from Steps 1 and 2 using the amplification primer pool;

[0056] Step 4: The reaction product obtained in step 3 is purified and amplified twice to obtain the tNGS sequencing library.

[0057] Step 5: Perform high-throughput sequencing on the sequencing library obtained in Step 4 to obtain the sequencing sequence;

[0058] Step 6: Perform bioinformatics analysis on the sequencing sequences from Step 5, compare them with pathogen sequences in the database, and determine the types of pathogens in the sample to be tested.

[0059] Example 1: Detection range and primer set for differential diagnosis of MTB, NTM and other pathogens

[0060] 1) Determining the scope of common pathogenic microorganisms causing pulmonary tuberculosis, extrapulmonary tuberculosis, and NTM disease. Through literature review and expert consensus in the field of infectious diseases, the scope of common pathogenic microorganisms was determined, ultimately identifying 81 pathogenic microorganisms for detection. Primer design and evaluation. High-quality pathogenic microorganism genomes were downloaded, a local genome database was constructed, and specific genomic sequences were determined using software to design primer pairs. Primer lengths were set between 18-25 bp, GC content was controlled between 40%-60%, and more than three consecutive identical bases were avoided. Software was used to evaluate dimerization and non-specific amplification.

[0061] 2) Single primer evaluation: Amplification is performed using single primers, and the amplification brightness is evaluated by agarose gel electrophoresis. Primers with similar brightness and amplification efficiency greater than 60% are selected.

[0062] 3) Wet experiment evaluation. Purchase standard strains, verify primer performance, and remove primers that cause primer dimers or have low amplification efficiency.

[0063] 4) Clinical sample evaluation: Using clinical samples, replace or delete primers that are not functioning.

[0064] The detection primer set for the target pathogenic microorganism was obtained through the above development process, as shown in Table 2 above.

[0065] Example 2: Detection Performance Verification

[0066] The detection performance of the kit was verified using simulated samples, and the detection limit of the kit was verified to be 10-50 copies / mL.

[0067] (1) Prepare simulated samples. The list of pathogens in the simulated samples is shown in Table 3 below. The concentration of human cells is 10. 5 The pathogen concentration was 500 copies / mL, and the samples were named P1-1 and P1-2.

[0068] Table 3 List of pathogens in simulated samples

[0069]

[0070] Use NC (containing only 10) for P1-1 and P1-2. 5 Human cells (cells / mL) were serially diluted 10-fold and 50-fold, and named P2-1, P2-2, P3-1, and P3-2, respectively.

[0071] (2) Sample pretreatment

[0072] Gently shake to mix the sample, take 800 μL of the corresponding sample, add 80 μL of lysis buffer L, transfer to a pre-filled grinding tube, tighten the cap to ensure no leakage during cell disruption.

[0073] (3) Automated nucleic acid extraction

[0074] 1) Place the Lysis Tube M containing the sample into the blender and run the blending program (recommended program: 6 m / s vibration intensity, 30 s grinding time, 30 s interval time, 6 cycles). After blending, centrifuge at 12,000 rpm for 3 min to remove foam.

[0075] 2) Transfer 600 μL of supernatant to well A in the deep well plate;

[0076] 3) Finally, add 40 μL of proteinase K to well A of the deep well plate;

[0077] 4) Insert the stirring sleeve into row B of the deep hole plate and place it on the worktable of the automated extractor;

[0078] 5) Start the pre-set program and enable automatic extraction. The running time is approximately 30 minutes.

[0079] 6) After the procedure is complete, remove the deep well plate, transfer the nucleic acid from well F to a 1.5 mL centrifuge tube and measure the Qubit concentration.

[0080] (4) Multiplex PCR

[0081] Add the corresponding PCR primers for the pathogen and the primers for the internal control IC to samples with different known concentrations from step 1) respectively to prepare a one-round multiplex PCR amplification system, as follows:

[0082] The amplification system consisted of 5 μL of primer panel mix, 5 μL of PCR enzyme, 4 μL of H2O, and 1 μL of internal control. The total amplification volume was 25 μL.

[0083] The first round of PCR enriches the target gene and internal reference sequence. The amplification system from step (2) is thoroughly mixed, briefly centrifuged, and then placed on a PCR instrument. Amplification is performed according to the following PCR reaction procedure (Table 4).

[0084] Table 4 PCR reaction procedure

[0085]

[0086] (5) Purify the first-round amplification products, screen for the target fragment, and remove non-specific amplifications such as dimers. After obtaining the target fragment from the first round of PCR, purify it using the Ampure XP magnetic bead purification kit or other equivalent kits to remove excess PCR primers and dimers, effectively enrich the target region fragment, and reduce the concentration of non-specific amplifications. The specific steps are as follows:

[0087] 1) Add 20 μL of AMPure XP Beads to the PCR reaction solution, and use a pipette to mix the amplification product with the AMPure XP Beads thoroughly. Let it stand at room temperature for 5 min.

[0088] 2) Use a magnetic rack to attract magnetic beads until the solution becomes clear.

[0089] 3) Carefully aspirate the supernatant with a pipette and discard it.

[0090] 4) Add 200 μL of 80% ethanol to wash the magnetic beads and discard the supernatant.

[0091] 5) Repeat the previous step.

[0092] 6) Let stand at room temperature for 5 minutes until the ethanol has completely evaporated.

[0093] 7) Add 25 μL ddH2O, vortex until homogeneous, and let stand at room temperature for 1-2 minutes.

