Mycobacterium tuberculosis complex group detection method based on probe method and multiple LAMP (loop-mediated isothermal amplification)

Abstract

The invention belongs to the technical field of microbial molecule detection, and particularly relates to a multiple LAMP mycobacterium tuberculosis complex group detection method based on a probe method, the LAMP multiple probe method is used for detecting mycobacterium tuberculosis complex flora IS6110 and IS1081 primer groups, the mycobacterium tuberculosis complex flora can be conveniently and rapidly detected at the same time, the positive detection rate of a sputum sample is increased, and the detection time is shortened. Meanwhile, non-specific amplification is weakened, false positive is reduced, the arrangement of the reference gene GAPDH can effectively monitor the sample collection / sample preservation / sample nucleic acid extraction process, and false negative is avoided; the specificity is high, and cross reaction with common strains is avoided. And the sensitivity can reach 10 < 0 > cop i es / u < l >.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial molecular detection, and particularly relates to a probe-based multiplex LAMP detection method for Mycobacterium tuberculosis complex. BACKGROUND

[0002] Tuberculosis is an ancient and serious infectious disease that harms human health caused by Mycobacterium tuberculosis complex (MTBC).

[0003] Mycobacterium tuberculosis complex (MTBC) mainly includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, etc., which have certain similarities but also differences in biological characteristics and pathogenicity. In the detection of Mycobacterium tuberculosis complex nucleic acid, the most commonly used detection target is IS6110, which is the most studied and most widely used insertion sequence in the genome of MTBC. It has multiple copies, and multiple copies (the copy number usually varies from 1 to 25) exist in most Mycobacterium tuberculosis strains, but the copy number is less or even missing in some strains such as Mycobacterium bovis. IS1081 is relatively conservative and stably exists in MTBC. It is found in Mycobacterium tuberculosis, Mycobacterium bovis and other MTBC members, and the copy number is relatively stable. Due to its conservatism, IS1081 can be used as a universal target for the detection of MTBC, especially for strains with low or missing IS6110 copy number. Therefore, simultaneous detection of IS6110 and IS1081 double targets can not only improve the sensitivity of detection, but also reduce false negative results.

[0004] Accurate and rapid detection of Mycobacterium tuberculosis complex is crucial for early diagnosis, treatment and prevention and control of tuberculosis. Early diagnosis can enable patients to receive timely treatment, improve cure rate and reduce disease transmission.

[0005] Technical advantages of probe-based LAMP based on DARQ principle: DARQ (Detection of Amplification Refractory to Quenching) principle is a new type of nucleic acid detection signal amplification technology, which combines the isothermal amplification advantage of LAMP technology and the DARQ signal amplification technology, and has the following advantages:

[0006] High specificity: DARQ probe has a unique structure and design, which can specifically bind to target nucleic acid sequences. When the probe is not combined with the target nucleic acid, the fluorescence signal is quenched; when combined with the target nucleic acid, the fluorescence signal is released and amplified. This specific binding mechanism can effectively avoid false positive problems caused by non-specific amplification, and improve the accuracy of detection.

[0007] High sensitivity: DARQ technology can significantly enhance the fluorescence signal through signal amplification mechanism. Even if the content of target nucleic acid in the sample is extremely low, it can be accurately judged through the detected fluorescence signal. Compared with traditional LAMP detection method, the probe-based LAMP based on DARQ principle has greatly improved in sensitivity.

[0008] Quantitative detection ability: This method can accurately quantify the content of target nucleic acid in the sample by detecting the intensity of fluorescence signal. This is of great significance for early diagnosis of diseases, disease monitoring and treatment effect evaluation, etc. For example, in the detection of Mycobacterium tuberculosis, the load of Mycobacterium tuberculosis in the sample can be accurately determined, providing more accurate basis for clinical treatment.

[0009] Strong anti-interference ability: DARQ probe has good anti-interference ability and can accurately identify target nucleic acid in complex sample background. Even if there are other nucleic acids, proteins and other impurities in the sample, it will not have obvious influence on the detection result, ensuring the stability and reliability of the detection.

[0010] Simple and fast operation: Probe-based LAMP based on DARQ principle still retains the advantages of isothermal amplification of LAMP technology, which does not require complex temperature cycling equipment and is relatively simple to operate. At the same time, the reaction time is short, generally within 30-60 minutes, which can complete amplification and detection, and quickly obtain results, meeting the needs of rapid diagnosis in clinic.

[0011] The existing detection methods of Mycobacterium tuberculosis complex have various limitations, and the traditional LAMP technology needs to be improved in specificity and quantitative detection, and multiple detection. Therefore, developing a probe-based multiple LAMP Mycobacterium tuberculosis complex detection kit has important clinical significance and application value. The kit can fully exert the isothermal amplification advantage of LAMP technology, combined with the high specificity of probe method, quantitative and multiple detection ability, realize the rapid, accurate and quantitative detection of Mycobacterium tuberculosis complex, and provide strong technical support for the early diagnosis and prevention and control of tuberculosis.

