Primer probe combination and kit for identifying Mycobacterium tuberculosis and Mycobacterium avium complex

Abstract

The invention belongs to the technical field of biology, and particularly relates to a primer probe combination and a kit for identifying a mycobacterium tuberculosis and mycobacterium avium complex. The composition for detecting mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 1 or SEQ NO: 2; the nucleotide sequence of the downstream primer is as shown in SEQ NO: 3 or SEQ NO: 4 or SEQ NO: 5; a probe having a nucleotide sequence as shown in SEQ NO: 6; the composition for detecting the Mycobacterium avium complex comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ NO: 7 or SEQ NO: 8 or SEQ NO: 9; the nucleotide sequence of the downstream primer is as shown in SEQ NO: 10 or SEQ NO: 11 or SEQ NO: 12; the nucleotide sequence of the probe is as shown in SEQ NO: 13. A new primer probe combination is screened out and used for differential diagnosis of Mtb and MAC, the primer probe combination has good specificity, the result is analyzed by a real-time fluorescent quantitative PCR instrument, the result is visual and continuous, operation is easy and rapid, and the false positive rate is low.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer probe combination and a kit for identifying a complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare. Background Art

[0002] Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the leading killer among infectious diseases that mainly affect human lungs. In recent years, the prevalence of nontuberculous mycobacteria (NTM) has been increasing worldwide. One of the most common NTM causing human lung diseases in China is Mycobacterium aviumintracellular complex (MAC). NTM is diverse and ubiquitous in the environment, and both humans and animals can become ill through environmental infection. Tuberculosis and NTM are very similar in clinical symptoms and imaging manifestations. Conventional acid-fast staining methods are positive for both Mtb and NTM, so the diagnosis of both is often misleading, complicating the diagnosis of tuberculosis. Due to differences in the treatment regimens for Mtb and NTM, conventional anti-tuberculosis treatment of NTM infection may even induce the production of NTM resistant strains. Correct differential diagnosis methods are an important part of controlling and eliminating clinical misdiagnosis. The main methods for differential diagnosis of Mtb and NTM currently used in clinical practice include p-nitrobenzoic acid selective culture method, linear probe hybridization technology, DNA sequencing, bacterial culture combined with mass spectrometry identification, etc. (1) p-nitrobenzoic acid selective culture method: ① It takes a long time, and it takes 4-8 weeks to obtain culture results; ② It is not possible to identify mycobacterial species. (2) Linear probe hybridization technology: ① It takes a long time, and the PCR amplification and hybridization process takes 1.5-2 hours to obtain culture results; ② It is costly, requires a laboratory testing platform and has high requirements for operating skills, which is not convenient for popularization in areas with limited resources, limiting its promotion and application in low- and middle-income countries; ③ The false positive rate is high, which may lead to excessive re-examination, chest X-ray examination, etc. (3) DNA sequencing: ① It is expensive, and it is not convenient for popularization in areas with limited resources, limiting its promotion and application in low- and middle-income countries; ② The technology is complex and the equipment is limited, and it has high requirements for laboratory testing platforms and operating skills. (4) Bacterial culture combined with mass spectrometry identification: The detection time takes about 1 month; multi-gene detection has high accuracy when identifying bacterial species, but it is time-consuming and the operation is relatively complicated. These technologies have high requirements on experimental conditions and testing personnel, and require further identification of the obtained pure bacteria on the basis of positive mycobacterium culture. The detection cycle is long and the cost is high. Therefore, it is particularly important to develop simple methods for differential diagnosis of Mtb and MAC, shorten the detection time, and increase the detection rate of mycobacteria for the rational treatment of patients and improve prognosis.

