Detection agent for nucleic acid marker used in diagnosis of mycobacterium tuberculosis infection, and use thereof

Abstract

The present invention relates to the technical field of clinical detection, and in particular, relates to a detection agent for a nucleic acid marker used in the diagnosis of Mycobacterium tuberculosis infection, and the use thereof. Nucleic acid segments as shown in SEQ ID NO: 1 and SEQ ID NO: 2 can be used as new molecular markers for the detection and diagnosis of Mycobacterium tuberculosis infection by means of an isothermal amplification reaction. Compared with commonly used detection segments disclosed in the prior art, the two segments exhibit great compatibility with the isothermal amplification reaction technique, and can be used in the preparation of preparations used for diagnosing Mycobacterium tuberculosis infection.

Description

Detection reagents for nucleic acid biomarkers used in the diagnosis of Mycobacterium tuberculosis infection and their applications

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202410934342.4, filed on July 12, 2024, entitled "Detection reagent for nucleic acid markers for diagnosing Mycobacterium tuberculosis infection and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of clinical testing technology, and more specifically, to detection reagents for diagnosing Mycobacterium tuberculosis infection and their applications. Background Technology

[0004] Tuberculosis is a major respiratory infectious disease that seriously endangers the health of the people. The pathogen of tuberculosis is Mycobacterium tuberculosis, mainly including Mycobacterium human tuberculosis, Mycobacterium bovis, Mycobacterium Africanum tuberculosis, and Mycobacterium murineum tuberculosis. It can enter the body through the respiratory tract, digestive tract, and broken skin and mucous membranes, invading a variety of tissues and organs and causing tuberculosis in the corresponding organs, especially pulmonary tuberculosis.

[0005] Research and development in tuberculosis diagnostic technologies is showing strong momentum, primarily covering areas such as tuberculosis screening, detection of latent tuberculosis infection, and diagnosis of drug-resistant tuberculosis. Tuberculosis diagnostics can be categorized into four types based on their intended use: Mycobacterium tuberculosis (MTB) detection reagents, drug susceptibility testing reagents, MTB + drug susceptibility testing reagents (dual-function), and bacterial identification reagents. Currently, there are numerous tuberculosis diagnostic technology platforms, such as enzyme-linked immunosorbent assay (ELISA), fluorescent PCR, colloidal gold assay, and mycobacterial culture. However, from a detection technology perspective, bacteriological and immunological detection technologies for tuberculosis suffer from long detection times, poor sensitivity or specificity, and the inability to distinguish between active tuberculosis and latent Mycobacterium tuberculosis infection, which to some extent restricts the early diagnosis and treatment of tuberculosis. With the continuous development of medical science and technology, molecular biology detection technologies have received widespread attention and application in assisting the early diagnosis of tuberculosis.

[0006] Currently approved molecular biology detection products for Mycobacterium tuberculosis are mainly based on quantitative real-time PCR technology, for example... Products such as the Xpert MTB / RIF series are available. However, these molecular biology detection technologies have limitations such as high platform requirements, complex operation, and expensive equipment, hindering their widespread adoption in primary healthcare institutions. To address these shortcomings, there is a need to develop a Mycobacterium tuberculosis detection technology that is more suitable for primary care and can be used for point-of-care testing (POCT).

[0007] Isothermal amplification combined with CRISPR detection technology, which can accurately identify single-base differences, ensures high specificity of the detection, enabling earlier and more convenient nucleic acid detection for tuberculosis. This approach will greatly improve the efficiency of early diagnosis and monitoring of tuberculosis and is expected to become an important technical support for tuberculosis prevention and control at the grassroots level and in the field. Currently, molecular detection methods for detecting Mycobacterium tuberculosis complexes are mainly based on PCR, including three steps: denaturation, annealing, and extension. However, isothermal amplification is a method of template amplification through the action of specific enzymes at a constant temperature. It is worth noting that not all amplification regions and biomarkers suitable for PCR detection are suitable for isothermal amplification. Therefore, the research in this disclosure mainly aims to discover biomarkers suitable for highly sensitive detection of Mycobacterium tuberculosis complexes, which are not only suitable for PCR detection systems but also for isothermal amplification systems. This disclosure hopes to upgrade and promote Mycobacterium tuberculosis detection technology by developing related kits, thereby contributing to the development of disease detection technology. Summary of the Invention

[0008] This disclosure covers the following technical solutions:

[0009] The first aspect of this disclosure relates to the use of nucleic acid biomarker detection reagents in the preparation of reagents or kits for diagnosing Mycobacterium tuberculosis infection;

[0010] The sequence of the nucleic acid biomarker is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2, and the detection reagent is used to detect the nucleic acid biomarker in a biological sample by isothermal amplification reaction.

[0011] Optionally, in the applications described above, the detection agent includes primers and / or probes targeting the nucleic acid biomarker.

[0012] Optionally, in the applications described above, the isothermal amplification reaction is one or more of the following: recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, multiple substitution amplification, cross-primer amplification, recombinase-mediated amplification, novel recombinase-dependent amplification, chain substitution amplification, and helicase-dependent amplification.

[0013] Optionally, in the applications described above, the reagent or kit contains a strand displacement DNA polymerase and / or recombinase system.

[0014] Optionally, in the applications described above, the reagent or kit further comprises an isothermal amplification reaction product detection reagent to determine the presence or absence of Mycobacterium tuberculosis.

[0015] Optionally, in the application described above, the isothermal amplification reaction product detection reagent includes at least one of the following: a CRISPR-Cas detection system, a dye and / or auxiliary agent for detecting DNA double strands, a fluorescent probe for hybridizing with the isothermal amplification reaction product, and a pH colorimetric reagent.

[0016] Optionally, in the applications described above, the CRISPR-Cas includes one or more of the following: a CRISPR-Cas12 detection system, a CRISPR-Cas13 detection system, and a Cas detection system having bypass cleavage activity similar to that of Cas12 and / or Cas13.

[0017] Optionally, in the applications described above, at least one component of the reagent or kit is a solid, the solid including at least one of lyophilized microspheres, lyophilized cakes, lyophilized powders, and spots that depend on the presence of a solid medium.

[0018] Optionally, in the application described above, the biological sample is selected from one or more of the following: tongue swab, sputum, saliva, serum, plasma, blood, urine, mucus, sweat, tears or other eye fluids, ear fluids, blisters, sores, gastric juice, gastric juice, bronchoalveolar lavage fluid, feces, pancreatic juice or juice, pharyngeal swab, nasal swab, conjunctival swab, semen, breast milk, cerebrospinal fluid, liquid bone marrow, and lymph.

[0019] The second aspect of this disclosure relates to reagents or kits as defined in the first aspect.

[0020] A third aspect of this disclosure relates to a method for detecting Mycobacterium tuberculosis in a sample, comprising:

[0021] a) Obtain isolated nucleic acids from the sample; b) Amplify potential nucleic acid biomarkers in the nucleic acids using an isothermal amplification reaction; wherein the sequences of the nucleic acid biomarkers are as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; and c) Detect the presence of the nucleic acid biomarkers.

[0022] Optionally, in the method described above, the isothermal amplification reaction includes one or more of the following: recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, multiple substitution amplification, cross primer amplification, recombinase-mediated amplification, novel recombinase-dependent amplification, chain substitution amplification, and helicase-dependent amplification.

[0023] Optionally, in the method described above, the detection method in c) is selected from at least one of CRISPR-Cas method, fluorescence method, and lateral flow immunochromatography.