[0012] At present, the methods for detecting Mycobacterium tuberculosis complex based on LAMP in clinic mainly include the following:

[0013] Traditional loop-mediated isothermal amplification (LAMP) detection method

[0014] LAMP (Loop-mediated isothermal amplification) probe method for detecting Mycobacterium tuberculosis is a detection method combining LAMP technology and probe technology, and its specific principle is as follows: LAMP technology relies on Bst DNA polymerase with strand displacement activity, and recognizes 6-8 specific regions on the target gene of Mycobacterium tuberculosis through 4-6 specific primers. The reaction process mainly includes the following steps: primer binding, strand displacement synthesis, loop structure formation and exponential amplification.

[0015] Traditional LAMP detection methods include turbidity method, visual colorimetric method and fluorescent dye method. The disadvantages are as follows: 1. Specificity problem: Although LAMP technology itself has certain specificity, in some cases, non-specific amplification may occur. Especially when there are complex background nucleic acids in the sample or the primer design is not optimized, non-specific amplification may lead to false positive results. 2. Unable to accurately quantify: Traditional LAMP detection methods are mostly qualitative detection, that is, to judge whether there is target nucleic acid in the sample, but cannot accurately determine the content of target nucleic acid. In some cases where the load of pathogen needs to be accurately understood, such as disease severity assessment, treatment effect monitoring, etc., the limitations of traditional methods are more obvious. 3. Easy to be affected by subjective factors: The result interpretation of visual colorimetric method depends on visual observation, and different operators may have different judgments on color changes, which is easy to be affected by subjective factors, resulting in reduced accuracy and reliability of the results. 4. Risk of cross contamination: Since LAMP reaction can produce a large amount of amplification product, if the protection measures are not proper, the amplification product is easy to contaminate the laboratory environment and subsequent samples, resulting in false positive results. 5. Unable to detect multiple targets. Turbidity method and visual colorimetric method are mainly used for qualitative detection, which can judge whether amplification reaction has occurred by observing the turbidity change or color change of the reaction solution. In multiple target detection, it is difficult to accurately distinguish the amplification of each target through single turbidity or color change due to the simultaneous amplification of multiple targets, resulting in difficult result interpretation. They lack the ability to specifically distinguish different target amplification products. They can only detect the overall amplification, and cannot accurately determine whether each target has truly amplified. For example, when non-specific amplification occurs, it is difficult to determine which target primer or reaction has a problem. In view of this, we propose a probe-based multiple LAMP Mycobacterium tuberculosis complex detection method. SUMMARY

[0016] The purpose of the present application is to provide a probe-based multiple LAMP Mycobacterium tuberculosis complex detection method to solve the problems raised in the above background art.

[0017] In view of the above, the present application provides a probe-based multiplex LAMP detection method for the Mycobacterium tuberculosis complex, which comprises target genes IS6110, IS1081 and GAPDH, and further comprises the following sequences:

[0018] IS6110 primers and probes:

[0019] F3: TCGAGTTGGCCACCGC (SEQ ID NO: 1)

[0020] B3: GTCGCCAACTCCCACAG (SEQ ID NO: 2)

[0021] FIP: CAGCCTCGAGTTCGACCGGGTTCAACCATCGCCGCCT (SEQ ID NO: 3)

[0022] BIP: TCAGAGAGTCTCCGGACTCACCCCGCCAACACCTTTCGC (SEQ ID NO: 4)

[0023] LF: TCGCCGCAGTACTGGTAG (SEQ ID NO: 5)

[0024] LB: CGGTTCAACCGCCAGTGA (SEQ ID NO: 6)

[0025] BIP-P: FAM-TCAGAGAGTCTCCGGACTCACCCCGCCAACACCTTTCGC (SEQ ID NO: 7)

[0026] BIP-FQ: GGTGAGTCCGGAGACTCTCTGA-BHQ1 (SEQ ID NO: 8)

[0027] IS1081 primers and probes:

[0028] F3: CGAGCCCGGATCTGCT (SEQ ID NO: 9)

[0029] B3: CGCGTTCAGCTCGCTTG (SEQ ID NO: 10)

[0030] FIP: TCGCTGCGTTCGCGGTAGCCTCGACGTTCATCGCCG (SEQ ID NO: 11)

[0031] BIP: ACCGCCACCGTGATTTCGACCGGGAAATAGCTGCCCTGG (SEQ ID NO: 12)

[0032] LF: GCTTCAGCCCCCATCAAGG (SEQ ID NO: 13)

[0033] LB: TGCCGCAACCATCGACG (SEQ ID NO: 14)

[0034] BIP-P: ROX-ACCGCCACCGTGATTTCGACCGGGAAATAGCTGCCCTGG (SEQ ID NO: 15)

[0035] BIP-FQ: TCGAAATCACGGTGGCGGT-BHQ2 (SEQ ID NO: 16)