[0003] Recombinase-mediated isothermal nucleic acid amplification (RAA) technology is a new in vitro rapid nucleic acid detection technology. Under normal temperature and constant temperature conditions, the reaction time is about 20 minutes, which can achieve the effect of PCR reaction of 1 to 2 hours. It does not require complex nucleic acid amplification instruments and has the advantages of short time consumption and diversified result reading. RAA has been widely used in the detection of various pathogens, but there are no reports on its application in the identification of mycobacteria in combination with real-time fluorescence technology. Therefore, it is necessary to explore the potential value of rapid detection technology based on dual real-time fluorescence RAA technology as a method for distinguishing Mtb and MAC. Summary of the invention

[0004] In view of the difficulty in the existing differential diagnosis of Mtb and MAC, the present invention uses the IS6110 gene of Mtb and the DT1 gene of MAC as templates, designs and screens primer probes, and constructs a dual RAA reaction system to quickly and efficiently detect Mtb and MAC, providing a detection method that is low in cost, simple to operate, and more suitable for clinical use, thereby improving the detection rate of pathogens in clinical practice and reducing misdiagnosis and missed diagnosis.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: a primer-probe combination for identifying the complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare,

[0006] The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:1 or SEQ NO:2; a downstream primer having a nucleotide sequence as shown in SEQ NO:3 or SEQ NO:4 or SEQ NO:5; and a probe having a nucleotide sequence as shown in SEQ NO:6;

[0007] The composition for detecting Mycobacterium avium intracellulare complex comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:7, SEQ NO:8 or SEQ NO:9; a downstream primer having a nucleotide sequence as shown in SEQ NO:10, SEQ NO:11 or SEQ NO:12; and a probe having a nucleotide sequence as shown in SEQ NO:13.

[0008] Furthermore,

[0009] The 29th base from the 5' end of SEQ NO:6 is modified with a fluorescent reporter group FAM, the 31st base from the 5' end is replaced with a dSpacer (tetrahydrofuran, THF) label, the 32nd base from the 5' end is modified with a fluorescent quencher group BHQ, and the probe end is modified with a C3-spacer.

[0010] Furthermore,

[0011] The 28th base from the 5' end of SEQ NO: 13 is modified with a fluorescent reporter group HEX, the 30th base from the 5' end is replaced with a dSpacer (tetrahydrofuran, THF) label, the 31st base from the 5' end is modified with a fluorescent quencher group BHQ, and the probe end is modified with a C3-spacer.

[0012] Furthermore,

[0013] The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:2; a downstream primer having a nucleotide sequence as shown in SEQ NO:3; and a probe having a nucleotide sequence as shown in SEQ NO:6;

[0014] The composition for detecting Mycobacterium avium intracellulare complex comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:7; a downstream primer having a nucleotide sequence as shown in SEQ NO:10; and a probe having a nucleotide sequence as shown in SEQ NO:13.

[0015] Furthermore,

[0016] The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 1; a downstream primer having a nucleotide sequence as shown in SEQ NO: 4; and a probe having a nucleotide sequence as shown in SEQ NO: 6;

[0017] The composition for detecting Mycobacterium avium intracellulare complex comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:7; a downstream primer having a nucleotide sequence as shown in SEQ NO:12; and a probe having a nucleotide sequence as shown in SEQ NO:13.

[0018] Furthermore,

[0019] The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:2; a downstream primer having a nucleotide sequence as shown in SEQ NO:4; and a probe having a nucleotide sequence as shown in SEQ NO:6;

[0020] The composition for detecting Mycobacterium avium intracellulare complex comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:8; a downstream primer having a nucleotide sequence as shown in SEQ NO:12; and a probe having a nucleotide sequence as shown in SEQ NO:13.

[0021] A kit for identifying a complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare, comprising the primer-probe combination described above,

[0022] DNA concentrate; DNA extract; lyophilized powder; buffer; positive standard; negative standard.

[0023] Furthermore,

[0024] The lyophilized powder includes Bst polymerase and single-stranded binding protein;

[0025] The positive standard is the genomic DNA of Mycobacterium tuberculosis and Mycobacterium avium intracellulare complex;

[0026] The negative standard is a PBS solution.