[0024] The fourth aspect of this disclosure relates to the use of nucleic acid biomarkers in the preparation of reagents or kits for diagnosing Mycobacterium tuberculosis infection, characterized in that the sequence of the nucleic acid biomarker is as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0025] This disclosure reveals that the nucleic acid segments shown in SEQ ID NO: 1 and SEQ ID NO: 2 can be used as novel molecular markers for isothermal amplification reaction detection and diagnosis of Mycobacterium tuberculosis infection. These two segments are derived from the IS6110 gene of Mycobacterium tuberculosis and, compared to commonly used detection segments disclosed in traditional techniques, exhibit excellent compatibility with isothermal amplification reaction technology, making them suitable for the preparation of formulations for diagnosing Mycobacterium tuberculosis infection. Developing formulations for diagnosing Mycobacterium tuberculosis infection using the molecular markers of this disclosure offers advantages such as higher sensitivity, convenience, speed, high specificity, and lower cost. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 shows the detection sensitivity (chromatographic method) results of different detection segments in a sputum sample provided in an embodiment of this disclosure;

[0028] Figure 2 shows the detection sensitivity (chromatographic method) results of different detection segments in a tongue swab sample provided in an embodiment of this disclosure;

[0029] Figure 3 shows the detection sensitivity (fluorescence method) of different detection segments in a sputum sample provided in an embodiment of this disclosure;

[0030] Figure 4 shows the detection sensitivity (fluorescence method) results of different detection segments in a tongue swab sample provided in an embodiment of this disclosure;

[0031] Figure 5 shows the detection sensitivity (RAA fluorescence method) results of different detection segments in a sputum sample provided in an embodiment of this disclosure;

[0032] Figure 6 shows the detection sensitivity (RAA fluorescence method) of different detection segments in a tongue swab sample provided in an embodiment of this disclosure;

[0033] Figure 7 shows the detection sensitivity (LAMP fluorescence method) results of different detection segments in a sputum sample provided in an embodiment of this disclosure; and

[0034] Figure 8 shows the detection sensitivity (LAMP fluorescence method) results of different detection segments in a tongue swab sample provided in an embodiment of this disclosure. Detailed Implementation

[0035] Reference will now be made to embodiments of this disclosure in detail, with one or more examples described below. Each example is provided for explanation and not for limitation of this disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to this disclosure without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0036] Unless otherwise stated, all terms used in the disclosure of this disclosure (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Further guidance is provided below for a better understanding of the teachings of this disclosure. The terminology used in this disclosure and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure.

[0037] In this disclosure, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this disclosure are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0038] The terms "and / or," "or / and," and "and / or" as used in this disclosure include any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this disclosure, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0039] The terms “containing,” “including,” and “comprise” as used in this disclosure are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0040] The range of values ​​represented by endpoints in this disclosure includes all values ​​and fractions contained within that range, as well as the endpoints referenced.

[0041] As used in this disclosure, "about" a value or parameter includes (and describes) an implementation for the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0042] The concentration values ​​mentioned in this disclosure include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may allow fluctuations within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are allowed. For example, 100mM may allow fluctuations within the ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations within ±10% are allowed.

[0043] As used in this disclosure, unless otherwise indicated, the singular articles “a,” “an,” and “the” include plural referents.

[0044] In this disclosure, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0045] In this disclosure, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0046] In this disclosure, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments and should be understood not to limit the scope of protection of this disclosure. In this disclosure, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" options appear in a technical solution, unless otherwise specified and without contradictions or mutual constraints, each "optional" option is independent.

[0047] The term "water" as used in this disclosure can be distilled water, purified water, filtered water, deionized water, etc.; it is preferably nucleic acid-free water, and even more preferably nuclease-free water.

[0048] As used in this disclosure, the term "primer" or "primer sequence" refers to a straight-chain oligonucleotide that hybridizes to a target DNA template to produce a target DNA:primer hybrid and initiate a DNA synthesis reaction. Upper and lower limits for primer length are determined empirically. The lower limit of primer length is the minimum length required to form a stable double strand when hybridizing with the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (typically less than 3 nucleotides long) will not form a thermodynamically stable double strand with the target nucleic acid under such hybridization conditions. The upper limit is often determined by the likelihood of double strand formation in regions other than the predetermined nucleic acid sequence in the target nucleic acid. Typically, suitable primer lengths range from about 3 nucleotides to about 40 nucleotides. The primers may be RNA oligonucleotides, DNA oligonucleotides, or chimeric sequences. Primers used in this disclosure are preferably primers suitable for isothermal amplification reactions.

[0049] The dNTP mixture used in this disclosure refers to a mixture of deoxyribonucleoside triphosphates, wherein N is a random nucleotide containing any one of A, C, G, or T / U.

[0050] As used in this disclosure, the term "reaction temperature" refers to the temperature maintained during the amplification reaction. Embodiments of this disclosure include isothermal amplification reactions, wherein the temperature of the reaction is constant. The entire isothermal amplification reaction is carried out at a reaction temperature, for example, about 30°C for isothermal amplification reactions using GenomiPhi. The reaction temperature varies with conditions, including but not limited to the use of different polymerases, primer or template sizes, or the use of additional salts or stabilizers. For RPA / RAA / RDA technologies, a suitable reaction temperature is preferably about 25°C to 40°C, more preferably 35°C to 40°C, and more preferably 37°C; for LAMP technologies, a suitable reaction temperature is preferably about 60°C to 65°C.

[0051] All documents mentioned in this disclosure are cited herein as if they were cited individually. Unless they conflict with the inventive purpose and / or technical solution of this disclosure, all cited documents are cited in their entirety and for all purposes. When citing documents in this disclosure, the definitions of relevant technical features, terms, nouns, phrases, etc., are also cited. When citing documents in this disclosure, examples and preferred embodiments of the relevant technical features may also be incorporated into this disclosure as reference, but only to the extent that they enable the implementation of this disclosure. It should be understood that when the cited content conflicts with the description in this disclosure, this disclosure shall prevail or modifications shall be made adaptively based on the description in this disclosure.

[0052] The first aspect of this disclosure relates to the use of nucleic acid biomarker detection reagents in the preparation of reagents or kits for diagnosing Mycobacterium tuberculosis infection;

[0053] The sequence of the nucleic acid biomarker is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2, and the detection reagent is used to detect the nucleic acid biomarker in a biological sample by isothermal amplification reaction.

[0054] This disclosure, through rigorous experiments and in accordance with relevant design principles, randomly designed a variety of primers and / or probes suitable for isothermal amplification reactions, which cover the nucleic acid sequences shown in SEQ ID NO: 1 and / or SEQ ID NO: 2, to verify their reliability as biomarkers for diagnosing Mycobacterium tuberculosis infection.

[0055] In this disclosure, unless otherwise stated, based on the general understanding of those skilled in the art, "nucleic acid biomarker," "target nucleic acid," "biomarker," "biomarker gene," "biochemical biomarker," "tuberculosis biomarker," or "Mycobacterium tuberculosis biomarker" refers to the full-length nucleotide sequence of the nucleic acid shown in SEQ ID NO: 1 and / or SEQ ID NO: 2, or a naturally occurring variant, or a fragment of the full-length sequence and variants, particularly a fragment that can be detected and identified as a specific sequence; their meanings may be interchangeable depending on the context. Specifically, it refers to the target nucleic acid to be used for analyzing biological samples of subjects.