[0036] GAPDH primers and probe:

[0037] F3: CAAGGTCATCCATGACAACT (SEQ ID NO: 17)

[0038] B3: CAGTGATGGCATGGACTG (SEQ ID NO: 18)

[0039] FIP: AGGAGCCAGTCTTGGATGAGAATTGGTATCGTGGAAGGACT (SEQ ID NO: 19)

[0040] BIP: GGTTCTGGGGACTGGCTTTCCATCACCCCTCTACCTCC (SEQ ID NO: 20)

[0041] LF: GAGCCTCAGTCCCATTCC (SEQ ID NO: 21)

[0042] LB: TTTCCTTTCAAGGTGGGGAG (SEQ ID NO: 22)

[0043] FIP-P: CY5-AGGAGCCAGTCTTGGATGAGAATTGGTATCGTGGAAGGACT (SEQ ID NO: 23)

[0044] FIP-FQ: TTTCTCATCCAAGACTGGCTCCT-BHQ2 (SEQ ID NO: 24).

[0045] In the above technical solution, further, the LAMP primers and probes include the following components:

[0046] 10x isothermal amplification buffer (1-2.5X, preferably 1X)

[0047] 100 mM MgSO4(2-8 mM, preferably 6 mM)

[0048] 8 U Bst DNA polymerase (0.12-0.32 U / μL, preferably 0.32 U / μL)

[0049] Betaine (0-0.4 M, preferably 0.2 M)

[0050] dNTP Mix (10 mM each, 0.2-2 mM each, preferably 1.4 mM each)

[0051] Sterile double-distilled water (to make up to 25 μL).

[0052] In the above technical solution, further, the working concentration of the primer and the probe is:

[0053] F3 / B3: 0.1-0.2 μM (preferably 0.2 μM)

[0054] F IP / B IP: 0.8-1.6 μM (preferably 1.6 μM)

[0055] LF / LB: 0.4-0.8 μM (preferably 0.8 μM)

[0056] Probe (BIP-P / FIP-P) and quenched probe (BIP-FQ / FIP-FQ): 0.4-0.8 μM (preferably 0.8 μM).

[0057] In the above technical solution, further, the following steps are included:

[0058] Step S1, extracting nucleic acid of the sample to be tested;

[0059] Step S2, preparing the reaction system of the kit according to claim 2 or 3, and the total reaction volume is 25 μL;

[0060] Step S3, performing isothermal amplification at 63°C, and collecting fluorescence signal every 20 seconds for 99 cycles;

[0061] Step S4, result interpretation: Ct value of IS6110 or IS1081 ≤ 60 is positive; Ct value of GAPDH ≤ 60 is effective internal standard, otherwise the sample needs to be prepared again.

[0062] In the above technical solution, further, IS6110, IS1081 and GAPDH can be simultaneously detected in steps S1 and S4, and the sensitivity reaches 10 0 copies / μL, and there is no cross-reaction with common non-tuberculous mycobacteria.

[0063] In the above technical solution, further, the fluorescent label of the primer and the probe is:

[0064] IS6110-BIP-P: 5' end FAM, 3' end BHQ1;

[0065] IS1081-BIP-P: 5' end ROX, 3' end BHQ2;

[0066] GAPDH-FIP-P: 5' end CY5, 3' end BHQ2.

[0067] The beneficial effects of the present application are:

[0068] 1. The multiplex LAMP detection method for Mycobacterium tuberculosis complex based on the probe method, the LAMP multiplex probe method for detecting IS6110 and IS1081 primer groups of Mycobacterium tuberculosis complex in the present application, which is convenient for rapid simultaneous detection of Mycobacterium tuberculosis complex, improves the positive detection rate of sputum samples, and reduces non-specific amplification and false positives.

[0069] 2. The multiplex LAMP detection method for Mycobacterium tuberculosis complex based on the probe method, the setting of the internal reference gene GAPDH can effectively monitor the sample collection / sample preservation / sample nucleic acid extraction process, and avoid false negatives;

[0070] Strong specificity, no cross reaction with common bacteria. The sensitivity can reach 10^0 copies / ul. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 IS6110 IS1081 GAPDH primer probe sequence information chart of the present application;

[0072] Figure 2 LAMP multiplex probe reaction system chart of the present application;

[0073] Figure 3 LAMP multiplex probe reaction system chart of the present application;

[0074] Figure 4 Bacteria used for cross reaction of Mycobacterium tuberculosis complex in the present application;

[0075] Figure 5 LAMP multiplex probe reaction system chart of the present application;

[0076] Figure 6 Plasmid used for sensitivity analysis of Mycobacterium tuberculosis complex in the present application;

[0077] Figure 7is a LAMP dye method reaction system chart of the present application;

[0078] Figure 8 is a sensitivity result chart of the present application;

[0079] Figure 9 is a LAMP multiplex probe method reaction system chart No. 4 of the present application;

[0080] Figure 10 is a LAMP dye method reaction system chart No. 2 of the present application;

[0081] Figure 11 is a clinical specimen detection result chart of the present application;

[0082] Figure 12 is a LAMP multiplex probe method reaction system chart No. 5 of the present application. DETAILED DESCRIPTION

[0083] The technical solutions in the embodiments of the present application will be clearly described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art belong to the scope of protection of the present application.

