A quantitative detection method for active Mycobacterium tuberculosis and its application

Abstract

The present invention relates to the field of medical detection technology, and provides a quantitative detection method for active Mycobacterium tuberculosis and its application. The label primer, reverse transcription primer, specific amplification primer and / or probe are designed with MTB 16S rRNA as the target gene, and the two target genes IS6100 and / or IC MM tmRNA are detected simultaneously to avoid the interference of genomic DNA. It can also be used to distinguish active tuberculosis from inactive tuberculosis. The amplification curve and CT value obtained by qPCR detection make the bacterial concentration and copy concentration correlated, and then the relative expression of active Mycobacterium tuberculosis is calculated by quantitative amplification. The detection result of the present invention has high sensitivity and specificity, and the detection sensitivity of active Mycobacterium tuberculosis can reach 10 1 copies / μL or 10 1 CFU / ml is suitable for reflecting the efficacy of medication and the infectiousness of tuberculosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and medical device detection technology, and in particular to a quantitative detection method for active Mycobacterium tuberculosis, a detection kit and applications thereof. Background Art

[0002] Pulmonary tuberculosis (also known as consumption) is the most common type of tuberculosis and is a chronic infectious disease. Mycobacterium tuberculosis (MTB), also known as Mycobacterium tuberculosis or Mycobacterium tuberculosis, infects the human body through inhalation of bacteria-containing droplets. After invading the human body, it mainly invades the lungs. Therefore, Mycobacterium tuberculosis is the main microorganism that causes pulmonary tuberculosis and poses a major threat to public health.

[0003] At present, the detection of Mycobacterium tuberculosis mainly relies on acid-fast staining (sputum smear), bacterial culture (or sputum culture), and nucleic acid detection represented by Xpert MTB / RIF. Both sputum culture and sputum smear (sensitivity is about 50%) have problems such as low sensitivity and poor specificity. Among them, although sputum culture is the gold standard for the diagnosis of tuberculosis, it takes a long time to get the results (2-8 weeks); and although sputum smear can get the results within 24 hours, the test is relatively fast and simple, but the sensitivity is relatively low, and its negative test results cannot completely rule out the possibility of tuberculosis. Nucleic acid detection methods for Mycobacterium tuberculosis, such as Xpert MTB / RIF with DNA as the target, can significantly improve the sensitivity and timeliness of the test, but due to the stability of DNA, the DNA template for in vitro amplification detection can come from live bacteria or dead bacteria, which is not suitable as a good detection marker for distinguishing active tuberculosis (ATB).

[0004] Compared with stable DNA, RNA mainly includes ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). Different types of RNA exist in different species with different stabilities. Messenger RNA (mRNA) is the blueprint for synthesizing proteins, which is related to gene expression and is most closely related to the activity and viability of Mycobacterium tuberculosis. However, there are literature reports that the detection method targeting Mycobacterium tuberculosis mRNA cannot avoid the false positive problem caused by genomic DNA (gDNA) contamination during amplification; the lack of gene expression of Mycobacterium tuberculosis does not mean that it is dead, and there are also inactive (dormant) but viable Mycobacterium tuberculosis. Although the dormant MTB does not reproduce, the rRNA in its cells still exists. Usually, rRNA will be rapidly degraded after the bacteria die. Therefore, in theory, rRNA is generally considered to be a molecular marker of live bacteria. For example, the TB-SAT test that targets MTB 16S rRNA, because MTB 16S rRNA is a structural RNA that is related to intact living cells, can be a suitable marker for detecting and quantifying live bacterial load. Compared with mRNA, MTB16S rRNA has a much longer half-life and stability. When Mycobacterium tuberculosis cells are killed by anti-tuberculosis treatment, the amount of MTB 16SrRNA will also decrease, which allows MTB 16S rRNA to reflect the activity of bacteria excretion in the human body and the therapeutic effect to a certain extent. However, the TB-SAT test that targets MTB 16S rRNA still has certain limitations and complexities: on the one hand, TB-SAT is used to distinguish between live and dead bacteria, as well as live bacteria in a dormant state, which is affected by many factors, such as the degradation rate after bacterial death, the compliance of sample collection, the stability of RNA during sample processing, etc., which may affect the accuracy of the test results. On the other hand, since TB-SAT is a constant temperature amplification technology based on T7 RNA polymerase, and T7 RNA polymerase is essentially a DNA-dependent RNA polymerase, its amplification target is still the DNA obtained after reverse transcription as a template, and these DNAs are used as templates to synthesize a large number of RNA copies. This process cannot directly reflect the actual RNA quantity of Mycobacterium tuberculosis, so it is impossible to calculate the relative expression level of the corresponding active Mycobacterium tuberculosis, and it is even more impossible to achieve quantitative detection of active Mycobacterium tuberculosis. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a rapid, sensitive, specific quantitative detection method that can truly distinguish active Mycobacterium tuberculosis and detect the relative expression amount of active Mycobacterium tuberculosis by using MTB 16S rRNA and genomic DNA as detection targets.

[0006] In order to achieve the above purpose or one of the purposes, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a primer composition for nucleic acid detection of active Mycobacterium tuberculosis. The primer composition with MTB 16S rRNA as the target gene comprises a reverse transcription primer and a specific amplification primer. The reverse transcription primer comprises a nucleotide sequence of a label primer and a nucleotide sequence for specific reverse transcription amplification of MTB 16S rRNA. The specific amplification primer comprises an upstream primer and a downstream primer. The nucleotide sequences of the label primer and the upstream primer are the same and are shown in SEQ ID NO: 3. The nucleotide sequence for specific reverse transcription amplification of MTB 16S rRNA is shown in SEQ ID NO: 2. The nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4.

[0008] According to a preferred embodiment of the present invention, in the primer composition, the nucleotide sequence of the reverse transcription primer is shown as SEQ ID NO: 1.

[0009] According to a preferred embodiment of the present invention, the primer composition further comprises specific amplification primers targeting IS6100 of Mycobacterium tuberculosis and MM tmRNA of Mycobacterium marinum, respectively, and the specific amplification primers respectively comprise corresponding upstream primers and downstream primers.

[0010] According to a preferred embodiment of the present invention, when qPCR amplification is performed based on specific amplification primers, the primer composition further includes corresponding probes with fluorescent groups labeled at the 5' end and quenching groups labeled at the 3' end.

[0011] Preferably, the fluorescent group is FAM, HEX, or ROX; the quenching group is BHQ1 or BHQ2;

[0012] More preferably, when the fluorescent group is FAM, the quenching group is BHQ1; when the fluorescent group is HEX or ROX, the quenching group is BHQ2.

[0013] In a second aspect, the present invention provides a method for detecting active Mycobacterium tuberculosis, using the above-mentioned primer combination to perform nucleic acid detection on Mycobacterium tuberculosis, and using MTB 16S rRNA as the target gene for reverse transcription amplification and specific amplification, respectively, the reverse transcription amplification includes step one and step two, and the specific amplification is qPCR amplification; wherein, step one includes adding reverse transcription system one to perform reverse transcription procedure one, step two includes briefly centrifuging reverse transcription system one after step one and then adding reverse transcription system two, and then performing reverse transcription procedure two, and the product after step two is the reverse transcription product; the template for qPCR amplification is the reverse transcription product, and the corresponding total volume of the qPCR reaction system is 50 μL; after performing the qPCR amplification reaction procedure, the amplification curve of the sample to be tested and the CT value of the positive amplification result can be obtained.

[0014] According to a preferred embodiment of the present invention, the preferred reaction conditions for reverse transcription amplification and specific amplification include: in reverse transcription amplification, the total volume of reverse transcription system one is 10 μL, including: 1 μL 10 mM dNTPs, 1 μL reverse transcription primer and 8 μL reverse transcription template; reverse transcription program one is: 50-65°C reaction for 5-10 min and then cooling on ice for 1-5 min; the total volume of reverse transcription system two is 10 μL, including: 2 μL 10× reverse transcription buffer, 4 μL 25 mM MgCl2, 2 μL 0.1 M DTT, 1 μL recombinant ribonuclease inhibitor and 1 μL reverse transcriptase; reverse transcription procedure 2: react at 45-55°C for 45-60 min, heat inactivate at 75-85°C for 5-10 min, and then cool at low temperature; qPCR reaction system includes: 25 μL 2× enhanced Tag enzyme premix, 1 μL each of upstream primer and downstream primer at a concentration of 5-20 pmol / μL, 2 μL of probe at a concentration of 5-20 pmol / μL, 2 μL of reverse transcription product, Add ddH2O to 50μL; the reaction procedure for qPCR amplification is as follows: the first step, pre-denaturation at 94℃~96℃ for 30sec~60sec; the second step, denaturation at 94~96℃ for 4sec~10sec, annealing and extension at 55℃~61℃ for 30sec~60sec, and this for 37~45 cycles; the third step, denaturation at 94~96℃ for 5~10sec, annealing and extension at 60~65℃ for 5~10sec, and denaturation at 94~96℃ for 5~10sec.

[0015] Preferably, in the reverse transcription system 1, the concentration of the reverse transcription primer is 2 pmol / mL; the reverse transcription procedure 1 is to react at 50°C for 5 min and then cool on ice for 1 min; in the reverse transcription system 2, the recombinant ribonuclease inhibitor is RNase OUT, the concentration is 40 U / μL, and the concentration of the reverse transcriptase is 200 U / μL; the reverse transcription procedure 2 is to react at 50°C for 50 min and then heat inactivate at 85°C for 5 min, cool at below 25°C for 10 min, and then cool on ice for standby use or store at -20°C for standby use; in the qPCR reaction system, the final concentrations of the primers and probes are unified as follows: each primer is 0.2 μM, and each probe is 0.4 μM; the reaction procedure of qPCR amplification is the first step, 95°C pre-denaturation for 30 sec; the second step, 95°C denaturation for 5 sec, 59°C annealing and extension for 30 sec, 40 cycles (fluorescence is collected at 59°C); the third step, denaturation at 95°C for 10 sec, 65°C annealing and extension for 5 sec, and 95°C denaturation for 5 sec in sequence.

[0016] According to a preferred embodiment of the present invention, in the method for detecting active Mycobacterium tuberculosis, the target gene for qPCR amplification of the same sample to be tested also includes IS6100 of Mycobacterium tuberculosis and / or MMtmRNA of Mycobacterium marinum, and the qPCR amplification reaction system and reaction procedure are the same, and amplification curves and amplification CT values ​​corresponding to different target genes can be obtained.

[0017] Preferably, different target genes have corresponding primer compositions that are different, but the qPCR amplification reaction system and reaction procedure are the same as the qPCR amplification conditions with MTB 16S rRNA as the target gene, and the amplification curves corresponding to different target genes and the CT values ​​of the positive amplification results are obtained under the same qPCR amplification conditions.