[0027] The present invention screened out a new primer-probe combination for differential diagnosis of Mtb and MAC. The primer-probe combination has good specificity, and the minimum detection limit of Mtb is 5.5×10 1 fg / μL, and the minimum detection limit of MAC was 7.8×10 3 fg / μL, the results are analyzed by real-time fluorescence quantitative PCR instrument, and the results are intuitive and continuous. Compared with the above popular methods on the market, this detection scheme is simple and fast to operate, with a low false positive rate, and can meet the detection applications of underdeveloped areas and large quantities of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The fluorescence response curve of the Mycobacterium tuberculosis (Mtb) probe finally determined in the embodiment of the present invention;

[0029] Figure 2 The fluorescence response curve of the Mycobacterium avium intracellulare complex (MAC) probe finally determined in the embodiment of the present invention;

[0030] Figure 3 The fluorescence reaction curve of the optimal Mtb and MAC primer combination screening by the real-time fluorescence RAA method according to the embodiment of the present invention;

[0031] Figure 4 The fluorescence reaction curve for screening the optimal Mtb and MAC primer concentrations by the real-time fluorescence RAA method according to the embodiment of the present invention;

[0032] Figure 5 The fluorescence response curve for screening the optimal Mtb and MAC probe concentrations by the real-time fluorescence RAA method according to an embodiment of the present invention;

[0033] Figure 6 The dual fluorescence RAA method system of the embodiment of the present invention is constructed;

[0034] Figure 7 This is the specific verification of the dual fluorescence RAA method of the embodiment of the present invention;

[0035] Figure 8 It is the sensitivity verification of the dual fluorescence RAA method of the embodiment of the present invention;

[0036] Fig. 9 This is the FAM fluorescence detection channel of clinical samples 1-8 in the embodiment of the present invention;

[0037] Fig.10 This is the 9-16FAM fluorescence detection channel for clinical samples in the embodiment of the present invention;

[0038] Fig.11 This is the HEX fluorescence detection channel of clinical samples 9-16 in the embodiment of the present invention;

[0039] Fig.12 This is the 17-24FAM fluorescence detection channel for clinical samples in the embodiment of the present invention;

[0040] Fig.13 This is the 25-32FAM fluorescence detection channel for clinical samples in the embodiment of the present invention;

[0041] Fig.14 This is the 33-40FAM fluorescence detection channel for clinical samples in the embodiment of the present invention;

[0042] Fig.15 This is the FAM fluorescence detection channel for clinical samples 41-48 in the embodiment of the present invention. DETAILED DESCRIPTION

[0043] The specific implementation manner will be further described below in conjunction with the accompanying drawings.

[0044] The reagents and instruments in the following examples are all conventional experimental reagents and instruments.

[0045] Embodiment 1:

[0046] A primer-probe combination for distinguishing Mycobacterium tuberculosis from Mycobacterium avium intracellulare complex,

[0047] Primers and probes were screened and a single-reaction system was initially constructed. The primer and probe concentrations of Mtb and MAC were optimized and a dual reaction system was constructed. The specificity and sensitivity were further verified. Finally, a rapid detection method for identifying Mtb and MAC was developed based on the constructed dual fluorescence RAA system.

[0048] 1. Design and screening of primers and probes

[0049] 1. Primer and probe design

[0050] Design upstream and downstream primers with reference to the IS6110 gene sequence of Mycobacterium tuberculosis and the DT1 gene sequence of Mycobacterium avium intracellulare published in Genbank. Design corresponding primers according to the design principles of RAA technology. The primer length is generally between 30-35bp (generally not recommended to be less than 27 or higher than 45bp), and the amplified template fragment is generally 80-500bp. Because the length of the amplified fragment has a direct impact on the reaction speed and sensitivity, the target fragment amplification is selected as short as possible according to the needs (110-140bp is the best). In addition, the TM value of the primer is not the main consideration. At the same time, in order to avoid the formation of internal secondary structures, continuous single-base repeat sequences or palindromic sequences as much as possible, Primer-BLAST, Primer Premier 5, and Oligo 7 are used to analyze the various parameters of the designed primers to comprehensively evaluate the quality of the primers. The specific parameters are: length 30-35nt, CG content 20%-70%, Tm value 50-100, and the maximum number of single-base repeats allowed: 5. The probe length is generally between 46-52bp. The probe sequence cannot overlap with the primer recognition site. There are four modification sites in total. A dSpacer (tetrahydrofuran, THF) is marked in the middle position ≥28-30bp away from the 5' end. THF serves as the recognition site for the exonuclease. A fluorescent group is marked upstream and a quenching group is marked downstream. The distance between the two groups is 1-5bp. THF is ≥15bp away from the 3' end. In addition, a modification group, such as an amine group, a phosphate group, biotin, biotin-TEG or C3-spacer, needs to be marked at the 3' end. It should also be noted that THF replaces rather than inserts a base, and the probe sequence should also avoid internal secondary structures and continuous repeated bases. The optimization and synthesis of the probe are the same as the primer design scheme. See Table 1 for details:

[0051] Table 1

[0052]

[0053] The 29th base from the 5' end of SEQ NO: 6 is modified with a fluorescent reporter group FAM, the 31st base from the 5' end is replaced with a dSpacer (tetrahydrofuran, THF) label, the 32nd base from the 5' end is modified with a fluorescent quencher group BHQ, and the probe end is modified with a C3-spacer.

[0054] The 28th base from the 5' end of SEQ NO: 13 is modified with a fluorescent reporter group HEX, the 30th base from the 5' end is replaced with a dSpacer (tetrahydrofuran, THF) label, the 31st base from the 5' end is modified with a fluorescent quencher group BHQ, and the probe end is modified with a C3-spacer.

[0055] 2. Nucleic acid extraction from cultured bacteria

[0056] (1) Use an inoculating loop to pick up a full loop of bacteria and add it to an EP tube containing 1 mL of normal saline to resuspend the bacteria. Place the tube in a metal bath at 80°C for 30 min to inactivate the bacteria.

[0057] (2) After slow cooling, centrifuge at 12,000 rpm for 5 min.

[0058] (3) After discarding the supernatant, add 200 μL of saline and 25 μL of 10 mg / mL lysozyme, shake to mix, and incubate at 37°C for 16 h.

[0059] (4) Add 5 μL RNase A, invert to mix, and let stand for 10 min.

[0060] (5) Add 75 μL 10% SDS and 5 μL 20 mg / mL proteinase K, mix by inversion, and incubate at 65°C for 10 min.

[0061] (6) Add 100 μL 5 mol / L NaCl and 100 μL CTAB / NaCl, vortex mix until a milky white flocculent precipitate appears in the liquid in the tube, and incubate at 65°C for 10 min.

[0062] (7) Add 750 μL of chloroform, mix thoroughly by inversion, and centrifuge at 12,000 rpm for 5 min. The liquid in the tube is now divided into three layers, with a white film in the middle. Carefully transfer 600 μL of the upper layer of liquid to a new EP tube, and then add 360 μL of pre-cooled isopropanol, 0.6 times the volume of the transferred supernatant.

[0063] (8) After standing at -20°C for 30 min, centrifuge at 12,000 rpm for 5 min.

[0064] (9) Discard the supernatant, wash the precipitate with 1 mL of 70% pre-cooled ethanol, and centrifuge at 12,000 rpm for 5 min.

[0065] (10) Dry at 37°C for 15 min, dissolve the precipitate in 20 μL of TE buffer or ddH2O, and store at -20°C until use.

[0066] 3. Screening of Primers and Probes

[0067] The optimal primer combination was screened based on the designed primer probes. While keeping other system components unchanged, the primers of the Mtb group were divided into Mtb-F1R1, Mtb-F1R2, Mtb-F1R3, Mtb-F2R1, Mtb-F2R2 and Mtb-F2R3, and the primers of the MAC group were divided into MAC-F1R1, MAC-F1R2, MAC-F1R3, MAC-F2R1, MAC-F2R2, MAC-F2R3, MAC-F3R1, MAC-F3R2 and MAC-F3R3. The optimal primer probes required for RAA reaction detection and able to be used were finally determined through the recommended reaction system.

[0068] The reaction system is as follows: add 25μL of Abuffer, 12.9μL of RNase-Free ddH2O, 2μL of upstream primer (10μM), 2μL of downstream primer (10μM), and 0.6μL of probe (10μM) to the unit reaction tube, shake it upside down 5-6 times, and centrifuge it instantly. Then add 5μL of template by putting the pipette tip below the liquid surface. Finally, add 2.5μL of B buffer to the inside of the reaction tube, cover the tube cap, and mix it upside down 5-6 times, centrifuge it instantly, repeat 3 times to fully mix it, and remember to operate this process in a light-proof environment. Machine program settings: 50μL system, select the "FAM HEX ROX" channel for fluorescence channel, and use the reaction temperature and number of reaction cycles recommended by the reagent. The specific machine parameter settings are as shown in Table 2:

[0069] Table 2

[0070]

[0071] Figure 1 The fluorescence response curve of the final Mycobacterium tuberculosis (Mtb) probe

[0072] 1: Mtb, 2: MAC, 3: negative control

[0073] Figure 2 The fluorescence response curve of the final determined Mycobacterium avium intracellulare complex (MAC) probe

[0074] 1: MAC, 2: Mtb, 3: negative control

[0075] Figure 3 Fluorescence reaction curve for screening the best Mtb and MAC primer combination by real-time fluorescence RAA method:

[0076] A: Fluorescence reaction curve of Mtb primer screening. 1: Mtb-F2R1, 2: Mtb-F1R2, 3: Mtb-F2R2, 4: Mtb-F1R1, 5: Mtb-F1R3, 6: Mtb-F2R3, 7: negative control;

[0077] B: Fluorescence reaction curve of MAC primer screening. 1: MAC-F1R1, 2: MAC-F2R3, 3: MAC-F1R3, 4-10: other MAC primer combinations and negative controls.

[0078] 2. Optimization of primer and probe concentrations

[0079] 1. Primer concentration optimization

[0080] The final concentrations of primers in the single-plex RAA reaction system were set to 0, 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, and 0.5 μmol / L, respectively, that is, the amounts of upstream and downstream primers added were 0 μL, 0.5 μL, 1 μL, 1.5 μL, 2 μL, and 2.5 μL. The reaction was carried out at 39°C for 30 min, and the optimal reaction primer concentrations of Mtb and MAC were determined by observing the changes in fluorescence intensity.

[0081] Figure 4 Fluorescence reaction curve for screening the optimal Mtb and MAC primer concentrations by real-time fluorescence RAA method:

[0082] A: Fluorescence reaction curve of the optimal primer concentration for Mtb. 1: 0.5 μmol / L, 2: 0.1 μmol / L, 3: 0.4 μmol / L, 4: 0.3 μmol / L, 5: 0.2 μmol / L, 6: 0;

[0083] B: Fluorescence reaction curve of the optimal primer concentration of MAC. 1: 0.4μmol / L, 2: 0.5μmol / L, 3: 0.3μmol / L, 4: 0.2μmol / L, 5: 0.1μmol / L, 6: 0.

[0084] 2. Probe Concentration Optimization

[0085] The final concentrations of the probes in the single-plex RAA reaction system were set to 0, 0.04 μmol / L, 0.08 μmol / L, 0.12 μmol / L, 0.16 μmol / L, and 0.2 μmol / L, respectively, that is, the amount of probe added was 0 μL, 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, and 1 μL. The reaction was carried out at 39°C for 30 min, and the optimal reaction probe concentrations of Mtb and MAC were determined by observing the changes in fluorescence intensity.

[0086] Figure 5The fluorescence response curve for screening the optimal Mtb and MAC probe concentrations for the real-time fluorescence RAA method;

[0087] A: Fluorescence response curve of the optimal probe concentration of Mtb. 1: 0.2 μmol / L, 2: 0.08 μmol / L, 3: 0.12 μmol / L, 4: 0.04 μmol / L, 5: 0.16 μmol / L, 6: 0;

[0088] B: Fluorescence response curves of the optimal probe concentrations of MAC. 1: 0.16 μmol / L, 2: 0.2 μmol / L, 3: 0.12 μmol / L, 4: 0.08 μmol / L, 5: 0.04 μmol / L, 6: 0.

[0089] 3. Establishment of dual fluorescence RAA reaction system

[0090] According to the optimization of the single RAA reaction system, a dual real-time fluorescent RAA for detecting Mtb and MAC was established. The reaction system was 50 μL, and the specific operation steps were the same as above. The construction of the multiple fluorescent RAA reaction system was judged based on the change in the intensity of the fluorescence reaction curve. The Mtb template, MAC template, and mixed template of Mtb and MAC were added to the dual RAA reaction system (containing two sets of specific primers) for amplification.