[0084] In the description of the present application, it should be noted that the terms used herein are only intended to describe the specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. For the convenience of description, the sizes of the various parts shown in the drawings are not drawn in accordance with the actual proportional relationship. The technology, methods and devices known to those skilled in the relevant art can not be discussed in detail, but in appropriate cases, the technology, methods and devices should be considered as part of the authorized description. In all examples shown and discussed herein, any specific value should be interpreted as merely exemplary, and not as a limitation. Therefore, other examples of exemplary embodiments can have different values. It should be noted that similar reference numerals and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0085] Embodiment 1:

[0086] Please refer to Figures 1-12 The present embodiment provides a multiplex LAMP detection method for Mycobacterium tuberculosis complex based on a probe method.

[0087] including target genes IS6110, IS1081 and GAPDH, also including the following sequences:

[0088] IS6110 primer and probe:

[0089] F3: TCGAGTTGGCCACCGC (SEQ ID NO: 1)

[0090] B3: GTCGCCAACTCCCACAG (SEQ ID NO: 2)

[0091] FIP: CAGCCTCGAGTTCGACCGGGTTCAACCATCGCCGCCT (SEQ ID NO: 3)

[0092] BIP: TCAGAGAGTCTCCGGACTCACCCCGCCAACACCTTTCGC (SEQ ID NO: 4)

[0093] LF: TCGCCGCAGTACTGGTAG (SEQ ID NO: 5)

[0094] LB: CGGTTCAACCGCCAGTGA (SEQ ID NO: 6)

[0095] BIP-P: FAM-TCAGAGAGTCTCCGGACTCACCCCGCCAACACCTTTCGC (SEQ ID NO: 7)

[0096] BIP-FQ: GGTGAGTCCGGAGACTCTCTGA-BHQ1 (SEQ ID NO: 8)

[0097] IS1081 primers and probes:

[0098] F3: CGAGCCCGGATCTGCT (SEQ ID NO: 9)

[0099] B3: CGCGTTCAGCTCGCTTG (SEQ ID NO: 10)

[0100] FIP: TCGCTGCGTTCGCGGTAGCCTCGACGTTCATCGCCG (SEQ ID NO: 11)

[0101] BIP: ACCGCCACCGTGATTTCGACCGGGAAATAGCTGCCCTGG (SEQ ID NO: 12)

[0102] LF: GCTTCAGCCCCCATCAAGG (SEQ ID NO: 13)

[0103] LB: TGCCGCAACCATCGACG (SEQ ID NO: 14)

[0104] BIP-P: ROX-ACCGCCACCGTGATTTCGACCGGGAAATAGCTGCCCTGG (SEQ ID NO: 15)

[0105] BIP-FQ: TCGAAATCACGGTGGCGGT-BHQ2 (SEQ ID NO: 16)

[0106] GAPDH primers and probe:

[0107] F3: CAAGGTCATCCATGACAACT (SEQ ID NO: 17)

[0108] B3: CAGTGATGGCATGGACTG (SEQ ID NO: 18)

[0109] FIP: AGGAGCCAGTCTTGGATGAGAATTGGTATCGTGGAAGGACT (SEQ ID NO: 19)

[0110] BIP: GGTTCTGGGGACTGGCTTTCCATCACCCCTCTACCTCC (SEQ ID NO: 20)

[0111] LF: GAGCCTCAGTCCCATTCC (SEQ ID NO: 21)

[0112] LB: TTTCCTTTCAAGGTGGGGAG (SEQ ID NO: 22)

[0113] FIP-P: CY5-AGGAGCCAGTCTTGGATGAGAATTGGTATCGTGGAAGGACT (SEQ ID NO: 23)

[0114] FIP-FQ: TTTCTCATCCAAGACTGGCTCCT-BHQ2 (SEQ ID NO: 24).

[0115] wherein the LAMP primers and probes comprise the following components:

[0116] 10x isothermal amplification buffer (1-2.5X, preferably IX)

[0117] 100 mM MgS04(2-8 mM, preferably 6 mM)

[0118] 8 U Bst DNA polymerase (0.12-0.32 U / µL, preferably 0.32 U / µL)

[0119] Betaine (0-0.4M, preferably 0.2M)

[0120] dNTP Mix (10mM each, 0.2-2mM each, preferably 1.4mM each)

[0121] Sterile double-distilled water (make up to 25μL).