[0018] In a third aspect, the present invention provides a standard for quantitative detection of active Mycobacterium tuberculosis, and the preparation of the standard mainly includes the preparation of active bacterial liquid and the preparation of the standard; wherein, the preparation of the active bacterial liquid includes: enriching and culturing the original strain of Mycobacterium tuberculosis H37Ra strain, and obtaining bacterial liquid of different concentrations after dilution of the cultured bacterial liquid to different degrees, and performing OD value detection, plate culture and colony counting respectively to obtain OD value and corresponding bacterial concentration; the preparation of the standard includes: extracting total RNA of the bacterial liquid, and using the above-mentioned primer composition and the extracted total RNA as a template to perform reverse transcription and RT-PCR amplification in sequence; wherein, the RT-PCR amplification uses the reverse transcription product obtained by reverse transcription as a template and MTB 16S rRNA as a target gene; constructing the RT-PCR amplification product into a gene cloning vector to form a recombinant plasmid containing the target gene, and the recombinant plasmid is diluted to different concentrations and then quantified, so as to obtain a standard of a series of concentrations of Mycobacterium tuberculosis H37Ra strain.

[0019] According to a preferred embodiment of the present invention, the gene cloning vector is preferably a pLB vector.

[0020] According to a preferred embodiment of the present invention, the standard product also includes a series of concentrations of marine mycobacterium standards prepared using marine mycobacterium as the original strain, and the preparation steps refer to the series of concentrations of the Mycobacterium tuberculosis H37Ra strain standard product.

[0021] Preferably, when preparing standards of a series of concentrations of marine mycobacterium, the reverse transcription primers are random primers, and MMtmRNA is used as the target gene.

[0022] Preferably, the standard involves two different strains, namely marine mycobacterium and mycobacterium tuberculosis H37Ra strain. The cultured bacterial solution is diluted by 1, 2, 4, 8, and 16 times, and a second dilution of 10 times is required before being used for plate colony counting. The series concentration of the standard is recorded according to the copy concentration, and the concentration range is preferably: 0.5×10 8 copies / μL~0.5×10 2 copies / μL.

[0023] In a fourth aspect, the present invention provides a method for quantitative detection of active Mycobacterium tuberculosis, the detection steps comprising: determining the quantitative relationship between bacterial concentration and copy concentration, performing nucleic acid detection of active Mycobacterium tuberculosis and quantitative analysis of the detection results; wherein the method for determining the quantitative relationship between bacterial concentration and copy concentration is: using a standard substance used for quantitative detection of active Mycobacterium tuberculosis and bacterial liquids of different concentrations obtained in the preparation process of the standard substance as detection objects, respectively, performing detection according to the detection method for active Mycobacterium tuberculosis, and establishing a quantitative relationship between bacterial concentration and copy concentration through a standard curve corresponding to the standard substance and CT values ​​corresponding to bacterial liquids of different concentrations; the method for performing nucleic acid detection of active Mycobacterium tuberculosis is: using a sample to be detected as a detection object, performing detection according to the detection method for active Mycobacterium tuberculosis, and obtaining amplification curves and amplification CT values ​​of MTB 16S rRNA, IS6100 of Mycobacterium tuberculosis and / or MM tmRNA of marine Mycobacterium as target genes; the method for quantitative analysis of the detection results is: MM of marine Mycobacterium The amplification results corresponding to tmRNA as the target gene are used to verify the credibility; the positive amplification results corresponding to IS6100 of Mycobacterium tuberculosis as the target gene are used to compare and distinguish the activity of Mycobacterium tuberculosis; the amplification results corresponding to MTB 16S rRNA as the target gene are used to calculate the bacterial concentration of active Mycobacterium tuberculosis based on the quantitative relationship between the amplification CT value and the copy concentration.

[0024] According to a preferred embodiment of the present invention, the quantitative relationship between bacterial concentration and copy concentration established with MM tmRNA of marine mycobacterium as the target gene is: bacterial concentration 1×10 6 The copy concentration corresponding to CFU / mL is 3.77×10 6 Copies / μL.

[0025] According to a preferred embodiment of the present invention, the quantitative relationship between bacterial concentration and copy concentration established with MTB 16S rRNA as the target gene is that a bacterial concentration copy concentration of 3.77 CFU / mL corresponds to a copy concentration of 1 copies / μL.

[0026] In a fifth aspect, the present invention provides a detection kit for active Mycobacterium tuberculosis, comprising the above-mentioned primer composition, supporting reagents, reverse transcriptase, DNA polymerase, positive quality control products and negative controls.

[0027] According to a preferred embodiment of the present invention, the DNA polymerase is an enhanced Tag enzyme;

[0028] According to a preferred embodiment of the present invention, the supporting reagents include 10× reverse transcription buffer and 2× enhanced Tag enzyme premix.

[0029] In a sixth aspect, the present invention provides the primer composition, the method for detecting active Mycobacterium tuberculosis, the standard for quantitative detection of active Mycobacterium tuberculosis, the detection kit for active Mycobacterium tuberculosis, and their use in preparing an active Mycobacterium tuberculosis detection product.

[0030] Beneficial effects of the present invention:

[0031] The present invention introduces a reverse transcription primer containing a molecular tag and a primer pair for specific amplification, which can not only quantitatively detect the MTB 16S rRNA as the main target gene, but also achieve the purpose of avoiding false positives caused by gDNA contamination through the "molecular tag" added to the reverse transcription product; the present invention optimizes the detection of MTB 16S rRNA on the basis of optimizing the detection of MTB 16S rRNA. The unified reaction conditions for specific amplification of two target genes, rRNA and IS6100, can better distinguish the activity of Mycobacterium tuberculosis by comparing the detection results of the two, and reflect the efficacy of Mycobacterium tuberculosis on the medication regimen; compared with the insufficient sensitivity and long detection cycle of classic sputum smear and sputum culture, the detection of active Mycobacterium tuberculosis in the present invention belongs to nucleic acid detection, which has the advantages of high sensitivity and specificity and rapid detection; the present invention can also quantitatively detect active Mycobacterium tuberculosis by introducing standard products by integrating the knowledge of basic pathogen biology and clinical laboratory diagnosis, more truly reflect the relative expression amount of active Mycobacterium tuberculosis, that is, the bacterial concentration, convert the nucleic acid amplification results into copy concentration (copies / μL) and the corresponding bacterial concentration (CFU / ml), evaluate the efficacy of tuberculosis drugs, establish an optimized medication course and the infectiousness classification of tuberculosis, and if the technical solution of the present invention can be implemented and transformed as soon as possible, it will have broad economic and social benefits.

[0032] Specifically, the present invention uses MTB 16S rRNA and genomic DNA as detection targets. First, a label primer with MTB 16s rRNA as the main target gene and a primer and probe composition containing the label primer sequence are provided. Among them, a barcodes index technology is used to introduce a molecular label (=label primer) to design specific reverse transcription and amplification primers, such as a marker sequence containing a molecular label, and a reverse transcription primer with both a molecular label and a specific sequence, an upstream primer and a downstream primer in specific amplification, and a probe that cooperates with the upstream primer and the downstream primer in qPCR. The use of the molecular label avoids the contamination of genomic DNA (gDNA) during amplification. Secondly, based on the preferred primers and primer-probe combinations, a method for detecting reverse transcription and specific nucleic acids for MTB 16S rRNA is established. Since the optimization process unifies the reaction conditions for nucleic acid detection, genomic DNA represented by IS6100 can be detected simultaneously to monitor the molecular load of active Mycobacterium tuberculosis (MTB) in the sample to be tested. IS6100 is an insertion repeat sequence unique to Mycobacterium tuberculosis, has a high copy number in the genome, and is often used as a specific target for detecting Mycobacterium tuberculosis DNA. The present invention can better reflect the efficacy of Mycobacterium tuberculosis on the medication regimen by simultaneously detecting at least two target genes, MTB 16S rRNA and IS6100, compared with the traditional single detection of MTB 16S rRNA and IS6100.

[0033] Comparison between molecular tags and traditional methods for preventing DNA contamination. In general, genomes are prone to false positive test results. There are two groups of traditional methods for preventing DNA contamination: one is to use kits such as RNeasy Plus Kits (QIA): gDNA Eliminator columns are used to remove DNA contamination during RNA extraction; the other is to digest gDNA with gDNARemover Mix before amplification. Although these two solutions for removing gDNA can reduce the contamination and interference of gDNA to a certain extent, they may not only cause the loss of sample nucleic acid, especially the loss of RNA with a low content, but also increase the complexity of sample pre-treatment in the detection operation, increase the detection cost, and ultimately cannot completely avoid gDNA contamination. The present invention uses molecular tags (barcodes index) in the second-generation sequencing to reverse transcribe and obtain labeled cDNA, and specifically selects MTB 16S rRNA as the target gene, which not only avoids pathogen genomic DNA contamination, but also makes the operation simpler. In addition, the present invention specifically introduces two reference standards: MTB genomic DNA (i.e., IS6100) and ICMM tmRNA of marine Mycobacterium marinum (M. marinum), which further avoids the false positive problem caused by genomic DNA contamination, distinguishes live bacteria from dead bacteria by comparing the amplification results of different target genes, accurately reflects the activity of bacterial excretion in the human body, realizes the distinction of active Mycobacterium tuberculosis, and is beneficial to the infectiousness classification of tuberculosis.

[0034] Conventionally, when determining the infectiousness grade of pulmonary tuberculosis, it is necessary to comprehensively consider multiple aspects such as the results of etiological tests (such as sputum culture), molecular biological test results (such as TB-SAT, X-pert and nucleic acid tests such as the present invention), imaging examinations and clinical symptoms. Although sputum culture is considered to be the gold standard for the diagnosis of tuberculosis, it has low sensitivity and a long culture time; although conventional nucleic acid testing is more suitable for early detection and discovery of drug-resistant tuberculosis due to its rapid detection and high sensitivity. However, the nucleic acid detection of active Mycobacterium tuberculosis in the present invention is a quantitative detection, which can obtain the relative expression amount of active Mycobacterium tuberculosis in the sample to be tested. Under the premise that the sample collection, transportation and detection are all true and effective, it can distinguish between active tuberculosis and inactive tuberculosis, and can more truly reflect the number of active Mycobacterium tuberculosis in the patient from whom the sample to be tested is derived, which is conducive to the classification and determination of the infectiousness of pulmonary tuberculosis. It is not only conducive to the formulation of protective measures corresponding to the degree of infectiousness, reducing the risk of infection, blocking the continued spread of MTB, and preventing further spread of the disease, but also helps to improve the detection accuracy of active Mycobacterium tuberculosis, and prospectively provide technical support for the prevention and treatment of infectious diseases such as pulmonary tuberculosis, and provide a technical basis for the planning and formulation of relevant rules such as isolation treatment.