[0091] Figure 6 It is constructed for the dual fluorescence RAA method system;

[0092] A: 1: Mtb, 2: negative control;

[0093] B: 1: MAC, 2: negative control;

[0094] C: 1: Mtb, 2: MAC, 3: negative control;

[0095] D: 1: MAC, 2: Mtb, 3: negative control

[0096] The optimized system of dual real-time fluorescence RAA method is shown in Table 3:

[0097] Table 3

[0098]

[0099] Embodiment 2:

[0100] A kit for identifying a complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare, comprising the primer-probe combination described above,

[0101] DNA concentrate; DNA extract; lyophilized powder; buffer; positive standard; negative standard.

[0102] The lyophilized powder includes Bst polymerase and single-stranded binding protein;

[0103] The positive standard is the genomic DNA of Mycobacterium tuberculosis and Mycobacterium avium intracellulare complex;

[0104] The negative standard is a PBS solution.

[0105] Steps for using the kit:

[0106] (1) Collect suspected clinical samples and process them. ① Sputum: Add 4 times the volume of 4% NaOH, shake well, and place at room temperature for about 30 minutes to liquefy; take 0.5mL to a centrifuge tube, add 0.5mL 4% NaOH, place at room temperature for 10 minutes, and centrifuge at 13000rpm for 5 minutes; discard the supernatant, add 1mL of normal saline, mix well, and centrifuge at 13000rpm for 5 minutes; repeat the washing once, remove most of the supernatant, retain about 50μL of liquid precipitate, and oscillate to mix well. ② Bronchoalveolar lavage fluid: Take 1mL to a 1.5mL centrifuge tube, centrifuge at 13000rpm for 5 minutes; discard the supernatant, add 1mL of sterile normal saline to the precipitate, mix well, centrifuge at 13000rpm for 5 minutes, and process in the same way as ①.

[0107] (2) Take 100 μL of liquid sample and add an equal amount of DNA concentrate, vortex to mix for 5 seconds, and centrifuge at 10,000 rpm for 10 minutes.

[0108] (3) Remove the supernatant, add 50 μL of DNA extraction solution to the precipitate, vortex to mix for 10 seconds, and centrifuge for a few seconds.

[0109] (4) The mixture was kept at 100°C for 15 min, and then centrifuged at 10,000 rpm for 5 min; the mixture was centrifuged at 10,000 rpm for 5 min, and then stored at -20°C for later use.

[0110] (5) Add 25 μL of Abuffer, 11.8 μL of RNase-Free ddH2O, 1.2 μL of upstream and downstream primers of Mtb (10 μM), 1 μL of upstream and downstream primers of MAC (10 μM), and 0.6 μL of probes of Mtb and MAC (10 μM) to the unit reaction tube containing the reaction dry powder, invert and shake gently 5-6 times, and then centrifuge instantly.

[0111] (6) Place the pipette tip below the liquid surface and add 5 μL of template. Finally, add 2.5 μL of LB buffer to the inside of the reaction tube. Cover the tube and invert it 5-6 times to mix. Centrifuge briefly. Repeat 3 times to mix thoroughly before loading onto the machine.

[0112] (7) Determine whether it is Mtb or MAC based on the change in the fluorescence response curve.

[0113] 1. Validation experiment of specificity and sensitivity:

[0114] 1. Specificity Experiment

[0115] The established dual fluorescence RAA reaction system was used to detect 9 mycobacteria, including Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium surga, Mycobacterium gordonii, Mycobacterium chelonae, Mycobacterium scrofula, Mycobacterium malmoe, Mycobacterium fortuitum, and Mycobacterium chimaera, and 6 common respiratory pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, and Acinetobacter baumannii. The specificity of the dual RAA system was determined based on the fluorescence intensity change results.

[0116] Figure 7 To verify the specificity of the dual fluorescence RAA method:

[0117] A: Mtb fluorescence change curve: Mtb, no fluorescence change curve: other pathogens;

[0118] B: MAC fluorescence change curve: MAC, no fluorescence change curve: other pathogens.

[0119] 2. Sensitivity experiment

[0120] The genomes of Mtb and MAC were extracted respectively, and the concentration and purity of the extracted bacterial genomes were detected. 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 The template genome was diluted and the sensitivity of the dual RAA system was analyzed based on the change in fluorescence intensity.