[0056] The target DNA amplified by the detection reagent preferably comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 consecutive nucleotides of the SEQ ID NO: 1 sequence; and / or, preferably comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, or 181 consecutive nucleotides of the SEQ ID NO: 2 sequence.

[0057] In this disclosure, the terms "biological sample," "sample," etc., refer to animal samples; tissues or organs, tissue lysates, or other biological samples that may be derived from animals (preferably including at least mammals, such as primates, including humans); cells (in vivo of a subject, directly taken from the subject, or held in a culture or derived from a cultured cell line), cell lysates (or portions of lysates), or cell extracts; solutions containing one or more molecules derived from cells, cellular material, or viral material (e.g., polypeptides or nucleic acids); or solutions containing naturally occurring or non-naturally occurring nucleic acids, which are or can be measured as described in this disclosure. In some embodiments, the sample contains nucleic acids. The sample may also be any bodily fluid or excretion containing one or more cells, cellular components, or nucleic acids, including but not limited to cellular, nuclear, or cell-free nucleic acids. In particular, the biological samples of this disclosure are preferably derived from bodily fluids, including liquids, semi-solids, aerated liquids, liquid-gas mixtures, etc., derived from animals. Such bodily fluids may include, but are not limited to, one or more of the following: tongue swabs, sputum, saliva, serum, plasma, blood, urine, mucus, sweat, tears or other ocular fluids, ear fluids, blisters, sores, gastric juice, gastric lavage fluid, bronchoalveolar lavage fluid, feces, pancreatic juice or fluid, pharyngeal swabs, nasal swabs, conjunctival swabs, semen, breast milk, cerebrospinal fluid, liquid bone marrow, and lymph. Biological samples may also be forensic samples or ancient samples. Preferred biological samples in this disclosure are selected from tongue swabs and sputum.

[0058] In some embodiments, the detection agent includes primers and / or probes that target the nucleic acid biomarker.

[0059] "Primers and / or probes targeting the nucleic acid biomarker" refers to primers capable of targeting and amplifying the nucleic acid biomarker, and / or probes targeting and binding to the nucleic acid biomarker. As used in this disclosure, the term "targeted amplification" refers to the process of amplifying a target nucleic acid. The target nucleic acid acts as a template in a DNA amplification reaction. A portion of the nucleic acid biomarker or an entire region of the target nucleic acid can be amplified by DNA polymerase in a DNA amplification reaction to generate an amplification product or amplicon. The amplicon may include multiple copies of the target nucleic acid or multiple copies of a sequence complementary to the target nucleic acid. The target nucleic acid can be obtained from a biological sample. The isolated nucleic acid biomarker can be dispersed in solution or immobilized on a solid support such as a speckle, array, slide, microtiter plate, beads, or ELISA plate.

[0060] Primers and probes may be modified, and modifications may be performed using known methods. Modified versions of these primer and / or probe sequences may include, by non-limiting examples, adding one or more nucleotides to the 5' end, adding one or more nucleotides to the 3' end, adding one or more nucleotides to both the 5' and 3' ends, adding a tail, shortening the sequence, lengthening the sequence, shifting the sequence upstream or downstream by several bases, or any combination thereof.

[0061] Base modifications, such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2'-deoxyadenosine, C-5-propynyl-deoxycytidine, C-5-propynyl-deoxyuridine, 2-amino-2'-deoxyadenosine-5'-triphosphate, 2,6-diaminopurine (2-amino-dA), reversed-dT, reversed-dideoxy-T, hydroxymethyl-dC, iso-dC, 5-methyl-dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNAs), including at least one mismatched base at one of the bases, or replacing at least one of the bases with an RNA base, can achieve, for example, increased nucleic acid interaction at the 3' end of mutant-specific primers to increase Tm. The addition of stable double-stranded base modifications has a positive effect on PCR, enabling it to be performed at higher temperatures, within which Taq polymerase is known to exhibit maximum activity. Modified probes should retain the ability to distinguish between the mutant and wild-type sites to be detected.

[0062] In some embodiments, the probe is labeled with a detectable signal substance. In some embodiments, the signal substance is a fluorophore, colorimetric label, colloidal gold, quantum dot, biotin, and other tag molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cycloalkenes for click reactions, and initiating groups for polymer labeling). It may also be selected from peptide / protein molecules, LNA / PNA, non-natural amino acids and their analogs (such as peptides), non-natural nucleic acids and their analogs (nucleotides), and nanostructures (including inorganic nanoparticles, NV-centers, aggregation / assembly-induced luminescent molecules, rare earth ion ligand molecules, polyoxometalates, etc.).

[0063] The articles by Lewis (1992, Genetic Engineering News 12:1-9) and Abramson and Myers (1993, Curr. Opin. Biotechnol. 4:41-47) provide excellent comprehensive reviews of amplification techniques. These techniques are primarily based on either methods requiring multiple cycles during amplification or methods performed at a single temperature. The main aspect of amplification in techniques performed at a single temperature (known as isothermal techniques) is that the amplification process occurs at a single temperature. Unlike PCR (where the reaction products are heated to separate the two strands so that other primers can bind to the template to repeat the process), isothermal techniques rely on strand displacement polymerases to separate / displace the two strands of the double helix and recopy the template. This well-known characteristic is the subject of many scientific papers (see, for example, Y. Masamute and C. C. Richardson, 1971, J. Biol. Chem. 246, 2692-2701; R. R. Lechner et al., 1983, J. Biol. Chem. 258, 11174-11184; or R. C. Lundquist and B. M. Livera, 1982, Cell 31, 53-60). The key difference in isothermal techniques lies in the method used to initiate a repetitive process.

[0064] Isothermal techniques in a broad sense can be further divided into the following methods: methods that rely on primer substitution to initiate repetitive template copying (examples below include HDA (helicase-dependent amplification), exonuclease-dependent amplification (EP1866434), recombinase polymerase amplification (RPA), recombinase-mediated amplification (RAA), novel recombinase-dependent amplification (RDA, see Chinese patent CN202010344702.7), loop-mediated amplification (LAMP), rolling circle amplification (RCA), multiple substitution amplification (MDA), and cross-primer amplification (CPA)); and methods that rely on the continuous reuse or de novo synthesis of single primer molecules (examples below include SDA (strand substitution amplification and nucleic acid-based amplification (NASBA and TMA)).

[0065] As a preferred embodiment, in this disclosure, the isothermal amplification reaction is one or more of the following: recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, multiple substitution amplification, cross-primer amplification, recombinase-mediated amplification, novel recombinase-dependent amplification, chain substitution amplification, and helicase-dependent amplification.

[0066] In some embodiments, the reagent or kit includes a strand displacement DNA polymerase and / or recombinase system.