[0094] 8) Aspirate 20 μL of supernatant for the next step.

[0095] (6) Perform a second round of PCR on the recovered products, ligating the corresponding sequencing adapters and barcodes. The second round of PCR reaction system is as follows: PCR Mix 25 μL, Index Primer 5 μL. Run the PCR reaction program as follows (Table 5):

[0096] Table 5 PCR reaction procedure

[0097]

[0098] (7) Purify the second round of PCR amplification products.

[0099] The obtained product was purified using the Ampure XP magnetic bead purification kit or other equivalent kits. The purification steps are as follows:

[0100] 1) Add 50 μL of AMPure XP Beads to the PCR product and pipette up and down to mix the recovered product with the AMPure XP Beads thoroughly. Let stand at room temperature for 5 min.

[0101] 2) Adsorb magnetic beads onto the magnetic rack for 2 minutes until the solution becomes clear.

[0102] 3) Carefully aspirate the supernatant with a pipette, discard the supernatant, and keep the magnetic bead.

[0103] 4) Add 200 μL of 80% ethanol to wash the magnetic beads and discard the supernatant.

[0104] 5) Repeat the previous step.

[0105] 6) Let stand at room temperature for 5 minutes until the ethanol has completely evaporated.

[0106] 7) Add 25 μL ddH2O, vortex until homogeneous, and let stand at room temperature for 1-2 minutes.

[0107] 8) Pipette 20 μL of supernatant into a new 1.5 mL centrifuge tube. The product can be used directly for subsequent experiments or stored at -20 °C. The remaining library in the PCR tube can be used for electrophoresis quality control.

[0108] (8) Document quality inspection

[0109] 1) Library electrophoresis: A portion of the library was diluted to 1 ng / μL, and Qsep was used to check the size of the library fragments. The average size of the correct library fragments was 300 bp.

[0110] 2) Library quantification: Take 1 μL of the library elution product, add 199 μL of Qubit dye (diluted to working concentration), mix thoroughly, incubate in the dark for 2 min, and then use the Qubit instrument for quantification.

[0111] (9) Mixed storage preparation and machine operation

[0112] All libraries were mixed at a 1:1 mass (ng) ratio according to the quantification results. The sequencing platform was BGI MGIseq200, and the sequencing kit was the MGISEQ-200RS high-throughput sequencing kit (FCL SE50).

[0113] The sequencing mode was SE50, and the data volume was 2M.

[0114] (10) Basic filtering analysis of offline data

[0115] The cutadapt software was used to remove sequencing adapters from the reads in the offline data. Reads with a Q20 of less than 85% and reads with a high N content were then used to match the reads. The non-specifically amplified reads were removed based on the matching results.

[0116] (11) Compare the data with the template to determine the number of reads of the target gene.

[0117] Based on pre-designed primers, the amplification product sequences of simulated PCR were constructed into a reference FASTA file for multiple target genes. The sequencing reads were then compared with the reference FASTA file using bwa software. Reads with mismatches not exceeding 10% of the total length were considered to have aligned to the target gene. After alignment, the number of aligned reads for each pathogen could be directly counted.

[0118] Table 6 Test Results

[0119]

[0120] Example 3: Consistency of Clinical Sample Testing

[0121] Twenty clinical samples were selected and tested using this kit. An approved qPCR reagent or an LDT product with the same methodology was used as a comparison reagent. The testing method was as described above. Analysis showed a 100% concordance rate for the clinical samples. The test results are shown in Table 7 below.

[0122] Table 7 Pathogen Detection Results

[0123]

[0124] The mutation detection results are shown in Table 8 below: Detection consistency rate 100%.

[0125] Table 8 Mutation Detection Results

[0126]

[0127] Example 4: An optimized kit with a multiplex amplification program using landing PCR, which can effectively improve the amplification efficiency of tuberculosis and its drug-resistant primers; the landing PCR program is shown in Table 9 below.

[0128] Table 9 Landing PCR Procedure

[0129]

[0130] For example, the detection results are...Figure 1 As shown, this demonstrates that landing PCR can effectively improve the detection rate of tuberculosis.

[0131] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A targeted sequencing primer set for mycobacterial identification and drug resistance detection, characterized in that, The primer set comprises primers with nucleotide sequences as shown in SEQ ID NO. 1 to SEQ ID NO.

206.

2. The targeted sequencing primer set according to claim 1, characterized in that, Each primer also contains a universal sequence at its 5' end, which can bind to the sequencing primers of the sequencing platform; The universal 5' sequence of the upstream primer in the primer set is CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT, and the universal 5' sequence of the downstream primer in the primer set is TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT.

3. A kit for mycobacterial identification and drug resistance detection, characterized in that, It includes the targeted sequencing primer set as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, It also contains reverse transcriptase, DNA polymerase, DNA purification magnetic beads, nuclease-free water, anhydrous ethanol, and Qubit quantitative reagent.

5. The reagent kit according to claim 3, characterized in that, The kit is used to detect MTBC, NTM, Staphylococcus aureus, Klebsiella pneumoniae, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, and Aspergillus flavus.

6. The use of the targeted sequencing primer set according to claim 1 or 2 or the kit according to any one of claims 3 to 5 in the preparation of products for the identification of mycobacteria, detection of drug resistance mutations in Mycobacterium tuberculosis, differential diagnosis of nontuberculous mycobacteria and other pathogens, or differentiation of mixed infections.

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