[0121] Figure 8 Sensitivity verification of dual fluorescence RAA method

[0122] A-Mtb:1:5.5×10 7 fg / μL, 2: 5.5×10 6 fg / μL, 3: 5.5×10 5 fg / μL, 4: 5.5×10 4 fg / μL, 5: 5.5×10 3 fg / μL, 6: 5.5×10 2 fg / μL, 7: 5.5×10 1 fg / μL, 8: 5.5×10 0 fg / μL;

[0123] B-MAC: 1:7.8×10 7 fg / μL, 2: 7.8×10 6 fg / μL, 3: 7.8×10 5 fg / μL, 4: 7.8×10 4 fg / μL, 5: 7.8×10 3 fg / μL, 6: 7.8×10 2 fg / μL, 7: 7.8×10 1 fg / μL, 8: 7.8×10 0 fg / μL.

[0124] II: Evaluation of the application value of the dual fluorescence RAA system

[0125] Based on the clinical diagnosis results, 48 ​​clinical samples such as sputum and bronchoalveolar lavage fluid were tested by acid-fast staining microscopy, liquid culture method and the dual fluorescence RAA method constructed in this study. The diagnostic efficacy and application value of the dual fluorescence RAA detection method were evaluated by statistical methods. The test results are shown in Figure 9-15 .

[0126] 1. Comparison of positive detection rates among three detection methods

[0127] In the clinical samples of 48 suspected pulmonary tuberculosis patients, the positive detection rates of dual fluorescence RAA method, liquid culture method and acid-fast stain microscopy method were 56.25% (27 / 48), 50.00% (24 / 48) and 35.42% (17 / 48), respectively. The positive detection rate of dual fluorescence RAA method was higher than that of liquid culture method and acid-fast stain microscopy method.

[0128] 2. Evaluation of the diagnostic efficacy of the three detection methods

[0129] Based on the clinical diagnosis of 48 clinical samples, the sensitivity of dual fluorescence RAA method, liquid culture method and acid-fast staining microscopy method were 72.22%, 66.67% and 47.22%, respectively. The sensitivity of dual fluorescence RAA method was higher than that of liquid culture method and acid-fast staining microscopy method. 2 The test yields χ 2 The difference was statistically significant. The Kappa value of the dual fluorescence RAA method was 0.51, indicating moderate consistency. The specificity, sensitivity, positive predictive value, negative predictive value, etc. are shown in Table 4 below.

[0130] Table 4

[0131]

[0132] 3. Evaluation of the detection efficiency of liquid culture method and dual fluorescence RAA method in acid-fast staining negative samples

[0133] Based on the clinical diagnosis results, among the 31 subjects with negative results in acid-fast staining microscopy, the positive detection rates of liquid culture and dual fluorescence RAA were 22.58% (7 / 31) and 32.26% (10 / 31), respectively. The positive detection rate of dual fluorescence RAA was higher than that of liquid culture, and the difference was statistically significant (χ 2 =5.71, P<0.05). The sensitivity of the dual fluorescence RAA method and liquid culture method were 47.36% and 36.84%, respectively. The sensitivity of the dual fluorescence RAA method was higher than that of the liquid culture method, and the difference was statistically significant (χ 2 =5.13, P<0.05). Other relevant evaluation parameters are shown in Table 5.

[0134] Table 5

[0135]

[0136]

[0137] 4. Evaluation of the detection efficiency of the dual fluorescence RAA method in acid-fast staining microscopy and liquid culture negative samples

[0138] Taking clinical diagnosis as the standard, among the 24 subjects whose acid-fast staining microscopy and liquid culture were negative, the positive detection rate of dual fluorescence RAA method was 12.50% (3 / 24). The relevant evaluation parameters are shown in Table 6 below.