[0067] The polymerases used in the methods of this disclosure are preferably those with strand displacement activity. This activity is a well-known characteristic of some DNA polymerases (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, pp. 5.33-5.35, Cold Spring Harbor Laboratory, Cold Spring Harbor). The characteristics of DNA polymerases, especially the strand displacement activity of some of them, are given in detail by Kornberg and Baker et al., DNA Replication, 2nd edition, pp. 113-225, Freeman, NY (1992). Strand displacement is not a characteristic common to all DNA polymerases, because some (e.g., T4 DNA polymerase) cannot perform strand displacement alone. In these cases, strand displacement can be achieved by adding polymerase helper proteins. Initially, the Klenow fragment of *E. coli* DNA polymerase I exhibited strand substitution activity (Masamune and Richardson, 1971, J. Biol. Chem. 246:2692-2701), conferring the enzyme the ability to initiate nucleic acid replication from the 3'OH end of the double-stranded DNA cleavage site. This strand substitution activity has also been observed in thermostable DNA polymerases such as Tli DNA polymerase (Kong et al., 1993, J. Biol. Chem. 268:1965-1975). In this case, a mutant form of the enzyme was shown to lack exonuclease 5'-3' activity, which results in a higher strand substitution capacity. This strand substitution activity has also been observed in T7 DNA polymerase (Lechner et al., 1983, J. Biol. Chem. 258:11174-11184) and HIV reverse transcriptase (Huber et al., 1989, J. Biol. Chem. 264:4669-4678). As an example, the strand substitution DNA polymerase is selected from at least one of phi29 DNA polymerase, Bsu DNA polymerase and Sau DNA polymerase.

[0068] Suitable polymerases include polI or polI fragments or variants, such as those from *E. coli*, *Bacillus subtilis*, *Bacillus stearothermophilus*, or T7 polymerases. Similarly, polymerase holoenzyme complexes such as those described for bacteriophage T4 can be used. Preferred polymerases are Klenow, exo-, T7 polymerases, or BST phi29 or *Bacillus subtilis* pol-I or holoenzymes. Bsu DNA polymerases or Sau DNA polymerases may also be used. In some embodiments, especially those using downstream elements or reverse complementation, preferred polymerases do not possess the same strong strand substitution activity as the Klenow fragment of *E. coli* polI. In other embodiments, strong strand substitution activity of the polymerase may be advantageous.

[0069] When recombinases are used in the chain insertion step, the system may require an energy source. Most of these enzymes utilize ATP as an energy source, but because magnesium ions are essential for the activity of ATP-collate enzymes, it is advantageous to provide an additional ATP regeneration system rather than increasing the ATP concentration. The ATP regeneration system preferably comprises ATP and reagents used in the ATP regeneration system. In some embodiments, the reagents used in the ATP regeneration system are selected from one or more of magnesium ions, creatine phosphate and its counterions, creatine kinase, myokinase, pyrophosphatase, sucrose, and sucrose phosphorylase.

[0070] In some embodiments, the strand substitution DNA polymerase is a strand substitution DNA polymerase suitable for loop-mediated isothermal amplification (LAMP). Strand substitution DNA polymerases suitable for LAMP are well known in the art, such as EquiPhi29, Bst DNA polymerase, and Bsm DNA polymerase. The LAMP reaction system may also contain dNTPs, Mg... 2+ It contains at least one of the following components: betaine, etc.

[0071] Various recombinase systems are known to those skilled in the art and have been extensively reviewed (e.g., Piero R. Bianco et al. Frontiers in Bioscience 3, d570-603, 1998, 570 DNA Strand Exchange Proteins: A Biochemical and Physical Comparison). Any recombinase system can be used in the methods of this disclosure, and detailed applications of recombinases for double-stranded nucleic acid insertion are known to those skilled in the art (Kodadek T et al., JBC 264, 1989; and Liu J, JBC Na Qian1, 281, 26308–26319, 2006).

[0072] The recombinase system may include components derived from yeast, bacterial bacteriophages, or mammals or other eukaryotes. The recombinase system may be mesophilic or thermophilic. For example, the recombinase may be derived from myoviridae bacteriophages. Myoviridae bacteriophages may be, for example, T4, T2, T6, Rb69, Aehl, KVP40, Acinetobacter spp. bacteriophage 133, Aeromonas spp. bacteriophage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rbl4, Rb32, Aeromonas spp. bacteriophage 25, Vibrio bacteriophage nt-1, phi-1, Rbl6, Rb43, bacteriophage 31, bacteriophage 44RR2.8t, Rb49, bacteriophage Rb3, or bacteriophage LZ2. In a preferred embodiment, the T4 recombinase UvsX may be used. Alternatively, the Rad system from eukaryotes, the recA-Reco system from Escherichia coli, or other prokaryotic systems can be used. Generally, the recombinase for RPA originates from T4 phages, such as Escherichia phage phT4A; the recombinase for RAA originates from bacteria or fungi.

[0073] Typically, recombinases polymerize on single-stranded oligonucleotides in a 5'-3' orientation. This disclosure relates to such recombinases as described herein. However, recombinases can also polymerize in a 3'-5' orientation, and such recombinases can also be used in the methods of this disclosure. In this case, and referring to the orientation of the component, the reverse orientation is applied.

[0074] Recombinase accessory proteins may be included in the system. In some embodiments, the recombinase system comprises a recombinase capable of binding single-stranded nucleic acids, a single-stranded DNA-binding protein (e.g., gp32), and a recombinase loader (e.g., UvsY). In a preferred embodiment, the recombinase system comprises T4 gp32, UvsX, and UvsY. The recombinase loader is used to alter the reversible reaction process of recombinase-primer complex dissociation and rebinding, directing the reaction towards a path more favorable for isothermal nucleic acid amplification.

[0075] Recombinases (e.g., UvsX), recombinase loaders (e.g., UvsY), and single-stranded DNA-binding proteins (e.g., gp32) can each be natural, heterozygous, or mutant proteins derived from the same or different myoviridae bacteriophage sources. Natural proteins can be wild-type or natural variants of the protein. Mutant proteins (also known as genetically modified proteins) are natural proteins with natural or artificial mutations such as insertions, deletions, substitutions, or combinations thereof at the N-terminus, C-terminus, or internally (between the N-terminus and C-terminus). Heterozygous proteins (also known as chimeric proteins) comprise sequences from at least two different organisms. For example, a heterozygous UvsX protein may contain amino acids from one species (e.g., T4) but also a DNA-binding loop from another species (e.g., T6). Heterozygous proteins may contain enhanced properties compared to natural proteins. These enhanced properties may be an increased or faster amplification rate, or a decreased or more controllable amplification rate.

[0076] Other components used to enhance the efficiency of the recombinase system may also be included in the reagents or kits disclosed herein, such as pH adjusters, dNTPs, molecular crowding agents, DTT, DMSO, betaine, proline, detergents, and one or more of water.

[0077] As used in this disclosure, the term "molecular crowding agent" refers to a reagent or molecule that alters the properties of other molecules in solution. Examples of molecular crowding agents include, but are not limited to, at least one of serum albumin (such as BSA and / or HSA), dextran, and polyethylene glycol (PEG). Typically, said molecular crowding agents have a high molecular weight or a large volume structure that creates a crowding environment in a solution containing other molecules. The molecular crowding agent reduces the volume of solvent available for other molecules in solution, thus causing molecular crowding. This molecular crowding can alter the reaction rate or equilibrium constant. In one or more embodiments, the molecular crowding agent used in the amplification reaction is selected from serum albumin, PEG, Ficoll, colloid, trehalose, and combinations thereof. In one embodiment, the molecular crowding agent comprises BSA and / or PEG with various molecular weight distributions. In one embodiment, the molecular crowding agent is selected from PEG 400, PEG 2000, PEG 6000, PEG 8000, and combinations thereof.

[0078] In some embodiments, the kit also includes an isothermal amplification reaction product detection reagent to determine the presence or absence of Mycobacterium tuberculosis.

[0079] The detection method for isothermal amplification reaction products may or may not depend on nucleic acid amplification. As used in this disclosure, the term "isothermal amplification reaction product detection reagent" refers to the further detection of amplification products generated by the nucleic acid detection reagents with sequences such as SEQ ID NO: 1 and / or SEQ ID NO: 2 provided in this disclosure, in order to increase the accuracy, specificity and / or convenience of Mycobacterium tuberculosis diagnosis.