[0035] Traditional nucleic acid testing cannot reflect the level of bacterial excretion activity in the human body. Generally, conventionally designed real-time fluorescence quantitative PCR needs to rely on standard curves or control samples for quantitative detection. Since the CT value in the test result indicates the number of cycles required to detect the target nucleic acid, which is inversely proportional to the initial amount of the virus in the sample, it is used to infer the amount of virus in the infected person. However, it is well known to those skilled in the art that the CT value is not a direct quantitative indicator and needs to be converted through a standard curve. In addition, the interpretation of the CT value also needs to consider multiple factors, such as sample quality, reaction efficiency, and even the dormant state of the bacteria. Therefore, the traditional nucleic acid quantitative test results cannot intuitively correspond to the concentration of active Mycobacterium tuberculosis in the original sample, or the relative expression of Mycobacterium tuberculosis target genes. The present invention selects MTB 16S rRNA as the main detection target, based on the representative strains of Mycobacterium tuberculosis H37Ra strain and marine Mycobacterium, preferably based on PLB cloning vector, constructs the standard products of the recombinant plasmids of the two strains and different concentrations of bacterial liquid, the two strains can verify each other, and the CT value is used as a bridge to establish the standard curve between the copy concentration copies / μL and the bacterial concentration (CFU / ml) corresponding to the standard products containing two target genes (MTB 16S rRNA and IC MM tmRNA), and obtain the correlation between the four standard curves. The target gene CT value is associated with the bacterial concentration and copy concentration of the active bacterial liquid, respectively, so that the copy concentration copies / μL and the corresponding bacterial concentration (CFU / ml) are associated, thereby realizing the quantitative detection of the relative expression of active Mycobacterium tuberculosis.

[0036] For most patients with pulmonary tuberculosis, conventional medication courses are chosen, which generally take at least half a year. Nucleic acid testing, with its advantages of simplicity, rapidity, and high specificity, can avoid the misdiagnosis and missed diagnosis problems that may exist in traditional testing methods; after the appropriate tuberculosis treatment plan reaches its efficacy, the patient's body will generally still have inactive tuberculosis DNA, resulting in false positive results for conventional nucleic acid tests. In addition, when choosing a course of medication, in order to avoid recurrence after discontinuation of medication or drug resistance caused by repeated medication, and also to ensure that Mycobacterium tuberculosis in the body is 100% completely eliminated, the choice of medication courses will be relatively conservative, and false positive test results may affect the choice of medication courses, which indirectly leads to extended medication time. Since the present invention provides a quantitative detection method that can more truly reflect the relative expression amount of the activity of Mycobacterium tuberculosis, the detection of active Mycobacterium tuberculosis is more sensitive, which is bound to assist doctors in optimizing treatment plans. For example, after truly reflecting the efficacy of medication, more appropriate choices with relatively short medication time can be made in a timely manner, shortening unnecessary medication courses, which is conducive to obtaining the best treatment effect within the shortest efficacy, and is also conducive to improving medication compliance, reducing adverse drug reactions caused by unnecessary medication, and reducing medication costs; when used for follow-up testing, it is conducive to monitoring drug efficacy, ensuring the effectiveness of medication, etc., which is of great significance.

[0037] The present invention uses molecular tags, reverse transcription primers, specific amplification primers and / or probes designed with MTB 16S rRNA as the target gene, and thereby establishes two targets, IS6100 and / or IC MMtmRNA, for simultaneous detection in conjunction with MTB 16S rRNA, thereby avoiding the interference of genomic DNA when detecting MTB 16S rRNA, and can also be used to distinguish active tuberculosis from inactive tuberculosis, thereby achieving quantitative detection of active Mycobacterium tuberculosis. Through the correlation between copy concentration (copies / μL) and bacterial cell concentration (CFU / ml), the CT value obtained through qPCR detection can quantitatively obtain the relative expression amount of active Mycobacterium tuberculosis. The quantitative detection method of the present invention is simple to operate, avoids false positives caused by gDNA, has good anti-interference, high sensitivity and specificity, and the detection sensitivity of active Mycobacterium tuberculosis can reach 10 1 copies / μL or 10 1CFU / ml; wherein the correlation standard curve between the copy concentration (copies / μL) and the bacterial concentration (CFU / ml) constructed by the present invention has R²>0.99 and good linearity; the present invention also establishes a standard substance based on the internal reference IC MM tmRNA, which is not only used to verify the feasibility of the present invention for quantitative detection of active Mycobacterium tuberculosis, but also can be used to reflect the real efficacy of Mycobacterium tuberculosis on the medication regimen, which is conducive to formulating a more appropriate medication regimen and shortening the effective medication course; the present invention is verified by follow-up detection of clinical samples, and the positive and negative consistency rate of the third-party nucleic acid detection reagent is greater than 90%, and the specific recognition efficiency of active Mycobacterium tuberculosis is higher than that of the commonly used nucleic acid detection reagents, which can be used as an auxiliary for the medication monitoring of tuberculosis and the infectiousness classification of tuberculosis, and plays an important role in public health prevention and control, scientific research and education, and escorts the promotion of the construction of a healthy China. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The linear regression line diagram of the OD value and bacterial concentration of marine mycobacterium after enrichment culture of the present invention is shown in FIG. 1 , wherein the X-axis represents the OD value of bacterial solution of different concentrations; the Y-axis represents the bacterial concentration of bacterial solution of different concentrations, indicating the activity of the bacterial solution, and the unit is 10 6 CFU / mL;

[0039] Figure 2 This is an amplification curve diagram of active Mycobacterium tuberculosis after qPCR quantitative detection of the present invention, wherein 1 is ICMM tmRNA, 2 is IS6100, and 3 is MTB 16S rRNA;

[0040] Figure 3 is an amplification curve diagram of qPCR reaction of bacterial solutions of marine mycobacteria of different concentrations of the present invention, wherein 1-5 represent that the concentrations of the bacterial solutions are diluted at a ratio of 1, 2, 4, 8, and 16 times, respectively;

[0041] Figure 4 The amplification curve and standard curve of the qPCR reaction of the plasmid standard of marine mycobacterium of different concentrations in the present invention are shown in Figure 1, wherein 1-6 represent the concentrations of the plasmid standard of marine mycobacterium, which are 3.77×10 6 Copies / μL to 3.77×10 1 copies / μL;

[0042] Figure 5 qPCR amplification curve of MTB16SrRNA standard for quantitative detection of active Mycobacterium tuberculosis of the present invention, wherein 1-8 represent different concentrations of the standard, 1 represents 10 8 copies / μL, 2 = 10 7copies / μL, 3 for 10 6 copies / μL, 4 = 10 5 copies / μL, 5 = 10 4 copies / μL, 6 for 10 3 copies / μL, 7 for 10 2 copies / μL, 8 to 10 1 copies / μL;

[0043] Figure 6 qPCR amplification curve of the internal reference standard (IC MM tmRNA) for quantitative detection of active Mycobacterium tuberculosis of the present invention, wherein 1-8 represent different concentrations of the standard, 1 represents 10 8 copies / μL, 2 = 10 7 copies / μL, 3 for 10 6 copies / μL, 4 = 10 5 copies / μL, 5 = 10 4 copies / μL, 6 for 10 3 copies / μL, 7 for 10 2 copies / μL, 8 to 10 1 copies / μL. DETAILED DESCRIPTION

[0044] Exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein the same or similar represents the same concept, such as Mycobacterium tuberculosis=MTB=TB, blank control=negative control, etc., genomic DNA=gDNA.

[0045] In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. The following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the present invention. Wherein, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources. The reverse transcription kit SuperScript III (Cat. No. 18080051) was purchased from Invitrogen-Thermo Fisher Scientific; 2× enzyme premix (2× Premix Ex Taq, Cat. No. RR390A and 2× Premix LA Taq, Cat. No. RR903A) was purchased from TaKaRa; pLB zero background rapid cloning kit (Cat. No. VT205) and plasmid mini-extraction kit (Cat. No. DP103) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. (TIANGEN); total RNA mini-extraction kit (RNasy Mini kit, Cat. No. 74104, including lysis buffer, centrifugal column, etc.) and gel recovery kit (QIAquick Gel Extraction Kit, Cat. No. 28704) were purchased from QIAGEN; competent cells E. coli DH5α were purchased from Shanghai Sangon Biotechnology Co., Ltd.; Mycobacterium Solid Culture Medium (Mycobacterium Solid Culture Medium, catalog number: GOMY0120) was purchased from Shanghai Jingnuo Biotechnology Co., Ltd.; marine mycobacterium (strain Mycobacterium marinum, referred to as M. marinum, catalog number: KL10611KRA) was purchased from Shanghai Kanglang Biotechnology Co., Ltd.; Mycobacterium tuberculosis H37Ra strain (strain M. tuberculosis H37Ra) is a sister strain of the global standard strain for Mycobacterium tuberculosis research (H37Rv strain, "v" means "toxic"), an attenuated strain of Mycobacterium tuberculosis, purchased from the ATCC Biological Standard Resource Center in the United States; CFX96 fluorescent quantitative PCR instrument was purchased from Bio-Rad, USA (model: 1855195-OG). However, it is obvious that one or more embodiments can also be implemented without these specific details. If the specific conditions are not indicated in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0046] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. Molecular biology experimental methods without special instructions are all carried out in accordance with the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd Edition) by J. Sambrook, or in accordance with the kit and product instructions. The detection object in the implementation of the present invention is a biological sample, not a living human or animal body, but an inanimate sample to be tested that has been separated from the human or animal body, such as sputum, blood, etc.; the direct purpose of the detection is to fully illustrate the construction process and beneficial effects of the quantitative detection method of active Mycobacterium tuberculosis of the present invention. The steps in the detection method of the present invention do not include the diagnosis results of the disease or the conclusion / step of the health status; the quantitative results of the detection can reflect the activity of active Mycobacterium tuberculosis in the sample to be tested to a certain extent, and assist doctors in combining clinical symptoms for the infectiousness classification of pulmonary tuberculosis. Therefore, the detection involved in the present invention does not belong to the diagnosis method of the disease, and meets the basic requirements of the Patent Law for the subject of patent protection.