[0139] Table 6

[0140]

[0141] Compared with traditional detection technologies such as PCR, the isothermal amplification technology based on recombinase has the advantages of simplicity, rapidity, and high efficiency. It does not require complex thermal cycling equipment, which greatly reduces the requirements for experimental environment and hardware conditions. At the same time, the real-time fluorescent RAA used in this patent adds a fluorescent marker to the conventional RAA, that is, a fluorescent probe with a tetrahydrofuran modification site. When the probe hybridizes with the target gene sequence in the amplified product, the THF site cleaves the probe under the action of the enzyme, thereby causing the fluorescent group to separate from the quenching group, generating a fluorescent signal, and realizing visual real-time monitoring of the amplified product. Secondly, we use the basic design software to set parameters and preliminarily screen high-quality primer probes, try to avoid the formation of secondary structures or palindromic sequences, and then eliminate primer probes with poor amplification effects through repeated verification.

[0142] The dual fluorescence RAA method constructed in the present application has a higher positive detection rate and sensitivity for clinical specimens than the acid-fast staining microscopy method and the liquid culture method. At the same time, the dual fluorescence RAA method also has a relatively high diagnostic efficacy for patients with pulmonary tuberculosis who are negative for acid-fast staining microscopy and liquid culture, which has a high application value for the clinical differential diagnosis of mycobacterial infection.

[0143] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer-probe combination for identifying a complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare, characterized in that: The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:1 or SEQ NO:2; a downstream primer having a nucleotide sequence as shown in SEQ NO:3 or SEQ NO:4 or SEQ NO:5; and a probe having a nucleotide sequence as shown in SEQ NO:6; The composition for detecting Mycobacterium avium intracellulare complex comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:7, SEQ NO:8 or SEQ NO:9; a downstream primer having a nucleotide sequence as shown in SEQ NO:10, SEQ NO:11 or SEQ NO:12; and a probe having a nucleotide sequence as shown in SEQ NO:

13.

2. The primer-probe combination for identifying the complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare according to claim 1, characterized in that: The 29th base from the 5' end of SEQ NO:6 is modified with a fluorescent reporter group FAM, the 31st base from the 5' end is replaced with a dSpacer label, the 32nd base from the 5' end is modified with a fluorescent quencher group BHQ, and the probe end is modified with a C3-spacer.

3. The primer-probe combination for identifying the complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare according to claim 1, characterized in that: The 28th base from the 5' end of SEQ NO: 13 is modified with a fluorescent reporter group HEX, the 30th base from the 5' end is replaced with a dSpacer label, the 31st base from the 5' end is modified with a fluorescent quencher group BHQ, and the probe end is modified with a C3-spacer.

4. The primer-probe combination for identifying the complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare according to claim 1, characterized in that: The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:2; a downstream primer having a nucleotide sequence as shown in SEQ NO:3; and a probe having a nucleotide sequence as shown in SEQ NO:6; The composition for detecting Mycobacterium avium intracellulare complex comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:7; a downstream primer having a nucleotide sequence as shown in SEQ NO:10; and a probe having a nucleotide sequence as shown in SEQ NO:

13.

5. The primer-probe combination for identifying the complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare according to claim 1, characterized in that: The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 1; a downstream primer having a nucleotide sequence as shown in SEQ NO: 4; and a probe having a nucleotide sequence as shown in SEQ NO: 6; The composition for detecting Mycobacterium avium intracellulare complex comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:7; a downstream primer having a nucleotide sequence as shown in SEQ NO:12; and a probe having a nucleotide sequence as shown in SEQ NO:

13.

6. The primer-probe combination for identifying the complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare according to claim 1, characterized in that: The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:2; a downstream primer having a nucleotide sequence as shown in SEQ NO:4; and a probe having a nucleotide sequence as shown in SEQ NO:6; The composition for detecting Mycobacterium avium intracellulare complex comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:8; a downstream primer having a nucleotide sequence as shown in SEQ NO:12; and a probe having a nucleotide sequence as shown in SEQ NO:

13.

7. A kit for identifying a complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare, characterized in that: A primer-probe combination comprising any one of claims 1 to 6, DNA concentrate; DNA extract; lyophilized powder; buffer; positive standard; negative standard.

8. The kit for identifying the complex of Mycobacterium tuberculosis and Mycobacterium avium intracellulare according to claim 7, characterized in that: The lyophilized powder includes Bst polymerase and single-stranded binding protein; The positive standard is the genomic DNA of Mycobacterium tuberculosis and Mycobacterium avium intracellulare complex; The negative standard is a PBS solution.

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