[0080] Isothermal amplification reaction product detection reagents can be based on any method for determining the nucleotide composition of the target nucleic acid, including but not limited to DNA sequencing methods, probe hybridization methods, detection after DNA is transcribed into RNA, structure-specific cleavage assays (e.g., INVADER assays, (Hologic, Inc.)) and described in, for example, U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamic Hev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and US2009 / 0253142, each incorporated herein by reference in its entirety for all purposes; enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Patent Nos. 6,110,684, 5,958,692, and 5,851,770, the entirety of which is incorporated herein by reference); the polymerases described above. Chain reaction (PCR); branching hybridization methods (e.g., Chiron, U.S. Patent Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, the entire contents of which are incorporated herein by reference); rolling circle replication (e.g., U.S. Patent Nos. 6,210,884, 6,183,960, and 6,235,502, the entire contents of which are incorporated herein by reference); NASBA (e.g., U.S. Patent No. 5,409,818, the entire contents of which are incorporated herein by reference). Molecular beacon technology (e.g., U.S. Patent No. 6,150,097, the entire contents of which are incorporated herein by reference); E-sensor technology (Motorola, U.S. Patent Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, the entire contents of which are incorporated herein by reference); cyclic probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769, and 5,660,988, the entire contents of which are incorporated herein by reference); Dade Behring signal amplification methods (e.g., U.S. Patent Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, the entire contents of which are incorporated herein by reference); ligation enzyme chain reactions (e.g., Baranay Proc. Natl. Acad. Sci USA 88,189-93 (1991)); sandwich hybridization methods (e.g., U.S. Patent No. 5,288,609, the entire contents of which are incorporated herein by reference); and the CRISPR-Cas12 detection system preferred herein.

[0081] In some preferred embodiments, the isothermal amplification reaction product detection reagent includes at least one of a CRISPR-Cas detection system, a dye and / or auxiliary agent for detecting DNA double strands, a fluorescent probe for hybridizing with the isothermal amplification reaction product, and a pH colorimetric reagent.

[0082] Based on the type of nucleic acid molecule being detected, CRISPR / Cas nucleic acid detection can be divided into DNA (CRISPR / Cas9, CRISPR / Cas12, and CRISPR / Cas12 recognize DNA sequences) and RNA (CRISPR / Cas13 recognizes RNA sequences) detection. Further, the CRISPR-Cas includes one or more of the following: a CRISPR-Cas12 detection system, a CRISPR-Cas13 detection system, and a Cas detection system with bypass cleavage activity similar to Cas12 and / or Cas13. Preferably, the CRISPR-Cas12 detection system is the CRISPR-Cas12a detection system, and the CRISPR-Cas13 detection system is the CRISPR-Cas13a detection system.

[0083] The dyes and / or auxiliary agents used to detect DNA double strands are components commonly used in conjunction with LAMP technology. The dyes may be selected from at least one of SYBR Green I, EvaGreen, PicoGreen, Peko Green, propidium iodide, calcein, or hydroxynaphthol blue. The auxiliary agents may be selected from at least one of betaine, L-proline, DTT, DMSO, Tween 20, or NP40.

[0084] Components within reagents or kits can be packaged in the form of solutions, solids, or test strips. In some preferred embodiments, at least one component of the reagent or kit is a solid, including at least one of lyophilized microspheres, lyophilized cakes, lyophilized powders, and spots dependent on a solid medium. Therefore, components required for nucleic acid amplification (and preferably nucleic acid detection), particularly various enzymes, nucleic acid components, and reaction buffer components, can be provided in lyophilized form. In this way, the nucleic acid amplification (and preferably nucleic acid detection) process can be directly initiated in a very user-friendly manner by adding the sample to be quantified and optionally other required components.

[0085] This disclosure also provides kits, which are the reagents or kits mentioned in the applications described above.

[0086] The kit disclosed herein comprises a detection reagent for detecting nucleic acid biomarkers in biological samples via isothermal amplification reactions, the sequences of which are shown in SEQ ID NO: 1 and / or SEQ ID NO: 2. Specifically, it comprises at least one of a detection reagent (primers and / or probes) containing nucleic acid biomarkers and a detection reagent for isothermal amplification reaction products (particularly a CRISPR-Cas detection system).

[0087] In this disclosure, the term "kit" may refer to any article (e.g., packaging or container) that includes at least one device and comprises the detection reagents as described in this disclosure. The kit may further include instructions for use, supplementary reagents, and / or components or parts used in the methods or steps described in this disclosure.

[0088] This disclosure also provides a diagnostic device for Mycobacterium tuberculosis, comprising one or more independent modules, at least one of which contains the reagents or kits described above, or components thereof.

[0089] In this diagnostic device, the modules and reagent kits can be packaged independently, or at least some components of the reagent kit (primers, probes, reaction buffers, enzymes, etc.) can be pre-filled into the nucleic acid detection device. The nucleic acid detection device is typically designed to facilitate nucleic acid detection, for example, by preventing contamination during the detection process, accelerating the detection speed (e.g., simultaneously detecting multiple samples), facilitating the preservation of components in the reagent kit during the detection process, or otherwise improving the convenience and / or accuracy of the detection. The use of these diagnostic devices is generally common practice among those skilled in the art, and exemplary diagnostic devices include any one or more of the devices described in CN202010571470.9, CN202021162806.8, CN202010769972.2, CN202021585862.2, and CN202210062076.1.

[0090] In some implementations, the standalone module includes one or more containment units, which are equipped with a nucleic acid marker amplification system and / or a detection system.

[0091] In some embodiments, at least one containment unit contains at least a portion of the components of the reagent or kit.

[0092] According to another aspect of this disclosure, a method for detecting Mycobacterium tuberculosis in a sample is also provided, comprising:

[0093] a) Obtain isolated nucleic acids from the sample; b) Amplify potential nucleic acid biomarkers in the nucleic acids using an isothermal amplification reaction; wherein the sequences of the nucleic acid biomarkers are as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; and c) Detect the presence of the nucleic acid biomarkers.

[0094] In some embodiments, the isothermal amplification reaction includes one or more of the following: recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, multiple substitution amplification, cross-primer amplification, recombinase-mediated amplification, novel recombinase-dependent amplification, strand substitution amplification, and helicase-dependent amplification.

[0095] In some embodiments, the detection method in c) is selected from at least one of CRISPR-Cas method, fluorescence method, and lateral flow immunochromatography.

[0096] In some implementations, step c) involves detection using an isothermal amplification reaction product detection reagent.

[0097] In some implementations, the targeted nucleic acid is an environmental sample or a biological sample.

[0098] Environmental samples can be from public or private places, indoors or outdoors (such as air, soil, water, etc.). Public places include hospitals, schools, bus stations, train stations, airports, etc.

[0099] In some embodiments, the method is used to diagnose Mycobacterium tuberculosis infection.

[0100] In some implementations, the meaning of at least one of the nucleic acid biomarker, isothermal amplification reaction, and isothermal amplification reaction product detection reagent is as described in the first aspect.

[0101] The embodiments of this disclosure will now be described in detail with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. For experimental methods in the following embodiments where specific conditions are not specified, reference should be made to the guidelines given in this disclosure, or to experimental manuals or conventional conditions in the art, or to other experimental methods known in the art, or to the conditions recommended by the manufacturer.