[0047] The invention provides a method for detecting active mycobacterium tuberculosis and an application thereof. According to the invention, the amplification result of MTB 16S rRNA can truly reflect the relative expression amount of active mycobacterium tuberculosis by using a molecular tag, a reverse transcription primer, a specific amplification primer and / or a probe designed with MTB16S rRNA as a target gene; further, by using MTB 16S rRNA as a main target gene and cooperating with two target genes, IS6100 and / or IC MM tmRNA, which are detected simultaneously, not only the interference of genomic DNA during the detection of MTB 16S rRNA is avoided, but also active tuberculosis and inactive tuberculosis can be distinguished, thereby realizing the quantitative detection of active mycobacterium tuberculosis; wherein, IC MM tmRNA can amplify an internal reference gene, which is used to verify the credibility of the entire active mycobacterium tuberculosis detection process, further improving the feasibility and stability of the detection result, thereby truly realizing the purpose of nucleic acid detection of active mycobacterium tuberculosis. In addition, the amplification curve and CT value obtained by qPCR detection make the bacterial concentration (CFU / ml) and the copy concentration (copies / μL) of the relevant standard prepared by the corresponding recombinant plasmid correlated, and then the relative expression of active Mycobacterium tuberculosis can be quantitatively obtained. The quantitative detection method of active Mycobacterium tuberculosis of the present invention is simple to operate, avoids false positives caused by gDNA, has good anti-interference, high sensitivity and specificity, and the detection sensitivity of active Mycobacterium tuberculosis can reach 10 1 copies / μL or 10 1CFU / ml; wherein, the present invention also establishes a standard substance based on the internal reference IC MM tmRNA, which is used to verify the credibility of the quantitative detection of active Mycobacterium tuberculosis by the present invention, and is also used to verify the correlation between the copy concentration copies / μL and the bacterial concentration (CFU / ml) constructed by the present invention, and its standard curve R²>0.99 and good linearity. The present invention is verified by follow-up detection of clinical samples, and the positive and negative consistency rate with the third-party nucleic acid detection reagent is greater than 90%, and the specific recognition efficiency of active Mycobacterium tuberculosis is higher than that of commonly used nucleic acid detection reagents. The specific embodiments of the present invention are described below, and the specific contents are as follows:

[0048] Example 1. Design and screening of primers

[0049] The genome sequences of representative bacterial groups were analyzed by comparative genomics. The 16S rRNA gene sequences of representative species or strains in the Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) were analyzed in detail, and primers were designed based on the MTB 16S rRNA gene sequences. At the same time, according to the references, amplification primer pairs and probes targeting IC (M. marinum) tmRNA, and amplification primer pairs and probes targeting the insertion sequence IS6100 unique to the Mycobacterium tuberculosis complex DNA were selected and used at the same time; among them, the introduction of IC (marine Mycobacterium marinum M. marinum, abbreviated as MM) tmRNA can not only be used as a quality control internal reference to verify the feasibility of active Mycobacterium tuberculosis detection, but also can be used as a detection target gene to monitor the compliance of the entire detection process; the introduction of MTB DNA IS6100 as a target can specifically identify MTB DNA, which can not only simplify the operation steps, avoid the interference of genomic DNA (gDNA), and reduce the occurrence of false positive results, but also can indirectly examine the quantitative situation of inactive MTB in the sample to be tested to a certain extent.

[0050] Primers designed with MTB 16S rRNA gene as the target include label primer, reverse transcription primer (Gene Specific Primer of reverse transcription, referred to as GSP), amplification primer and probe. Among them, the reverse transcription primer is formed by combining the sequence of the label primer and the specific primer (Specific primer, referred to as SP), that is, in terms of sequence: reverse transcription primer = label primer + specific primer; amplification primers include upstream amplification primers and downstream amplification primers, wherein the upstream amplification primer is also called the upstream primer or forward primer, and the downstream amplification primer is also called the downstream primer or reverse primer. In short, the amplification primer is composed of a pair of upstream primers and downstream primers. The amplification primer uses the reverse transcription product cDNA with the sequence characteristics of the label primer as a template, and uses the paired upstream primer and downstream primer to locate the target fragment between the starting point and the end point of the amplification for amplification. The amplification product is a double-stranded DNA structure. It is particularly noted that the nucleotide sequences of the upstream primer and the tag primer are the same, that is, the upstream primer = the tag primer; the downstream primer and the specific primer are designed based on the sequence of the specific region of the 16S rRNA gene of the Mycobacterium tuberculosis complex, and the preferred sequence length for specific recognition and binding is controlled at 15 bp to 20 bp; therefore, the specific implementation method for primer design and screening of the MTB 16S rRNA gene sequence is as follows:

[0051] 1) Specific primers

[0052] According to the core objective of the present invention, the target fragment of specific reverse transcription is mainly located on the gene sequence of MTB 16S rRNA, and a specific primer (SP) for specific identification of MTB 16S rRNA sequence is designed after multiple sequence alignment analysis. Specifically, the candidate region of the specific target fragment is a regional sequence with 1-3 SNP sites on the 3'RACE end of the conservative region of the MTB 16S rRNA gene sequence. The design steps include: first using software such as MEGAX and Primer premier5 to design specific primers for reverse transcription of MTB 16S rRNA; then using software to screen and analyze the sequence characteristics of the preliminary designed specific primers, focusing on analyzing the specificity of the sequence and annealing temperature and other properties; after preliminary design and screening analysis, 6 specific primers that can be used for reverse transcription are obtained, which are paired and synthesized with the tag primers designed later for specific experimental verification.

[0053] 2) Index primer (=upstream primer)

[0054] According to the barcodes index technical principle and the above-screened specific primers, a molecular label sequence was designed at the 5' end of the specific primer sequence as a label primer (label primer, also called Barcode primer or Barcode primer). The length of the label primer was controlled at ±25bp. The sequence characteristics of the preliminarily designed label primers were screened and analyzed. The requirements were: the nucleic acid sequence of the label primer should take into account the principle of base balance to ensure that the proportion of the four bases A, T, C, and G is approximately 25%; the annealing temperature of the label primer should be much higher than that of the specific primer, and the annealing temperature is preferably 18°C ​​to 28°C higher than that of the specific primer. After the preliminary design, 6 label primers were screened.

[0055] 3) Reverse transcription primer

[0056] The complete sequence formed by pairing the above-mentioned label primer and specific primer is used as a reverse transcription primer (GSP). The preferred length of the reverse transcription primer is controlled at ±40bp. The sequence of the specific primer on the reverse transcription primer is used to specifically identify, bind and reverse transcribe the MTB 16S rRNA on the Mycobacterium tuberculosis complex. The obtained reverse transcription product is a cDNA with a label primer at 5', that is, the MTB 16S rRNA is identified from the sample and reversely transcribed into a reverse transcription product cDNA with a molecular label.

[0057] 4) Amplification primers and probes

[0058] Specific amplification primers were designed according to the target sequence of Mycobacterium tuberculosis 16S rRNA. One pair of amplification primers included an upstream primer and a downstream primer, wherein the upstream primer = tag primer, and the downstream primer adopted a design and screening method similar to that of specific primers. The specific design and preliminary screening steps of downstream primers and probes included: designing using software such as MEGAX and Primer premier5, preliminarily analyzing the specificity, annealing temperature, presence or absence of primer dimers and hairpin structures and other essential sequence characteristics of the designed primers and probes, and preliminarily screening out 4 downstream primers and 4 probes that can be paired with the above-mentioned tag primers.

[0059] The reverse transcription primers (tag primers + specific primers) and amplification primers (tag primers, downstream primers) preliminarily designed in the present invention all need to be verified by experiments to verify the usability of the primers. The contents of the experimental verification include the sequence fitness of the tag primer and the specific primer in the reverse transcription primer, the fitness of the reverse transcription primer and the amplification primer pair, the fitness of the upstream tag primer and the downstream primer in the amplification primer, the reaction conditions of reverse transcription and amplification (primer concentration gradient, template concentration, reverse transcription system, reverse transcription program, specific amplification system, amplification program, number of amplification cycles, temperature / time gradient in transcription / amplification), preparation of standard products and positive quality control products, and other factors affecting the reaction. factors, wherein the tag primer is not only used as a reverse transcription primer to introduce a molecular tag, but also needs to be an upstream primer of an amplification primer. The specific experimental verification requirements are: it is not easy to cause nonspecific transcription and amplification during the reverse transcription and amplification processes, it will not form primer dimers with paired primers, and the melting curve formed by amplification is not easy to have dimer peaks, etc. The present invention conducts verification experiments of multi-factor arrangement and combination and layer-by-layer optimization, and completes more than 200 experimental designs and experimental verifications. According to the results of the verification experiments, the influencing factors that lead to nonspecific amplification, no standard amplification curve, and double peaks (primer dimers) are eliminated step by step, and the preferred primer combination that can be finally used in the present invention is screened out. Finally, after specific experimental verification, one with relatively better specificity was screened out from the six specific primers, recorded as SP1, and its nucleotide sequence is shown in SEQ ID NO: 2; after specific experimental verification, one with relatively better amplification effect was screened out from the six label primers, recorded as Label 1, and its nucleotide sequence is shown in SEQ ID NO: 3; a relatively better reverse transcription primer was formed by combining the preferred label primer and the preferred specific primer, recorded as GSP1, and its nucleotide sequence is shown in SEQ ID NO: 1; after specific experimental verification, one with relatively better specificity was screened out from the four downstream primers and the four probes, respectively recorded as Mtb 16s Reverse and Mtb 16s probe, and their nucleotide sequences are shown in SEQ ID NO: 4-5; wherein, the 5' end of the probe Mtb 16s probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group, wherein the fluorescent group is preferably FAM, and the quenching group is preferably BHQ1.

[0060] 5) Quality control primers

[0061] The quality control primers include primer pairs and probes targeting MTB DNA IS6100 (IS6100 623F, IS6100673R, IS6100 656Probe); the quality control internal standard (IC) is a primer pair and probe targeting MM tmRNA (IC MMtmRNA F, IC MM tmRNA R, IC MM tmRNA probe), whose nucleotide sequences are shown in SEQ ID NO: 6-11, and their specific sequences are from the reference (Gillespie, H., Stephen, SW, Oravcova, K.Diagnostic Bacteriology Methods and Protocols[M]. New York, USA: HumanaPress,2017.). The 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group. The fluorescent group labeled with IS6100656Probe is preferably HEX, and the fluorescent group labeled with IC MM tmRNA probe is preferably ROX. The quenchers labeled with IS6100656Probe and IC MM tmRNA probe are preferably BHQ2.

[0062] The final preferred primer sequences of the present invention are shown in the following table:

[0063] Table 1: Primer sequence list

[0064]

[0065] Example 2: Quantitative detection of bacterial liquid activity concentration and nucleic acid extraction

[0066] 1) Quantitative detection of bacterial activity concentration

[0067] Mycobacterium tuberculosis H37Ra strain was used as the source for the preparation of the activity quantitative detection standard of Mycobacterium tuberculosis, and marine Mycobacterium marinum (M. marinum) was used as the quality control product to verify the compliance of the detection process. The preliminary preparation work included the enrichment culture of the original strain, the quantitative detection of the activity concentration of the bacterial solution and the extraction of nucleic acid. The quantitative method of the activity concentration adopted the bacterial concentration obtained by the plate colony counting method, and the unit was CFU / mL.

[0068] The enrichment culture includes the enrichment culture of marine mycobacterium (M. marinum). Take the preserved bacterial liquid of marine mycobacterium (M. marinum), use a small amount of physiological saline or non-selective broth medium to dissolve and suspend the strain to activate the strain; then inoculate the bacterial liquid onto the mycobacterium solid culture medium by streaking or coating, and carry out enrichment culture at 30°C for 4 to 5 days.