[0122] Wherein, the working concentration of the primers and probes is:

[0123] F3 / B3: 0.1-0.2μM (preferably 0.2μM)

[0124] F IP / B IP: 0.8-1.6μM (preferably 1.6μM)

[0125] LF / LB: 0.4-0.8μM (preferably 0.8μM)

[0126] Probes (BIP-P / FIP-P) and quenching probes (BIP-FQ / FIP-FQ): 0.4-0.8μM (preferably 0.8μM).

[0127] Further comprising the following steps:

[0128] Step S1, extracting the nucleic acid of the sample to be tested;

[0129] Step S2, preparing the reaction system of the kit of claim 2 or 3, with a total reaction volume of 25μL;

[0130] Step S3, performing isothermal amplification at 63℃, with 99 cycles, and collecting fluorescence signals every 20 seconds;

[0131] Step S4, result interpretation: Ct value of IS6110 or IS1081 ≤60 is positive; Ct value of GAPDH ≤60 is valid internal standard, otherwise the sample needs to be prepared again.

[0132] Wherein, IS6110, IS1081 and GAPDH can be simultaneously detected in steps S1 and S4, with a sensitivity of 10 0 copies / μL, and no cross-reaction with common non-tuberculous mycobacteria.

[0133] Wherein, the fluorescence labels of the primers and probes are:

[0134] IS6110-BIP-P: 5' end FAM, 3' end BHQ1;

[0135] IS1081-BIP-P: 5' end ROX, 3' end BHQ2;

[0136] GAPDH-FIP-P: 5' end CY5, 3' end BHQ2.

[0137] Example 2:

[0138] The embodiment provides a probe-based multiplex LAMP detection method for the Mycobacterium tuberculosis complex, in addition to the technical solutions in the above embodiment, further has the following technical features.

[0139] The method comprises the following steps:

[0140] Extracting nucleic acid of a sample to be detected:

[0141] The experiment sets a control group and an experimental group, the control group is a sputum sample of a normal person, the experimental group is inactivated BCG freeze-dried bacteria purchased, the strain is obtained through a public channel, and the sputum sample of the normal person is diluted to 104 copies / mL;

[0142] Sputum liquefaction:

[0143] 1-2 volumes of 4% NaOH are added to the sputum, shaken, and placed at room temperature for about 30 minutes for liquefaction; 0.5 mL to 1.5 mL of 4% NaOH is added to a 0.5 mL centrifuge tube, and the sputum is placed at room temperature for 10 minutes for full liquefaction; centrifugation is performed at 13000 rpm for 5 minutes, the supernatant is discarded, and the lower precipitate is collected for use;

[0144] Nucleic acid extraction and purification:

[0145] A nucleic acid extraction or purification kit (Wuhan Dechen Biological Technology Co., Ltd.) is used, and DNA is extracted according to the kit instructions;

[0146] Lysis and combination:

[0147] A new 1.5 mL centrifuge tube is taken, 300 μL of sputum liquefaction sample, 600 μL of lysis solution, and 20 μL of magnetic bead solution (fully vortexed before use) are added, fully shaken and mixed, heated at 80°C for 5 min (inverted several times every 1 min), then cooled at room temperature for 1 min, and then magnetically absorbed to the solution to be clear (about 1 min), and the supernatant is discarded;

[0148] Washing:

[0149] (1) 700 μL of washing solution I is added, and the magnetic beads are slowly blown and beaten with a pipette gun for 10 times or vortexed for 10 s, so that the magnetic beads are fully resuspended, magnetically absorbed to the solution to be clear (about 1 min), and the supernatant is discarded;

[0150] (2) 800 μL of washing solution II is added, and the magnetic beads are slowly blown and beaten with a pipette gun for 10 times or vortexed for 10 s, so that the magnetic beads are fully resuspended, magnetically absorbed to the solution to be clear (about 1 min), and the supernatant is discarded;

[0151] (3) Add 800 μL of wash solution III, resuspend without magnetic beads, quickly magnetize to the solution clear (about 1 min), discard the supernatant;

[0152] Elution:

[0153] Add 60-100 μL of elution solution, heat at 80°C for 5 min, then resuspend the magnetic beads by blowing with a pipette gun for 10 times or vortexing for 1 min, magnetize to the solution clear, transfer the supernatant to a new 1.5 mL centrifuge tube, which is the purified nucleic acid, which can be used immediately or stored at -20°C or below;

[0154] Prepare the LAMP multiplex reaction system and perform the loop-mediated isothermal amplification reaction;

[0155] Amplification procedure: 63°C, 99 cycles, 20s acquisition of FAM channel fluorescence signal once; 85°C, 5 min;

[0156] Detection result analysis: according to the automatic threshold line interpretation result, Ct value ≤70 is effective amplification; if Figure 1 As shown, the results show that the LAMP multiplex probe method can accurately detect the IS6110, IS1081 genes of Mycobacterium tuberculosis, can detect the human-derived internal standard GAPDH gene, and can report positive detection in about 10 minutes, which greatly shortens the detection time compared with fluorescent quantitative PCR.