[0102] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0103] Example 1: Novel molecular markers for diagnosing Mycobacterium tuberculosis infection

[0104] IS6110 is a commonly used gene for detecting Mycobacterium tuberculosis. This disclosure provides two novel molecular markers for diagnosing Mycobacterium tuberculosis infection. The sequences of the molecular markers correspond to segments 1 and 2, respectively:

[0105] Segment 1 begins at base 544 of the IS6110 gene and extends to base 663, with the following specific sequence:

[0106] Segment 2 begins at base 1090 of the IS6110 gene and extends to base 1270, with the following specific sequence:

[0107] For comparison, the commonly used molecular markers for IS6110 in the prior art are listed as segment 3 and segment 4:

[0108] Segment 3 is described in Chinese patents CN202311778526.8 and CN201310009018.3 of Sansure Biotech. It is the middle segment, starting from the 665th base of the IS6110 gene and extending to the 870th base, with the specific sequence as follows:

[0109] Segment 4 is the detection segment found in Cepheid's literature (The New Xpert MTB / RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing. mBio. 2017 Aug 29;8(4):e00812-17. doi:10.1128 / mBio.00812-17.PMID:28851844;PMCID:PMC5574709.). Starting from the 1010th base of the IS6110 gene and extending to the 1120th base, the specific sequence is as follows:

[0110] Example 2: Diagnostic efficacy of novel molecular biomarkers—based on PCR detection technology

[0111] This embodiment uses PCR detection technology and, based on different sample types (plasma samples, tongue swab samples, sputum samples, and bronchoalveolar lavage fluid), compares the diagnostic efficacy of the biomarkers provided in this disclosure with common existing biomarkers in Mycobacterium tuberculosis infection.

[0112] The experimental procedure and method of this embodiment are as follows.

[0113] 1. Sample collection

[0114] 1.1 Plasma Collection: Whole blood was collected using EDTA blood collection tubes and centrifuged at room temperature for 1 hour to obtain plasma. Centrifugation conditions: 1500g, 10 minutes, supernatant collected, centrifuged again: 12000g, 10 minutes, supernatant collected, which is the separated plasma, sample volume 2mL.

[0115] 1.2 Tongue swab sample collection: Extend your tongue and hold it in place. Place the swab on the back of your tongue, press along your cheek and roll the swab for about 10 seconds (7-8 rolls to collect). Store the swab in a preservation tube.

[0116] 1.3 Sputum Sample Collection: After waking up in the morning, the patient rinses their mouth repeatedly with water to reduce contamination by normal flora, and forcefully coughs up the first sputum from the trachea into a sterile sampling tube. The sputum sample volume is 2 mL.

[0117] 1.4 Bronchoalveolar lavage fluid collection method: Following the operating procedure, sterile saline was injected into the local alveoli using a bronchoscope, and the lavage fluid was collected under negative pressure into a sterile sampling tube. The sample volume of bronchoalveolar lavage fluid was 2 mL.

[0118] 2. Sample processing

[0119] 2.1 Processing of plasma / tongue swabs / bronchoalveolar lavage fluid / sputum samples: Samples were extracted using Jiangsu Weizhen nucleic acid extraction and purification reagent.

[0120] 3. Nucleic acid testing

[0121] (1) PCR primers and probes

[0122] The following is a detailed list of the detection primers and probes designed for each detection segment, as shown in Table 1:

[0123] Table 1 Different detection primer and probe sequences

[0124] (2) PCR reaction system

[0125] Table 2 PCR reaction system

[0126] (3) PCR reaction procedure

[0127] Pre-denaturation at 95℃ for 5 min, followed by 50 cycles of 95℃ for 10 s, 60℃ for 30 s, and 40℃ for 20 s.

[0128] 4. Clinical sample testing

[0129] Sixty patients with clinically diagnosed pulmonary tuberculosis and non-pulmonary tuberculosis were included in this study, and four different types of samples were collected (plasma, tongue swabs, sputum, and bronchoalveolar lavage fluid). All clinical samples underwent medical ethics review, patient informed consent review, and corresponding data privacy and security audits.

[0130] 5. PCR test results of different segments

[0131] Table 3. Detection results for different detection sections

[0132] Table 4 Analysis of PCR detection results for different detection segments

[0133] The analysis results of the detection data in Table 4 show that the PCR detection performance of IS6110 in segments 1 and 2 is consistent with the PCR detection performance of segments 3 and 4 reported in the prior art across different sample types.

[0134] Example 3: Diagnostic efficacy of novel molecular biomarkers—based on isothermal amplification + CRISPR detection technology

[0135] In this embodiment, the analytical performance (using quality control materials) and clinical performance (using clinical samples) of segments 1-4 were evaluated based on isothermal amplification combined with CRISPR-Cas12a or CRISPR-Cas13a detection technology.

[0136] 1. Preprocessing

[0137] Quality control samples: Reference samples S2-S4 were extracted using the extraction kit described in 2.1 of Example 2;

[0138] Reference products S2-S4 are Chinese national standard reference products for Mycobacterium tuberculosis PCR detection kits purchased from the China National Institutes for Food and Drug Control. The approval number for reference product S2-S4 is (2010) Guo Sheng Can Zi 0042, batch number: 230030-202205. Reference products S2-S4 are used to detect the minimum detection limit. Specific specifications are as follows:

[0139] Test subject: Single-cell suspension of Mycobacterium tuberculosis (CMCC 93009);

[0140] S2 specification: 100 bacteria / mL;

[0141] S3 specification: 10 bacteria / mL;

[0142] S4 specification: 1 bacterium / mL.

[0143] Clinical samples: Sputum and tongue swab samples were collected from 20 patients with pulmonary tuberculosis and 20 patients without pulmonary tuberculosis. The samples were extracted using the procedures in 1.2-1.3 of Example 2.

[0144] 2. Marker detection

[0145] Based on primer and crRNA design principles, each of the four segments was detected using RAA, CRISPR fluorescence, and chromatography with 10 pairs of primers per segment. Primer positions were randomized to ensure that each group of 10 primer pairs covered the entire segment.

[0146] (1) Reaction system

[0147] The CRISPR detection system is shown in Tables 5-1 and 5-2 below.

[0148] Table 5-1 CRISPR-Cas12a detection system

[0149] Table 5-2 CRISPR-Cas13a Detection System

[0150] CRISPR detection primers are shown in Tables 5-3 to 5-10 below.

[0151] Table 5-3 CRISPR-Cas12a detection primers (segment 1)

[0152] Table 5-4 CRISPR-Cas12a detection primers (segment 2)

[0153] Table 5-5 CRISPR-Cas12a detection primers (segment 3)

[0154] Table 5-6 CRISPR-Cas12a detection primers (segment 4)

[0155] Table 5-7 CRISPR-Cas13a detection primers (segment 1)

[0156] Table 5-8 CRISPR-Cas13a detection primers (segment 2)

[0157] Table 5-9 CRISPR-Cas13a detection primers (segment 3)

[0158] Table 5-10 CRISPR-Cas13a detection primers (segment 4)

[0159] (2) Reaction procedure:

[0160] After mixing and centrifuging the above products, the mixture was placed in a 37°C water bath for reaction. 50 μL of each product was then used for fluorescence CRISPR detection and isothermal amplification test strip detection. The test strips and isothermal amplification reagents were purchased from Shanghai Liangrun.