[0069] Dilution of bacterial solution concentration: scrape the strains (referred to as M. marinum) cultured on the mycobacterium solid culture medium, scrape 3 loops of each strain with an inoculation loop, transfer to a 15mL centrifuge tube containing 6mL sterile saline to prepare 2 kinds of original bacterial solutions, shake and mix the original bacterial solutions and let them stand for 30min, dilute the original bacterial solutions of the strains by 1, 2, 4, 8, and 16 times, respectively, and place them in the corresponding wells of the ELISA plate, set 3 detection replicate wells for each dilution, use an ultraviolet spectrophotometer to detect the absorbance (OD value) of the bacterial solutions of different dilution concentrations at λ=450nm, repeat the detection for each well twice, and calculate the average OD value of each dilution of the strain according to the detection results; in order to obtain plate colonies that are easy to count, each dilution after the above-mentioned multiple dilution is further diluted by 10 times, and a total of 5 concentration gradient dilutions are made for plate colony counting.

[0070] Plate colony count: Take 100 μL of the bacterial suspension corresponding to the selected secondary dilution concentration after 1, 2, 4, 8, and 16 times dilution, and spread them on the mycobacterium solid culture medium respectively. After the bacterial suspension corresponding to the M. marinum strain is placed at 37℃ and 30℃ for 3-4 weeks, the plate colony count is performed. According to the preliminary experimental results, the bacterial suspension diluted at 1, 2, and 4 times and diluted at 10 times, the final secondary dilution concentration selected for counting is 10 -4 The bacterial suspension was diluted 8 and 16 times, and then diluted twice at a ratio of 10. The final secondary dilution concentration selected for counting was 10 -3 .

[0071] Relationship between OD value and bacterial concentration: Based on the results of plate colony counting of the above bacterial suspension, the bacterial concentration (CFU / mL) corresponding to the bacterial suspension of different dilution concentrations was obtained. Combined with the OD values ​​obtained after the initial dilution of 1, 2, 4, 8, and 16 times, and the corresponding secondary dilution multiples, the OD value is related to the bacterial concentration, where the OD value is the independent variable and the bacterial concentration is the dependent variable. Using Graphpad Prism8 software, the linear regression analysis method was selected to draw a univariate linear regression line graph between the OD value of a certain concentration and the bacterial concentration corresponding to the concentration, and thus obtain the univariate linear regression equation of the OD value and the bacterial concentration. According to the test results, the OD value and bacterial concentration of marine Mycobacterium marinum (M. marinum) after bacterial enrichment culture are shown in Table 2, and the univariate linear regression line graph drawn based on this is shown in Figure 1 As shown, the linear regression equation is: Y=13.23X+0.04948, R 2 =0.997, which shows that there is a linear correlation between the absorbance (OD value) of bacterial solutions of different concentrations and the bacterial concentration (CFU / mL) indicating the activity of the bacterial solution, and the correlation is good.

[0072] Table 2 OD values ​​and bacterial concentrations of M. marinum

[0073]

[0074] Preparation for nucleic acid extraction: Before establishing a quantitative detection method for active Mycobacterium tuberculosis, it is necessary to prepare standards and process samples to be tested. These operations require nucleic acid extraction, that is, nucleic acid extraction of relevant bacterial liquid and samples to be tested. The main steps of nucleic acid extraction are carried out according to the product instructions of the relevant nucleic acid extraction kit. For example, the specific steps for extracting total RNA include: take a 1.5ml or 2ml nuclease-free EP tube, add 280μL bacterial liquid or sample to be tested, 625μL lysis buffer RLT, 700μL 70% ethanol, put the EP tube into a water bath sonicator, and perform ultrasound at 120W for 10 seconds and then 30 seconds, for a total of 3 cycles; oscillate the EP tube until the solution is fully mixed and then let it stand for 5 minutes; after the above-mentioned wall-breaking treatment, a nucleic acid mixture is obtained, which is added to the centrifuge column in 2-3 times, and centrifuged at 12000rpm for 15 seconds. The sample volume does not exceed 720μL each time, and the waste liquid is discarded and the centrifuge column is placed on a new collection tube. Take 700μL of buffer RW1 and add it to the centrifuge column and centrifuge at 12000rpm for 15 seconds, discard the waste liquid and place the centrifuge column on a new collection tube; When transferring the sample to the centrifuge column, be careful not to tilt it to avoid touching the waste liquid at the bottom. Take 500 μL to 700 μL of eluent and add it to the centrifuge column to clean the membrane of the centrifuge column. Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid and place the centrifuge column on a new collection tube. Take the eluent and repeat the above cleaning and centrifugation steps once, and keep the eluent in the collection tube for later use. Place the centrifuge column on a new collection tube again, centrifuge at 13000 rpm for 2 minutes, open the cover and air dry for 2 minutes, and place the centrifuge column on a new 1.5 ml nuclease-free EP tube. Slowly add 30 μL of Use nuclease-free water (or enzyme-free sterile water, or DEPC-treated ddH2O) to allow RNA to react in water for 1 minute to fully dissolve, then centrifuge at 13000rpm for 1 minute; take 30μL of the retained eluate and slowly add it above the adsorption membrane of the centrifugal column, then place it on a nuclease-free EP tube, let it stand for 1 minute, and then centrifuge at 12000rpm for 1 minute. The collected liquid is the total RNA extract, and then add 1μL of nuclease inhibitor. The final nucleic acid extract can be used directly, or the nucleic acid extract can be stored at -80℃ for later use.

[0075] Example 3: Establishment of a quantitative detection method for active Mycobacterium tuberculosis

[0076] Based on the above primer design and screening, the screening detection conditions were optimized layer by layer to verify whether it can be used for quantitative detection of active Mycobacterium tuberculosis, including reverse transcription, specific amplification (real-time fluorescence RT-PCR and qPCR), preparation of standards and analysis of test results. The specific experimental contents of establishing a quantitative detection method for active Mycobacterium tuberculosis are as follows:

[0077] 1) Reverse transcription:

[0078] Two different reverse transcription primers are used to perform reverse transcription on the samples to be tested, and the reverse transcription system and reverse transcription procedure are divided into step one and step two.

[0079] Step 1: The total volume of the reverse transcription system 1 is 10 μL, including: 1 μL 10mM dNTPs, 1 μL reverse transcription primer and 8 μL reverse transcription template; wherein, the reverse transcription primers are GSP1 and random primers (Random primer), respectively, and the concentration is 2pmol / μL~50pmol / μL, and the final concentration of the reverse transcription primers (GSP1 and random primers) configured in this way is 2pmol / μL~50pmol / μL; the reverse transcription template is the total RNA extracted from the sample to be tested by nucleic acid, and the sample to be tested can be the two strains of Mycobacterium tuberculosis H37Ra strain and marine Mycobacterium (MTB and M. marinum) cultured above; the reverse transcription program 1 is set to react at 50~65℃ for 5~10min and then cooled on ice for 1~5min. Based on the other reaction conditions in the fixed reverse transcription process, a series of experiments were set by changing a single condition to optimize and verify the reverse transcription system 1 and reverse transcription program 1.

[0080] Optimization of step one: After reverse transcription, the optimal conditions of reverse transcription system one and reverse transcription program one in step one were screened out by fixing the reaction conditions of step two and specific amplification. The reaction conditions and their combinations were screened by comprehensively considering the absence of non-specific amplification bands in the experimental results, the stability of repeated experimental results, and the principle of minimizing reaction time and cost. Finally, the optimal reaction conditions for step one were: the concentration of reverse transcription primer was 2 pmol / mL, the reaction was carried out at 50°C for 5 min, and then cooled on ice for 1 min.

[0081] Step 2: After the reverse transcription system 1 that has completed the reverse transcription procedure 1 in step 1 is briefly centrifuged, the reverse transcription system 2 is continued to be added to the reverse transcription system until the total volume is 20 μL, wherein the reverse transcription system 2 includes: 2 μL 10× reverse transcription buffer, 4 μL 25mM Mgcl2, 2 μL 0.1 M DTT, 1 μL recombinant ribonuclease inhibitor and 1 μL reverse transcriptase; after the reverse transcription system 2 is added, the reverse transcription procedure 2 is performed: after reacting at 45-55°C for 45-60 minutes, heating inactivation at 75-85°C for 5-10 minutes, and then cooling at low temperature to complete the reverse transcription process. Among them, since the reverse transcription primer GSP1 carries a "tag primer", the reverse transcription product MTB 16S rRNA cDNA obtained by GSP1 will also carry the sequence of the "tag primer", and the reverse transcription product obtained by reverse transcription using random primers and M. marinum as a template is the reverse transcription product M. marinum cDNA.

[0082] Similar to the optimization of step one, on the basis of fixing other reaction conditions, the optimal conditions of step two were screened and verified from the set series of experiments only by changing a single condition in step two. The final optimal reaction conditions of step two were: the recombinant ribonuclease inhibitor in reverse transcription system two was RNase OUT, the concentration was 40U / μL, and the concentration of reverse transcriptase was 200U / μL; reverse transcription procedure two was 50℃ reaction for 50min, followed by heating inactivation at 85℃ for 5min, low temperature cooling below 25℃ for 10min, and then cooling on ice for use or storing at -20℃ for later use.

[0083] 2) RT-PCR amplification:

[0084] RT-PCR: The two reverse transcription products (MTB 16S rRNA cDNA and M. marinum cDNA) are used as templates for specific amplification. Real-time fluorescence PCR (RT-PCR) is preferred for specific amplification. The RT-PCR amplification products using MTB 16S rRNA cDNA as template are used to prepare standard products, and the RT-PCR amplification products using M. marinum cDNA as template are used to prepare internal references for quality control products, which can also be called internal reference standards. Among them, the screening and verification of the RT-PCR reaction system and amplification procedure in specific amplification are as follows:

[0085] Reaction system: total volume 50 μL, including 25 μL of 2× long fragment Tag enzyme premix, upstream primer and downstream primer preferably used in an amount of 1 μL each, template is the above reverse transcription product, the amount of reverse transcription product is preferably 2 μL, and ddH2O is added to a total volume of 50 μL. Among them, 2× long fragment Tag enzyme premix is ​​LA Taq premix, a mixture of Taq high-fidelity enzyme suitable for long fragment amplification, including LA Taq, Buffer, dNTP Mixture, pigment Marker, etc. Among them, since the template and the specific amplification primer pair of RT-PCR correspond to the two strains, the reaction systems of RT-PCR are configured based on MTB 16S rRNA and M. marinum respectively: when the template is the reaction system configuration containing MTB 16S rRNA cDNA, the upstream primer and the downstream primer are added with primers Label 1 and MTB 16s Reverse, respectively, and when the template is the reaction system configuration containing M. marinum random cDNA, the upstream primer and the downstream primer are added with primers IC MM tmRNA F and IC MM tmRNA R. That is, primers Label 1, MTB 16s Reverse, template MTB 16S rRNA cDNA and their corresponding RT-PCR reaction systems are configured separately, and primers IC MM tmRNA F, IC MM tmRNA R, template M. marinum cDNA and their corresponding RT-PCR reaction systems are configured separately.