[0157] Example 3:

[0158] The present embodiment provides a Mycobacterium tuberculosis complex detection method based on multiplex LAMP probe method, in addition to the technical solutions of the above-mentioned embodiments, it also has the following technical features.

[0159] Comprising the following steps:

[0160] Extract the nucleic acid of the sample to be tested:

[0161] The strains for cross-interference analysis in this experiment are as shown in Figure 4 All strains are obtained by public access, and normal human sputum samples are diluted to 5x106cfu / mL;

[0162] Sputum liquefaction:

[0163] Add 1-2 volumes of 4% NaOH to the sputum, shake well, and place at room temperature for about 30 minutes for liquefaction; take 0.5 mL to 1.5 mL centrifuge tube, then add 0.5 mL of 4% NaOH and place at room temperature for 10 minutes to fully liquefy; centrifuge at 13000 rpm for 5 minutes, discard the supernatant, and collect the lower precipitate for use;

[0164] Nucleic acid extraction and purification:

[0165] DNA was extracted using a nucleic acid extraction or purification kit (Wuhan Dechen Biological Technology Co., Ltd.) according to the kit instructions;

[0166] Lysis and binding:

[0167] Take a new 1.5 mL centrifuge tube, add 300 μL of sputum liquefaction sample, 600 μL of lysis solution, 20 μL of magnetic bead solution (mix well before use), shake well, heat at 80°C for 5 min (invert several times every 1 min), then cool at room temperature for 1 min, then magnetically attract the solution to clarify (about 1 min), discard the supernatant;

[0168] Washing:

[0169] (1) Add 700 μL of washing solution I, slowly blow the magnetic beads with a pipette gun for 10 times or vortex for 10 s to fully resuspend the magnetic beads, magnetically attract the solution to clarify (about 1 min), discard the supernatant;

[0170] (2) Add 800 μL of washing solution II, slowly blow the magnetic beads with a pipette gun for 10 times or vortex for 10 s to fully resuspend the magnetic beads, magnetically attract the solution to clarify (about 1 min), discard the supernatant;

[0171] (3) Add 800 μL of washing solution III, without fully resuspending the magnetic beads, quickly magnetically attract the solution to clarify (about 1 min), discard the supernatant;

[0172] Elution:

[0173] Add 60-100 μL of elution solution, heat at 80°C for 5 min, then slowly blow the magnetic beads with a pipette gun for 10 times or vortex for 1 min to fully resuspend the magnetic beads, then magnetically attract the solution to clarify, transfer the supernatant to a new 1.5 mL centrifuge tube, which is the purified nucleic acid, which can be used immediately or stored at -20°C or below

[0174] Prepare the LAMP multiplex reaction system and perform loop-mediated isothermal amplification reaction;

[0175] Amplification program: 63°C, 99 cycles, 20 s acquisition of FAM / VIC / CY5 channel fluorescence signal; 85°C, 5 min;

[0176] Detection result analysis: according to the automatic threshold line interpretation result, Ct value ≤70 is effective amplification; the detection result is shown in Figure 2 The results show that the cross-reacted strains are not amplified, and the LAMP multiplex probe method has strong specificity and no cross-reaction with common bacteria.

[0177] Example 4:

[0178] The embodiment provides a Mycobacterium tuberculosis complex detection method based on a multiplex LAMP probe method, and has the following technical features in addition to the technical solutions in the above embodiment.

[0179] The method comprises the following steps:

[0180] Preparation of nucleic acid of a sample to be detected;

[0181] The nucleic acid used for sensitivity analysis in the experiment is a DNA plasmid containing the Mycobacterium tuberculosis IS6110 gene, the IS1081 gene and the GAPDH internal standard; the preparation steps of the plasmid are as follows: a plasmid containing a target gene fragment is constructed in Shanghai Sangon; the A260 is measured by a spectrophotometer to quantify and dilute to prepare P1-P6 plasmids, and the concentration is shown in Table 1. Figure 5 -20℃ preservation;

[0182] Preparation of a LAMP multiplex reaction system and a LAMP dye method system, and carrying out a loop-mediated isothermal amplification reaction;

[0183] Multiplex probe method amplification procedure: 63℃, 99 cycles, 20s acquisition of FAM / VIC / CY5 channel fluorescence signal once; 85℃, 5min; dye method amplification procedure: 63℃, 99 cycles, 20s acquisition of FAM channel fluorescence signal once; 85℃, 5min;

[0184] Result interpretation: dye method: automatic threshold line is used, Ct≤70, and the detection result is positive; multiplex probe method: automatic threshold line is used, IS6110 or IS1081 Ct≤70, and the detection result is positive; if the GAPDH Ct≤70, the sample needs to be prepared again;

[0185] The detection result is shown in Table 2. Figure 8 The results show that the LAMP multiplex probe method has higher sensitivity than the dye method, and can detect 10^0 copies / ul.