[0161] 3. Test Results

[0162] Table 6. Test results of RAA-CRISPR-Cas13a quality control materials and samples in different sections of IS6110 (chromatographic method)

[0163] Table 7. Detection results (fluorescence method) of RAA-CRISPR-Cas12a quality control samples and samples from different sections of IS6110.

[0164] 4. Results Analysis

[0165] Table 8. Comparison of analytical performance of four detection zones based on RAA-CRISPR chromatography and fluorescence method.

[0166] The results of TB-CRISPR-Cas12a fluorescence assay and TB-CRISPR-Cas13a chromatography showed that detection segments 1 and 2 exhibited a consistently high positive detection rate of 75%-95% in sputum and tongue swab samples from pulmonary tuberculosis, significantly superior to other detection segments (see Figures 1-4). This indicates that detection segments 1 and 2 are superior biomarkers for isothermal amplification detection, possessing better detection performance and accuracy.

[0167] Example 4: Diagnostic efficacy of novel molecular biomarkers – based on RAA fluorescence technique

[0168] In this embodiment, an IS6110 detection system based on RAA fluorescence method was constructed. Ten detection systems were designed for each of the four segments in Example 1 according to primer design principles, and the system performance was tested.

[0169] 1. Test Methods

[0170] Twenty-five sputum samples and 25 tongue swab samples were collected from individuals with clinically confirmed tuberculosis, 25 tongue swabs from healthy individuals, and 25 sputum samples from non-tuberculosis patients. Samples were extracted according to the method in Example 2, and RAA fluorescence assay was used for detection.

[0171] 2. Detection Method

[0172] (1) Reaction system and primer sequence

[0173] The reaction system is shown in Table 9-1.

[0174] Table 9-1 RAA-fluorescence reaction system

[0175] Primer sequences are shown in Table 9-2.

[0176] Table 9-2 RAA-fluorescent primers (segment 1)

[0177] Table 9-3 RAA-fluorescent primers (segment 2)

[0178] Table 9-4 RAA-fluorescent primers (segment 3)

[0179] Table 9-5 RAA-fluorescent primers (segment 4)

[0180] (2) Reaction program: 42℃ for 1 min for 30 cycles, and finally cooling down to 4℃ for 2 min.

[0181] 3. Test Results

[0182] The test results are shown in Table 10 and Figures 5-6. According to the test results in Figures 5-6, RAA fluorescence detection showed superior performance in detection segments 1 and 2 in 25 tongue swab and sputum samples.

[0183] (1) Comparison of analytical performance of four detection sections

[0184] Table 10 Comparison of analytical performance of four detection zones based on RAA fluorescence method

[0185] (2) The clinical performance comparison results of the four detection segments are shown in Figures 5 and 6.

[0186] Example 5: Diagnostic efficacy of novel molecular biomarkers—based on LAMP technology

[0187] This embodiment constructs an IS6110 fluorescence detection system based on LAMP amplification, and the specific process is shown below.

[0188] 1. Based on primer design principles, four different primer sequences were designed for LAMP amplification.

[0189] Table 11-1 LAMP reaction primers (segment 1)

[0190] Table 11-2 LAMP reaction primers (segment 2)

[0191] Table 11-3 LAMP reaction primers (segment 3)

[0192] Table 11-4 LAMP reaction primers (segment 4)

[0193] 2. Detection Method

[0194] (1) The LAMP reaction mixture was prepared as shown in Table 11-5.

[0195] Table 11-5 LAMP reaction system

[0196] (2) After preparation, place at 65℃ for 30 min.

[0197] 3. Test Results

[0198] (1) The results of LAMP fluorescence detection are shown in Tables 12 and 13 and Figures 7-8.

[0199] Table 12 Detection results of LAMP fluorescence control samples and samples

[0200] Table 13 Comparison of analytical performance of four detection zones based on LAMP fluorescence method

[0201] According to the detection results in Tables 12-13 and Figures 7-8, the detection effects of LAMP amplification fluorescence detection segments 1 and 2 are better, and the sensitivity of the detection reference is better. The detection sensitivity in sputum and tongue swab samples is ≥87%, which is far superior to detection segments 3 and 4.

[0202] In Examples 2-5 above, this disclosure found that detection regions 1 and 2 of the IS6110 gene exhibit significantly higher detection sensitivity in both quality control samples and paired sputum and tongue swab samples, far superior to detection regions 3 and 4 reported by conventional techniques. This demonstrates that our detection method has significant advantages in accuracy and reliability, and provides a more reliable option for TB diagnosis.

[0203] Example 6: A kit for diagnosing Mycobacterium tuberculosis infection (isothermal amplification-CRISPR)

[0204] 1. Novel Molecular Marker Reagent Kit

[0205] By using novel molecular markers—segment 1 and segment 2—and an isothermal amplification and CRISPR detection system constructed according to Example 3, a kit for diagnosing Mycobacterium tuberculosis infection was established (CRISPR fluorescence method). Furthermore, by combining an integrated sealing device and lyophilization technology (CN 114058489 A / CN 116103377 B / CN 116496869 A), a kit for diagnosing Mycobacterium tuberculosis infection was established (CRISPR chromatography method).

[0206] According to the results of Example 3, the detection limits of 9 out of 10 systems in Section 1 and Section 2 were as low as reference S4 (1 bacterium / ml), and the positive detection rates of clinical sputum samples and tongue swab samples were ≥75%. Therefore, the systems randomly selected from each section were prepared into kits for comparison with commercial kits.

[0207] 2. Research Methods

[0208] Collect the following samples with clinical diagnostic results:

[0209] (1) Tongue swab samples from 20 patients with pulmonary tuberculosis and 10 patients without pulmonary tuberculosis were collected. The above 30 samples were tested using the RAA-CRISPR fluorescence detection kit constructed in Section 1 (using System 3 of Table 5-3) and Section 2 (using System 5 of Table 5-4), the RAA-CRISPR chromatography detection kit constructed in Section 1 (using System 1 of Table 5-3) and Section 2 (using System 1 of Table 5-4), the Sansure PCR commercial kit (Section 3), and the Cepheid GeneXpert commercial kit (Section 4).

[0210] (2) Sputum samples from 20 patients with pulmonary tuberculosis and 10 patients without pulmonary tuberculosis were analyzed using Section 1 (using System 5 of Table 5-3) and Section 2 (using System 6 of Table 5-4) as described above; bronchoalveolar lavage fluid samples from 10 patients with pulmonary tuberculosis were analyzed using Section 1 (using System 7 of Table 5-3) and Section 2 (using System 9 of Table 5-4) as described above; and plasma samples from 10 patients with pulmonary tuberculosis were analyzed using Section 1 (using System 10 of Table 5-3) and Section 2 (using System 8 of Table 5-4) as described above. The above 50 samples were tested using different RAA-CRISPR fluorescence detection kits constructed using Section 1 and Section 2, as well as the Sansure PCR commercial kit (Section 3) and the Cepheid GeneXpert commercial kit (Section 4).

[0211] 3. Test Results

[0212] Table 14. Results of tongue swab sample testing

[0213] Table 15. Detection results of sputum samples

[0214] Table 16 Detection Results of Bronchoalveolar Lavage Fluid Samples

[0215] Table 17 Plasma Sample Detection Results

[0216] 1. Analysis of test results

[0217] Table 18 Comparison of detection rates between CRISPR chromatography and fluorescence assay kits for tongue swabs and commercial kits.