[0086] The two sets of RT-PCR reaction systems were configured with the same RT-PCR amplification program. After the preliminary experiment, the screening and verification conditions for the RT-PCR amplification program were as follows: the first step, 94~96℃ pre-denaturation for 3min~5min; the second step, 94~96℃ denaturation for 10sec~20sec, 55~60℃ annealing for 30sec~60sec, 68℃~72℃ extension for 30sec~2min, and so on for 37~45 cycles; the third step, 68℃~72℃ final extension for 5min~10min. A similar method was used to verify the optimization of the reaction conditions in the reverse transcription process mentioned above, that is, only a series of experiments with a single condition change in the amplification program were used to screen and verify the optimal parameters, and other reverse transcription and specific amplification conditions were fixedly selected. Finally, the RT-PCR amplification program was uniformly optimized as follows: 94℃ pre-denaturation for 3min; 94℃10sec, 59℃30sec, 72℃1min, and so on for 30 cycles; 72℃ final extension for 10min.

[0087] 3) Preparation of standard products

[0088] Molecular cloning of the target fragment of MTB 16S rRNA. Gel recovery: Based on the primer combination designed with MTB 16S rRNA as the target, the PCR product (106bp) that meets the size of the target fragment after reverse transcription and RT-PCR amplification is cut into the gel, and the gel recovery operation is performed according to the instructions of the gel recovery kit. The specific steps are as follows: cut the target band; weigh (weight after cutting the gel into the tube - weight of the EP tube before cutting the gel = weight of the recovered gel block containing the target fragment, add an appropriate amount of buffer QG to the recovery EP tube according to the ratio of 0.1g gel plus 300μL buffer QG, gel weight> 2g plus 6 times the volume of buffer QG, incubate at 50℃ every 2-3min, check every 2-3min, until the gel is completely dissolved (about 10min); check the color of the dissolved mixture. If the color is orange or purple, add 10μL sodium acetate to adjust the pH to 5.0 to turn the mixture to yellow; add isopropanol according to the ratio of glue: isopropanol = 1:1 (0.1g glue plus 100μL isopropanol), mix well and load onto the adsorption column, centrifuge at 8000rpm for 1min; add 500μL buffer QG to the adsorption column and centrifuge at 8000rpm for 1min; add an appropriate volume of anhydrous ethanol to the adsorption column and then add 750μL buffer PE, let it stand for 2-5min and then centrifuge at 8000rpm for 1min; centrifuge at 13000rpm for 2min; place the adsorption column in a new centrifuge tube and add 50μL After incubation in Buffer EB or ddH2O at room temperature for 5 minutes, centrifuge at 13000rpm for 1 minute. The recovered DNA is directly used for ligation reaction or stored at -20℃ for later use. Ligation: The DNA recovered from the gel is prepared according to the pLB vector instructions for the ligation system. The Taq enzyme amplification product needs to be blunt-ended before ligation. The blunt-end reaction system includes: 5μL reaction buffer (2×Reaction Solution), 0.5μL blunt-end enzyme (Blunting Enzyme), and DNA is added to 8μL; flick the centrifuge tube to mix the blunt-end reaction solution, centrifuge, place it at 20℃ for 2min, 70℃ for 5min, and cool; after the blunt-end is completed, add 1μL pLB vector (35ng / μL) and 1μL T4 DNA ligase (3U / μL) to form a mixed reaction solution (a total of 10μL), place it at room temperature (or 22℃) for 30min (or place it overnight), and the recovered MTB 16S rRNA target DNA can be inserted into the pLB vector.Transformation: Take 10 μL of the ligation product (after the 10 μL mixed reaction solution above completes the ligation reaction) and add 50~100 μL of competent cells Ecoli DH5α and then gently mix, ice bath for 30 minutes; quickly cool in ice bath for 2-3 minutes after 60-90 seconds in 42℃ water bath; add 700μL of sterile LB liquid culture medium without antibiotics, culture at 37℃ shaker (150rpm) for 45-60 minutes to revive the bacteria; centrifuge at 5000rpm for 3 minutes and discard the supernatant, keep 200μL to mix the precipitate by blowing, draw 60μL / 80μL and spread on LB agar plate (containing 1:1000 ampicillin) preheated at 37℃ for 20 minutes, and place incubator for culture at 37℃ for 12h-18h after the plate is completely dry. The positive colony is initially assumed to be the strain of bacteria that the ligation product has been successfully transformed into the host cell. Pick a single colony and place it in 5ml LB liquid culture medium containing ampicillin (1:1000) and culture it at 37℃ shaker (150rpm) for 12h-16h, then take the amplified bacterial solution for PCR verification. Plasmid extraction: transfer the PCR-positive bacterial solution to a 15ml centrifuge tube, centrifuge at 3500rpm for 5min, and remove the supernatant as much as possible; add 250μL solution P1 (containing RNase A) to the centrifuge tube with bacterial precipitate, mix by blowing or vortexing until the bacterial precipitate is completely suspended (if the bacterial block is not thoroughly mixed, it will affect the lysis, resulting in low extraction volume and purity); add 250μL solution P2, gently mix upside down 6-8 times until the bacterial solution becomes clear and viscous (avoid violent shaking, because it will interrupt the genomic DNA, causing the extracted plasmid to be mixed with genomic DNA fragments; if the liquid does not become clear, it may be that there are too many bacteria and the lysis is not complete), so that the bacteria are fully lysed, and the time should not exceed 5min; add 350μL solution P3, immediately mix gently upside down 6-8 times until a white flocculent precipitate appears (if there is a tiny white precipitate remaining in the supernatant, centrifuge again and take the supernatant), so that the bacterial solution is fully lysed. After mixing, centrifuge at 12000rpm for 10min; equilibrate the adsorption column CP3 in advance for use: add 500μL equilibration solution BL to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid, transfer the supernatant to the equilibrated adsorption column, try not to precipitate the supernatant after transfer, centrifuge at 12000rpm for 30~60sec, and discard the waste liquid; add 600μL rinsing solution PW (containing anhydrous ethanol) to the adsorption column, centrifuge at 12000rpm for 30~60sec, discard the waste liquid, repeat the rinsing step once; centrifuge at 12000rpm for 2min, open the lid and dry at 37℃ for about 5min; add 40μL EB or ddH2O to the center of the membrane of the adsorption column, let it stand at room temperature for 5min, centrifuge at 12000rpm for 2min, and you can collect about 40μL of recombinant plasmid in the centrifuge tube.Concentration of recombinant plasmid: The measured mass concentration (ng / μL) was converted into copy concentration (copies / μL) according to Avogadro's constant. The conversion formula is: (6.02×10. 23 )×(plasmid concentration ng / μL×10 -9 ) / (DNA length×660), and the plasmid with mass concentration determined by concentration detection or gradient dilution was used as the standard.

[0089] Selection of standard concentration: Based on the calculated recombinant plasmid copy concentration, dilute the recombinant plasmid with nuclease-free water to 8 concentration gradients as standard. The copy concentrations of the standard are: 0.5×10 8 copies / μL, 0.5×10 7 copies / μL, 0.5×10 6 copies / μL, 0.5×10 5 copies / μL, 0.5×10 4 copies / μL, 0.5×10 3 copies / μL, 0.5×10 2 copies / μL, 0.5×10 1 copies / μL (which can be abbreviated as 8, 7, 6, 5, 4, 3, 2, 1 respectively). After each copy concentration standard is packaged, it can be stored at -20℃ for later use. According to the above reverse transcription and RT-qPCR detection, the CT values ​​corresponding to the standards of different mass concentrations are obtained, and they are plotted into a standard curve. The corresponding copy concentration (copies / μL) can also be calculated according to the mass concentration (ng / μL).

[0090] 4) qPCR

[0091] The detection object is a sample to be tested, and the sample to be tested includes a standard. After reverse transcription is performed separately according to the preferred reverse transcription conditions above, two reverse transcription products of the same sample to be tested (MTB 16S rRNA cDNA and cDNA reverse transcribed by random primers) are obtained. The cDNA of the reverse transcription product is used as a template for specific amplification. The specific amplification method is preferably real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qPCR). Different templates are composed of different primer and probe combinations. The qPCR reaction system: when the template is MTB 16S rRNA cDNA, the upstream / downstream primers in the corresponding qPCR reaction system are Label1 and MTB 16s Reverse, and the probe is MTB 16s probe; when the template is cDNA reverse transcribed by random primers, the upstream / downstream primers in the corresponding qPCR reaction system are primers IC MM tmRNA F, IC MM tmRNA R, and probe IC MMtmRNAProbe respectively; it also includes directly using nucleic acid DNA extracted from the sample to be tested as a template, and the upstream / downstream primers in the corresponding qPCR reaction system are primers IS6100 and IS6100. 623F, IS6100 623R, probe IS6100 656Probe; firstly, the reaction system was configured with different target genes to optimize the reaction conditions. The amplification procedures of different target genes were unified during the optimization process. Finally, the feasibility of qPCR detection in the same tube of the same sample was further verified. The screening and verification of the qPCR reaction system and amplification procedure are as follows:

[0092] Prepare the qPCR reaction system. The total volume is 50μL, including 25μL of 2× Enhanced Tag enzyme premix, 1μL of upstream primer and downstream primer (5~20pmol / μL), 2μL of probe (5~20pmol / μL), 2μL of template or standard (reverse transcription product) (add 2μL of standard, each system has exactly 1× copy concentration, reducing the error of sample addition), and add ddH2O to 50μL; among them, 2× Enhanced Tag enzyme premix is ​​a premixed fluorescent dye Tag enzyme mixture, which is suitable for high-efficiency amplification of taqman probe-based qPCR. The reaction procedure to be optimized and verified is as follows: the first step, pre-denaturation at 94°C~96°C for 30sec~60sec; the second step, denaturation at 94~96°C for 4sec~10sec, annealing and extension at 55°C~61°C for 30sec~60sec, and this for 37~45 cycles; the third step, denaturation at 94~96°C for 5~10sec, annealing and extension at 60~65°C for 5~10sec, and denaturation at 94~96°C for 5~10sec.

[0093] On the basis of fixed reverse transcription and other amplification program conditions, a series of specific experiments were carried out by changing a single condition to optimize and verify the qPCR reaction system and reaction conditions. The results of the preferred qPCR reaction system and amplification program are as follows. The preferred qPCR reaction system is that the preferred concentrations of the upstream primer, downstream primer and probe are all 10pmol / μL, corresponding to the preferred final concentrations of 0.2μM and 0.4μM in the qPCR reaction system, respectively; the preferred qPCR amplification program is: the first step, 95℃ pre-denaturation for 30sec; the second step, 95℃ denaturation for 5sec, 59℃ annealing and extension for 30sec, 40 cycles (collecting fluorescence at 59℃); the third step, sequentially denaturation at 95℃ for 10sec, 65℃ annealing and extension for 5sec, and 95℃ denaturation for 5sec. After the preferred qPCR amplification, the amplification curve of the sample to be tested and the cycle threshold value (CT value) of the positive amplification result can be obtained, among which the bacterial solution concentration is related to the CT value obtained by qPCR detection, which can be used as the basis for drawing the MTB activity loading standard curve.