[0186] Embodiment 5

[0187] The embodiment provides a Mycobacterium tuberculosis complex detection method based on a multiplex LAMP probe method, and has the following technical features in addition to the technical solutions in the above embodiment;

[0188] The method comprises the following steps:

[0189] Preparation of nucleic acid of a sample to be detected:

[0190] Liquefaction of sputum:

[0191] Add 1-2 times the volume of 4% NaOH to the sputum, shake well, and let it stand at room temperature for about 30 minutes to liquefy; take 0.5 mL into a 1.5 mL centrifuge tube, add 0.5 mL of 4% NaOH, and let it stand at room temperature for 10 minutes to fully liquefy; centrifuge at 13000 rpm for 5 minutes, discard the supernatant, and collect the lower precipitate for later use;

[0192] Nucleic acid extraction and purification:

[0193] DNA was extracted using a nucleic acid extraction or purification kit (Wuhan Dechen Biotechnology Co., Ltd.) according to the kit instructions;

[0194] Cleavage and Binding:

[0195] Take a new 1.5 mL centrifuge tube, add 300 μL of sputum liquefaction sample, 600 μL of lysis buffer, and 20 μL of magnetic bead solution (vortex thoroughly before use), shake thoroughly to mix, heat at 80°C for 5 min (invert several times every 1 min), then cool at room temperature for 1 min and magnetically absorb until the solution is clear (about 1 min), and discard the supernatant;

[0196] washing:

[0197] (1) Add 700 μL of washing solution I, pipette slowly to the magnetic beads 10 times or vortex for 10 seconds to fully resuspend the beads, magnetically absorb until the solution is clear (about 1 minute), and discard the supernatant;

[0198] (2) Add 800 μL of washing solution II, slowly pipette the magnetic beads 10 times or vortex for 10 seconds to fully resuspend the magnetic beads, magnetically absorb until the solution is clear (about 1 minute), and discard the supernatant;

[0199] (3) Add 800 μL of washing solution III. Without fully resuspending the magnetic beads, quickly magnetically aspirate until the solution is clear (about 1 min), and discard the supernatant.

[0200] Elution:

[0201] Add 60-100 μL of elution buffer and heat at 80°C for 5 minutes. Use a pipette to slowly blow the magnetic beads 10 times or vortex for 1 minute to fully resuspend the beads. Then magnetically absorb until the solution is clear. Transfer the supernatant to a new 1.5 mL centrifuge tube. This is the purified nucleic acid, which can be used immediately or stored at -20°C or below.

[0202] Prepare LAMP multiplex reaction system and LAMP dye method system to perform loop-mediated isothermal amplification reaction;

[0203] Multiplex probe method amplification procedure: 63 DEG C, 99 cycles, 20s acquisition of FAM / VIC / CY5 channel fluorescence signal once; 85 DEG C, 5min; Dye method amplification procedure: 63 DEG C, 99 cycles, 20s acquisition of FAM channel fluorescence signal once; 85 DEG C, 5min;

[0204] Result interpretation: dye method: automatic threshold line is used, Ct<=70, and the detection result is positive; multiplex probe method: automatic threshold line is used, IS6110 or IS1081 Ct<=70, and the detection result is positive; if GAPDH Ct<=70, the sample needs to be prepared again; the detection result is as shown in the table, and the results show that, for the detection of clinical specimens, the coincidence rate of the LAMP multiplex probe method and the Daan detection kit is 100%, and the coincidence rate of the dye method is only 39%; the probe method adds one more detection target gene IS1081, improves the detection rate of positive samples, weakens the non-specific amplification of the dye method, and reduces false positives. Figure 11

[0205] The LAMP multiplex probe method for detecting IS6110 and IS1081 primer groups of Mycobacterium tuberculosis complex in the application can be used for quickly and simultaneously detecting Mycobacterium tuberculosis complex, improves the positive detection rate of sputum samples, and weakens non-specific amplification and reduces false positives;

[0206] The setting of the internal reference gene GAPDH can effectively monitor the sample collection / sample preservation / sample nucleic acid extraction process, and avoid false negatives;

[0207] The specificity is strong, and there is no cross reaction with common bacteria.

[0208] The embodiments of the application are described above in combination with the drawings, and the embodiments in the application and the features in the embodiments can be combined with each other without conflict, the application is not limited to the above specific embodiments, and the above specific embodiments are only illustrative but not restrictive, and a person of ordinary skill in the art can make many forms under the guidance of the application without departing from the purpose of the application and the scope protected by the claims, and all the forms belong to the protection of the application.​