[0094] The above-mentioned specific amplification includes RT-PCR and qPCR. The optimization and verification process of the reaction conditions are based on fixing other reaction conditions, and only optimization and verification are performed by changing a single condition. The optimization screening also includes the selection of internal reference genes, as well as the optimized pairing of primers and probe compositions, primer concentrations, reaction systems, reaction procedures, etc.

[0095] According to the reaction conditions optimized by qPCR, the corresponding quantitative amplification curves of the three target genes, MTB 16S rRNA, IC MM tmRNA and IS6100, were obtained. The amplification curves of different concentrations of Mycobacterium tuberculosis H37Ra strain were as follows: Figure 2 As shown, it can be seen that using the same optimized reaction conditions (for example, the final concentrations of primers and probes corresponding to the three target genes in the reaction system are unified as: 0.2μM primers, 0.4μM probes) to detect the three target genes, standard "S-shaped" amplification curves can be obtained, and there is no non-specific amplification. The CT values ​​of the three groups of amplification curves in the figure are 24.18, 21.54, and 24.22, respectively.

[0096] 5) Quantitative analysis

[0097] The above has verified that there is a linear correlation between the absorbance (OD value) of different concentrations of Mycobacterium tuberculosis bacterial solution and the bacterial concentration (CFU / mL) indicating the activity of the bacterial solution. After extracting nucleic acid from the bacterial solution of marine Mycobacterium marinum diluted 1, 2, 4, 8, and 16 times after the above enrichment culture, the RNA of the bacterial solution with different dilutions was reverse transcribed and qPCR reacted using the optimized reaction conditions and reaction system. The amplification curve is shown in the figure. Figure 3As shown, it can be seen that the qPCR reaction amplification curve of the original bacterial solution of marine mycobacterium after 1, 2, 4, 8, and 16 times dilution as the sample to be tested is a standard "S-type". In the preferred 50μL reaction system, the CT values ​​corresponding to the bacterial solutions of different concentrations after dilution are 21.5, 22.33, 23.68, 24.52, and 25.84, respectively. There is an inverse relationship between the CT value and the starting concentration, that is, the higher the starting concentration, the smaller the CT value; the lower the starting concentration, the larger the CT value. Moreover, the bacterial concentration and copy concentration of the sample to be tested can also be converted according to the CT value, and the results are shown in Table 3 below.

[0098] Table 3 Bacterial concentration and copy concentration

[0099]

[0100] It can be seen that the corresponding copy concentrations of bacterial solutions of different concentrations were calculated according to the standard curve obtained by qPCR amplification, and then converted into the active concentration of the colony (bacterial concentration). There is a corresponding relationship between the copy concentration and the bacterial concentration, and the average value of this corresponding relationship is 1×10 6 The copy concentration corresponding to the CFU / mL bacterial concentration is 3.77×10 6 Copies / μL. Based on this, the amplification curve and standard curve with bacterial concentration as the unit can be established. Further, the nucleic acid was extracted from the bacterial solution of marine mycobacterium after bacterial enrichment culture as described above and prepared into 3.77×10 6 copies / μL to 3.77×10 1 After qPCR amplification in a 50 μL reaction system, the amplification curves of the six copy concentration standards all showed a standard "S-shaped" ( Figure 4 ), when the number of amplification cycles was within 40, the fluorescence amplification signal could be detected, and the minimum detection limit was 3.77×10 1 copies / μL, and the sensitivity of the corresponding activity detection can reach 10 1 CFU / mL, CT value and the logarithm of the starting copy concentration (Log Starting Quantitiy) showed a good linear correlation, and the standard curve equation was Y=-3.036X+39.914, R 2 The amplification efficiency was 113.5%.

[0101] Therefore, based on the correlation between the recombinant plasmid prepared by PLB vector of marine mycobacterium (M. marinum) and the bacterial concentration for quantitative detection of bacterial activity concentration, the two detection targets of MTB 16S rRNA and IS6100, the standard curves amplified by MTB and M. marinum 2, 4, 8, and 16-fold diluted bacterial RNA and plasmid standards, and the CT values ​​of three different concentrations of standards, the consistency and correlation between the bacterial concentration CFU / ml at different OD values ​​and the recombinant plasmid copies / μL calculated at different OD values ​​were analyzed. The CT value of MTB 16S rRNA can be detected by qPCR, and the positive internal reference (preferably with a bacterial concentration of 10 4 CFU / mL of marine mycobacterium) and the CT values ​​corresponding to them are mutually verified, and finally the quantitative detection results of active mycobacterium tuberculosis (or the load of active mycobacterium tuberculosis) are obtained. Among them, the internal reference gene IC MM tmRNA is used as the detection target, and the efficiency and performance of each link of the experiment can also be monitored. Among them, for the qualitative detection results of the core target genes, the interpretation of the final quantitative detection results of active mycobacterium tuberculosis is shown in Table 4.

[0102] Table 4 Interpretation of the results of quantitative detection of active Mycobacterium tuberculosis

[0103] ;

[0104] Note: "+" means positive; "-" means negative; in the interpretation results: "+a" means the qPCR test result is positive, but the result is unreliable; "+b" means the qPCR test result is reliable, but the result is inactive Mycobacterium tuberculosis; "Invalid" means the qPCR test result is invalid.

[0105] The judgment standard for the quantitative detection of active Mycobacterium tuberculosis by the detection results obtained by qPCR is mainly based on the quantitative detection result of MTB 16SrRNA, and the positive and negative results of the qPCR amplification of MM tmRNA and IS6100 are combined to determine whether the quantitative detection result of MTB16S rRNA is valid, and only when the qPCR amplification results of IC MM tmRNA are all positive, the quantitative detection of active Mycobacterium tuberculosis by the present invention is effective, and the judgment standard of effectiveness and positive and negative is: if the qPCR amplification results of MTB 16SrRNA, IC MM tmRNA and IS6100 are all positive, the quantitative detection result of MTB 16S rRNA is valid and positive; if the qPCR amplification results of IC MM tmRNA and IS6100 are positive and only the qPCR amplification result of MTB 16S rRNA is negative, it can be determined that the present invention is effective for the quantitative detection of active Mycobacterium tuberculosis, but the Mycobacterium tuberculosis present in the sample to be tested is inactive Mycobacterium tuberculosis. If the qPCR amplification results of IC MM tmRNA are all negative, the quantitative detection results of active Mycobacterium tuberculosis of the present invention are invalid or unreliable. The specific situations include two kinds: one is that at least one of the qPCR amplification results of MTB 16S rRNA and / or IS6100 is positive, then the positive results of the quantitative detection of MTB 16S rRNA are determined to be unreliable, and re-testing or re-testing by other methods are required; the other is that the qPCR amplification results of MTB 16SrRNA and / or IS6100 are all negative, then the quantitative detection results of active Mycobacterium tuberculosis of the present invention are invalid, and there may be errors in the detection process.

[0106] Example 4. Quantitative detection kit for active Mycobacterium tuberculosis

[0107] Referring to Example 3, the preferred reaction conditions for reverse transcription and specific amplification and the prepared standard products, the main components of the kit include but are not limited to:

[0108] 1) The preferred primer combination or the combination of primers and / or probes required for amplifying three target genes (MTB 16S rRNA, IC MM tmRNA and IS6100) in Example 1;

[0109] 2) Reverse transcriptase and reverse transcription reagents;

[0110] 3) DNA polymerase and specific amplification reagents;

[0111] 4) Standard products;

[0112] 5) Quality control products: positive control, negative control;

[0113] The preferred concentration of the reverse transcription primer is 2pmol / μL, and the preferred loading amount of the upstream primer and downstream primer for specific amplification is 1μL. The final concentration of the primer in the reaction system is adjusted by adjusting the concentration of the primer; in the qPCR reaction system, the preferred final concentration of the primer is 0.2μM, and the preferred final concentration of the probe is 0.4μM. The standard products are mainly MTB16SrRNA series standards (10 1 ~10 8 copies / μL) and the qPCR amplification curves of this series of standards are shown in Figure 5 As shown in the figure, the amplification curve of MTB16SrRNA showed a standard "S-shaped" pattern. In the preferred 50 μL reaction system, when the number of amplification cycles was within 40, the fluorescence amplification signals of the eight concentrations of the standard samples could be detected, that is, the detection sensitivity could reach 10 1 copies / μL, the CT value and the logarithm of the starting copy concentration showed a good linear correlation, and the standard curve equation was Y=-3.035X+41.099, R 2 =0.995, the amplification efficiency is 113.5%; the standard can also include an internal reference standard, that is, a series of concentrations of IC MM tmRNA fragment clone plasmids (10 1 ~10 8 copies / μL) and the qPCR amplification curves of this series of standards are shown in Figure 6 As shown in the figure, the amplification curve of IC MM tmRNA presents a standard "S-shaped" curve. In the preferred 50 μL reaction system, when the number of amplification cycles is within 40, the fluorescence amplification signals of the internal reference standards at 8 concentrations can all be detected, that is, the detection sensitivity can reach 10 1 copies / μL, the CT value and the logarithm of the starting copy concentration showed a good linear correlation, and the standard curve equation was Y=-2.927X+40.005, R 2 =0.998, the amplification efficiency is 119.6%. The positive control is a synthetic plasmid containing the target fragment (MTB 16S rRNA), the concentration is preferably 10 3 ~10 5 Copies / μL; the positive control also includes a positive internal reference, specifically a synthetic plasmid containing IC MM tmRNA, with a preferred concentration of 10 3 ~10 5 Copies / mL; negative control was diethylpyrocarbonate (DEPC)-treated H2O (ddH2O).

[0114] Example 5: Detection of clinical samples

[0115] The quantitative detection kit for active Mycobacterium tuberculosis and the preferred quantitative detection method for active Mycobacterium tuberculosis were used to follow up 10 clinically confirmed patients for more than half a year. The results showed that compared with the conventional detection method and the third-party detection reagents, the conventional detection method included acid-fast staining (referred to as sputum smear) and Mycobacterium tuberculosis culture method (referred to as sputum culture), and the third-party reagents included the Mycobacterium tuberculosis nucleic acid detection kit (RNA constant temperature amplification) (referred to as TB-SAT) purchased from Shanghai Rendu Biotechnology Co., Ltd. and the Mycobacterium tuberculosis and rifampicin resistance gene detection kit (referred to as X-pert) purchased from Cepei, the quantitative detection of active Mycobacterium tuberculosis by the present invention can better reflect the changes in active Mycobacterium tuberculosis in clinically confirmed patients from the same sputum sample source. The following Tables 5-6 exemplify the follow-up detection results of 2 patients, and Table 7 shows the quantitative values ​​of the follow-up detection by the present invention, wherein the detection results of IC MM tmRNA in the quantitative detection results of active Mycobacterium tuberculosis by the present invention are all positive and effective.

[0116] Table 5 Follow-up test results of two samples from the same source using different test methods - Follow-up 1

[0117] ;

[0118] Note: “+” indicates a positive test result; “-” indicates a negative test result; “*” indicates that no test was performed due to lack of samples.