Claims

1. A method for detecting Mycobacterium tuberculosis complex based on a multiplex LAMP probe method, characterized in that: IS6110, IS1081 and GAPDH, see the following information: IS6110 mounting plate: F3:TCGAGTTGGCCACCGC(SEQ ID NO:1) B3:GTCGCCAACTCCCACAG(SEQ ID NO:2) FIP:CAGCCTCGAGTTCGACCGGGTTCAACCATCGCCGCCT(SEQ ID NO:3); BIP:TCAGAGAGTCTCCGGACTCACCCCGCCAACACTTTCGC(SEQ ID NO:4) LF:TCGCCGCAGTACTGGTAG(SEQ ID NO:5) LB:CGGTTCAACCGCCAGTGA(SEQ ID NO:6) BIP-P:FAM-TCAGAGAGTCTCCGGACTCACCCCGCCAACACCTTTCGC(SEQ ID NO:7)BIP-FQ:GGTGAGTCCGGAGACTCTCTGA-BHQ1(SEQ ID NO:8) IS1081 specifications: F3:CGAGCCCGGATCTGCT(SEQ ID NO:9) B3:CGCGTTCAGCTCGCTTG(SEQ ID NO:10) FIP:TCGCTGCGTTCGCGGTAGCCTCGACGTTCATCGCCG(SEQ ID NO:11) BIP:ACCGCCACCGTGATTTCGACCGGGAAATAGCTGCCCTGG(SEQ ID NO:12) LF:GCTTCAGCCCCCATCAAGG(SEQ ID NO:13) LB:TGCCGCAACCATCGACG(SEQ ID NO:14) BIP-P:ROX-ACCGCCACCGTGATTTCGACCGGGAAATAGCTGCCCTGG(SEQ ID NO:15)BIP-FQ:TCGAAATCACGGTGGCGGT-BHQ2(SEQ ID NO:16) GAPDH scientists: F3:CAAGGTCATCCATGACAACT(SEQ ID NO:17) B3:CAGTGATGGCATGGACTG(SEQ ID NO:18) FIP:AGGAGCCAGTCTTGGATGAGAATTGGTATCGTGGAAGGACT(SEQ ID NO:19)BIP:GGTTCTGGGGACTGGCTTTCCATCACCCCTCTACCTCC(SEQ ID NO:20) LF:GAGCCTCAGTCCCATTCC(SEQ ID NO:21) LB:TTTCCTTTCAAGGTGGGGAG(SEQ ID NO:22) FIP-P:CY5-AGGAGCCAGTCTTGGATGAGAATTGGTATCGTGGAAGGACT(SEQ ID NO:23) FIP-FQ: TTTCTCATCCAAGACTGGCTCCT-BHQ2 (SEQ ID NO: 24).

2. The method for detecting Mycobacterium tuberculosis complex based on multiple LAMP probes according to claim 1, characterized in that: The LAMP primers and probes include the following components: 10× isothermal amplification buffer (1-2.5×, preferably 1×) 100 mM MgSO4 (2-8 mM, preferably 6 mM) 8U Bst DNA polymerase (0.12-0.32U / μL, preferably 0.32U / μL) Betaine (0-0.4M, preferably 0.2M) dNTP Mix (10mM each, 0.2-2mM each, preferably 1.4mM each) Sterile double-distilled water (make up to 25 μL).

3. The method for detecting Mycobacterium tuberculosis complex based on multiple LAMP probes according to claim 1, characterized in that: The working concentrations of the primers and probes are: F3 / B3: 0.1-0.2 μM (preferably 0.2 μM) F IP / B IP: 0.8-1.6 μM (preferably 1.6 μM) LF / LB: 0.4-0.8 μM (preferably 0.8 μM) Probe (BIP-P / FIP-P) and quencher probe (BIP-FQ / FIP-FQ): 0.4-0.8 μM (preferably 0.8 μM).

4. The method for detecting Mycobacterium tuberculosis complex based on multiple LAMP probes according to claim 1, characterized in that: The following steps are also included: Step S1, extracting nucleic acid from the sample to be tested; Step S2, preparing the reaction system of the kit according to claim 2 or 3, with a total reaction volume of 25 μL; Step S3, performing isothermal amplification at 63°C for 99 cycles, collecting fluorescence signals every 20 seconds; Step S4, result interpretation: a Ct value of IS6110 or IS1081 ≤ 60 is considered positive; a Ct value of GAPDH ≤ 60 is considered a valid internal standard, otherwise the sample needs to be re-prepared.

5. The method for detecting Mycobacterium tuberculosis complex based on multiple LAMP probes according to claim 4, characterized in that: In steps S1 and S4, IS6110, IS1081 and GAPDH can be detected simultaneously with a sensitivity of 10 copies / μL, and there is no cross-reaction with common non-tuberculous mycobacteria.

6. The method for detecting Mycobacterium tuberculosis complex based on multiple LAMP probes according to claim 1, characterized in that: The fluorescent labels of the primers and probes are: IS6110-BIP-P: FAM at 5′ end, BHQ1 at 3′ end; IS1081-BIP-P: ROX at 5′ end, BHQ2 at 3′ end; GAPDH-FIP-P: CY5 at the 5′ end and BHQ2 at the 3′ end.

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