[0119] Table 6 Follow-up test results of 2 samples from the same source using different test methods - Follow-up 2

[0120] ;

[0121] Note: “+” indicates a positive test result; “-” indicates a negative test result; “*” indicates that no test was performed due to lack of samples.

[0122] Table 7 Quantitative detection values ​​of active Mycobacterium tuberculosis

[0123] ;

[0124] Note: “-” indicates a negative result; “*” indicates that the test was not performed due to lack of samples. Copy concentration (copies / μL) / 3.77≈bacterial concentration (CFU / mL).

[0125] By comparing the quantitative detection results of active Mycobacterium tuberculosis of the present invention with at least 3 parallel detection results, firstly, compared with traditional sputum smear and sputum culture, the nucleic acid detection methods including the present invention have higher detection sensitivity; secondly, compared with the currently commonly used nucleic acid detection reagents, the detection results of the present invention have a positive and negative consistency rate with the third-party reagents greater than 90%. At the same time, the detection results of the present invention can show that with the extension of treatment time, active Mycobacterium tuberculosis in the body can no longer be detected. After the effective medication course is completed, Mycobacterium tuberculosis DNA fragments (or inactive Mycobacterium tuberculosis) may still exist in the patient's body. Therefore, the detection result of the third-party detection reagent (such as the X-pert test performed by follow-up 2 in May) is still positive. The present invention uses MTB 16S rRNA as the detection target, which belongs to the quantitative detection of active Mycobacterium tuberculosis. According to the judgment standard of the present invention, the qPCR detection result of MTB 16S rRNA of follow-up 2 in May is: the result is credible and negative, and the detection result can reflect that the patient's medication is effective. It can be seen that compared with the current detection methods, the present invention not only has high detection sensitivity and can more truly reflect the activity level of Mycobacterium tuberculosis in the sample to be tested (related to infectivity), but also can calculate the relative expression level of active Mycobacterium tuberculosis, which is conducive to more truly reflecting the effect of medication (especially suitable for follow-up detection during the medication process), judging the rationality of the drug treatment plan, and is also conducive to grading the infectiousness of pulmonary tuberculosis in patients, avoiding the inevitable false positive results caused by DNA in the body.

[0126] The present invention provides a quantitative detection method for active Mycobacterium tuberculosis and its application, wherein MTB 16SrRNA is used as the main target gene, and the reaction conditions of specific amplification are unified, combined with auxiliary detection of two target genes, IC MMtmRNA and / or IS6100, under the same conditions, to simplify the operation, avoid the interference of genomic DNA when detecting MTB 16S rRNA, and can also be used to distinguish active tuberculosis from inactive tuberculosis, truly realizing the quantitative detection of active Mycobacterium tuberculosis. If the relevant products of the present invention can be used for drug monitoring of tuberculosis and infectious grading of tuberculosis after landing, they can also be used to reflect the more real efficacy of Mycobacterium tuberculosis on the medication regimen, which is conducive to formulating a more suitable medication regimen and shortening the effective medication course.

[0127] The specific embodiments of the present invention have been described in detail so that those skilled in the art can easily understand them. However, according to all the descriptions disclosed, some details therein may be modified or replaced in different ways, and these changes are within the scope of protection of the present invention, which is given by the attached claims and any equivalents thereof.

Claims

1. A primer composition, characterized in that: The primer composition for nucleic acid detection of active Mycobacterium tuberculosis with MTB 16SrRNA as the target gene comprises a reverse transcription primer and a specific amplification primer, wherein the reverse transcription primer comprises a nucleotide sequence of a tag primer and a nucleotide sequence for specific reverse transcription amplification of MTB 16S rRNA, and the specific amplification primer comprises an upstream primer and a downstream primer; wherein, The nucleotide sequences of the tag primer and the upstream primer are the same, which is shown in SEQ ID NO: 3; the nucleotide sequence for specific reverse transcription amplification of MTB 16S rRNA is shown in SEQ ID NO: 2; the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4; It also includes a primer pair and a probe with MTB DNA IS6100 as a target, which are quality control primers, wherein the IS6100 is used as a specific target for detecting Mycobacterium tuberculosis DNA; and / or a primer pair and a probe with MM tmRNA as a target, which are quality control internal standards. When the amplification result of the quality control internal standard is positive, it is used to determine that the nucleic acid detection result of active Mycobacterium tuberculosis is valid.

2. The primer composition according to claim 1, characterized in that: The nucleotide sequence of the reverse transcription primer is shown in SEQ ID NO:

1.

3. The primer composition according to claim 1, characterized in that: When qPCR amplification is performed based on specific amplification primers, the primer composition also includes corresponding probes with fluorescent groups labeled at the 5' end and quenching groups labeled at the 3' end.

4. A method for detecting active Mycobacterium tuberculosis, characterized in that: Using the primer combination described in any one of claims 1 to 3 to detect nucleic acid of active Mycobacterium tuberculosis, using MTB 16S rRNA as the target gene for reverse transcription amplification and specific amplification, the reverse transcription amplification includes step 1 and step 2, and the specific amplification is qPCR amplification; wherein, The step 1 includes adding reverse transcription system 1 to perform reverse transcription procedure 1, the step 2 includes adding reverse transcription system 2 after short centrifugation of reverse transcription system 1 after step 1, and then performing reverse transcription procedure 2, and the product after step 2 is the reverse transcription product; The template for qPCR amplification is the reverse transcription product, and the corresponding total volume of the qPCR reaction system is 50 μL; after executing the qPCR amplification reaction program, the amplification curve of the sample to be tested and the CT value of the positive amplification result can be obtained; the detection method does not include the diagnosis and treatment of the disease.

5. The method for detecting active Mycobacterium tuberculosis according to claim 4, characterized in that: The preferred reaction conditions for the reverse transcription amplification and the specific amplification include: In the reverse transcription amplification, the total volume of the reverse transcription system 1 is 10 μL, including: 1 μL 10mM dNTPs, 1 μL reverse transcription primer and 8 μL reverse transcription template; the reverse transcription procedure 1 is: react at 50-65°C for 5-10min and then cool on ice for 1-5min; the total volume of the reverse transcription system 2 is 10 μL, including: 2 μL 10× reverse transcription buffer, 4 μL 25mM MgCl2, 2 μL 0.1 MDTT, 1 μL recombinant ribonuclease inhibitor and 1 μL reverse transcriptase; the reverse transcription procedure 2 is: react at 45-55°C for 45-60min, heat inactivate at 75-85°C for 5-10min, and then cool at low temperature; The qPCR reaction system includes: 25 μL of 2× enhanced Tag enzyme premix, 1 μL of upstream primer and downstream primer with a concentration of 5 to 20 pmol / μL, 2 μL of probe with a concentration of 5 to 20 pmol / μL, 2 μL of reverse transcription product, and ddH2O supplemented to 50 μL; the reaction procedure of the qPCR amplification is: the first step, pre-denaturation at 94°C to 96°C for 30 seconds to 60 seconds; the second step, denaturation at 94°C to 96°C for 4 seconds to 10 seconds, annealing and extension at 55°C to 61°C for 30 seconds to 60 seconds, and 37 to 45 cycles; the third step, denaturation at 94°C to 96°C for 5 to 10 seconds, annealing and extension at 60°C to 65°C for 5 to 10 seconds, and denaturation at 94°C to 96°C for 5 to 10 seconds.

6. The method for detecting active Mycobacterium tuberculosis according to claim 4, characterized in that: The target genes for qPCR amplification of the same sample to be tested also include IS6100 of Mycobacterium tuberculosis and / or MMtmRNA of Mycobacterium marinum. The qPCR amplification reaction system and reaction procedure are the same, and the amplification curves and amplification CT values ​​corresponding to different target genes can be obtained.

7. A standard for quantitative detection of active Mycobacterium tuberculosis, characterized in that: The preparation of standard products mainly includes the preparation of active bacterial solution and the preparation of standard products; Preparation of active bacterial solution: The original strain of Mycobacterium tuberculosis H37Ra strain was cultured, and the cultured bacterial solution was diluted to different degrees to obtain bacterial solutions of different concentrations, and OD value detection, plate culture and colony counting were performed to obtain OD value and corresponding bacterial concentration; The preparation of the standard comprises: extracting total RNA from bacterial liquid, using the primer composition described in claim 2 and the extracted total RNA as a template to sequentially perform reverse transcription and RT-PCR amplification; wherein the RT-PCR amplification uses the reverse transcription product obtained by reverse transcription as a template and MTB 16S rRNA as a target gene; constructing the RT-PCR amplification product into a gene cloning vector to form a recombinant plasmid containing the target gene, and quantifying the recombinant plasmid after diluting it to different concentrations, thereby obtaining a standard of corresponding concentration.

8. A method for quantitative detection of active Mycobacterium tuberculosis, characterized in that: The detection steps include: determining the quantitative relationship between bacterial concentration and copy concentration, performing nucleic acid detection of active Mycobacterium tuberculosis and quantitative analysis of the detection results; wherein, The method for determining the quantitative relationship between bacterial concentration and copy concentration is as follows: the standard for quantitative detection of active Mycobacterium tuberculosis according to claim 7 and bacterial solutions of different concentrations obtained in the preparation process of the standard are respectively used as detection objects, and detection is performed according to the method for detecting active Mycobacterium tuberculosis according to claim 6, and the quantitative relationship between bacterial concentration and copy concentration is established through the standard curve corresponding to the standard and the CT values ​​corresponding to bacterial solutions of different concentrations; The method for performing nucleic acid detection of active Mycobacterium tuberculosis is as follows: using a sample to be tested as a detection object, performing detection according to the method for detecting active Mycobacterium tuberculosis according to claim 6, and obtaining amplification curves and amplification CT values ​​of MTB 16S rRNA, IS6100 of Mycobacterium tuberculosis and / or MM tmRNA of Mycobacterium marineus as target genes, respectively; The quantitative analysis method of the detection results is as follows: the amplification results corresponding to the MM tmRNA of marine mycobacterium as the target gene are used to verify the credibility; the amplification results corresponding to the IS6100 of Mycobacterium tuberculosis as the target gene are used to compare and distinguish the activity of Mycobacterium tuberculosis; the amplification results corresponding to the MTB 16S rRNA as the target gene are used to calculate the bacterial concentration of active Mycobacterium tuberculosis based on the quantitative relationship between the amplification CT value and the copy concentration; the detection method does not include the diagnosis and treatment methods of the disease.

9. A detection kit for active Mycobacterium tuberculosis, comprising the primer combination according to any one of claims 1 to 3, supporting reagents, reverse transcriptase, DNA polymerase, positive quality control and negative control, wherein the DNA polymerase is an enhanced Tag enzyme.

10. Use of the primer composition according to any one of claims 1 to 3, the method for detecting active Mycobacterium tuberculosis according to any one of claims 4 to 6, the standard for quantitative detection of active Mycobacterium tuberculosis according to claim 7, and the detection kit for active Mycobacterium tuberculosis according to claim 9 in the preparation of an active Mycobacterium tuberculosis detection product.

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