[0218] Table 19 Comparison of detection rates between CRISPR fluorescence assay kits and commercial kits in sputum / bronchoalveolar lavage fluid / plasma

[0219] Analysis of Tables 14-19 shows that the Mycobacterium tuberculosis complex detection kit (CRISPR immunochromatography, fluorescence method) based on TB-CRISPR detection segments 1 and 2 established in this disclosure has a detection sensitivity of 85%-95% and a specificity of 100% in tongue swab samples, which is significantly better than commercially available detection kits. The same applies to the Mycobacterium tuberculosis complex detection kit (CRISPR fluorescence method) in sputum samples / bronchoalveolar lavage fluid / plasma.

[0220] Example 7: A kit for diagnosing Mycobacterium tuberculosis infection (isothermal amplification fluorescence method)

[0221] 1. Reagent kit

[0222] A kit for diagnosing Mycobacterium tuberculosis infection was established by using any one of the 10 detection systems for RAA and LAMP fluorescence detection constructed according to Examples 4 and 5, based on novel molecular markers - Segment 1 and Segment 2.

[0223] Among them, the RAA fluorescence detection section 1 of reference sample S4 adopts system 3 of Table 9-2, the detection section 2 adopts system 5 of Table 9-3, and the RAA fluorescence detection section 1 of tongue swab sample adopts system 4 of Table 9-2, the detection section 2 adopts system 7 of Table 9-3.

[0224] For sputum samples, RAA fluorescence detection in segment 1 used system 7 of Table 9-2, and detection in segment 2 used system 10 of Table 9-3; for bronchoalveolar lavage fluid and plasma samples, RAA fluorescence detection in segment 1 used system 2 of Table 9-2, and detection in segment 2 used system 8 of Table 9-3.

[0225] For reference sample S4, LAMP fluorescence detection segment 1 uses system 5 of Table 11-1, and detection segment 2 uses system 7 of Table 11-2. For tongue swab samples, LAMP fluorescence detection segment 1 uses system 3 of Table 11-1, and detection segment 2 uses system 9 of Table 11-2. For sputum samples, LAMP fluorescence detection segment 1 uses system 8 of Table 11-1, and detection segment 2 uses system 3 of Table 11-2. For bronchoalveolar lavage fluid and plasma samples, LAMP fluorescence detection segment 1 uses system 9 of Table 11-1, and detection segment 2 uses system 1 of Table 11-2.

[0226] 2. Research Methods

[0227] Analytical performance: Detects quality control samples (reference sample S4 diluted proportionally).

[0228] Collect the following samples with clinical diagnostic results:

[0229] (1) Tongue swab samples from 15 patients with pulmonary tuberculosis and 15 patients without pulmonary tuberculosis; (2) Sputum samples from 15 patients with pulmonary tuberculosis and 15 patients without pulmonary tuberculosis; (3) Bronchoalveolar lavage fluid samples from 10 patients with pulmonary tuberculosis; (4) Plasma samples from 10 patients with pulmonary tuberculosis. The above 80 samples were detected using the fluorescence detection kit constructed in sections 1 and 2 above, as well as the Sansure PCR commercial kit (section 3) and the Cepheid GeneXpert commercial kit (section 4).

[0230] 3. Analysis of test results

[0231] (1) Analysis performance

[0232] Table 20 Test Results of Reference Sample S4

[0233] (2) Clinical performance

[0234] Table 21 Results of RAA and LAMP fluorescence detection in tongue swabs

[0235] Table 22 Detection results of RAA and LAMP fluorescence samples in sputum samples

[0236] Table 23 Detection results of RAA and LAMP fluorescence samples in bronchoalveolar lavage fluid samples

[0237] Table 24 Results of RAA and LAMP fluorescence detection in plasma samples

[0238] Table 25 Comparison of sensitivity of different detection kits for RAA and LAMP fluorescent samples.

[0239] According to the results in Tables 20-25, based on RAA and LAMP fluorescence detection technologies, we compared detection segment 1 and detection segment 2. Both detection references S4 and S4 after proportional dilution were detectable. Furthermore, we found that our detection method showed significantly better detection sensitivity than commercially available kits in tongue swabs and sputum / bronchoalveolar lavage fluid / plasma samples.

[0240] Specifically, the results show that the RAA and LAMP fluorescence detection kits disclosed in this publication can stably detect the reference sample at 0.5 bacteria / ml after serial dilution in S4, while commercially available kits cannot meet this standard, and their sensitivity is significantly lower than that of our kit. In 15 tongue swab samples, the sensitivity of our kit reached 87%-93%, exceeding that of the commercially available Cepheid GeneXpert and Sansure PCR detection kits, which were 67% and 60%, respectively. The same trend was observed in other sample types, such as sputum samples (93% vs 87% vs 80%), bronchoalveolar lavage fluid (80% vs 70% vs 70%), and plasma samples (70%-80% vs 60% vs 60%), where our kit demonstrated higher sensitivity and reliability. These results indicate that our kit provides a powerful tool for the early diagnosis and treatment of tuberculosis.

[0241] Example 8 uses other isothermal amplification methods to verify the clinical detection availability of segment 1 and segment 2.

[0242] 1. Research Methods

[0243] Using novel molecular markers – segment 1 and segment 2 – highly sensitive rolling circle amplification (HiRCA) and multiple substitution amplification (MDA) cross primer amplification (CPA) methods, and following primer design principles, different detection systems were designed to detect samples with clinical diagnostic results:

[0244] (1) Tongue swab samples from 20 patients with pulmonary tuberculosis and 20 patients without pulmonary tuberculosis. (2) Sputum samples from 20 patients with pulmonary tuberculosis and 20 patients without pulmonary tuberculosis. Samples were extracted using the procedures in 1.2-1.3 of Example 2.

[0245] 2. Analysis of test results

[0246] Table 26 Summary of Clinical Sample Detection Performance of Different Detection Methods (HiRCA / MDA / CPA Method) for Sections 1 and 2

[0247] As shown in Table 26, in 20 tongue swabs and 20 sputum samples, the detection sensitivity of different detection methods (HiRCA / MDA / CPA) for segments 1 and 2 ranged from 85% to 95%, with a specificity of 100%. Our results indicate that our detection segments 1 and 2 exhibited high detection sensitivity under different isothermal amplification methods. This means that regardless of the isothermal amplification method used, we can efficiently detect the target substance, providing a more reliable and stable basis for our experiments.

[0248] Example 9: Testing of Environmental Samples

[0249] 1. Research Methods

[0250] This experiment used the test kits described in Examples 6, 7, and 8 to test different environmental samples. The test results were compared with those from a hospital laboratory to verify the effectiveness of the test kits consisting of segments 1 and 2 in testing environmental samples.

[0251] 2. Results Analysis

[0252] Table 27 Summary of Environmental Sample Detection Results Using Different Constant Temperature Detection Methods

[0253] According to the results in Table 27, the test kit formed by segments 1 and 2 of the isothermal detection method in this embodiment is not only suitable for the detection of bodily fluid samples such as human swabs or sputum, but also suitable for the detection of Mycobacterium tuberculosis in different environmental samples, thus its applicability is more extensive.

[0254] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the disclosure. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the scope of protection of this disclosure. Therefore, the scope of protection of this patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

Application of nucleic acid biomarker detection reagents in the preparation of reagents or kits for the diagnosis of Mycobacterium tuberculosis infection; in, The sequence of the nucleic acid biomarker is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2, and the detection reagent is used to detect the nucleic acid biomarker in a biological sample by isothermal amplification reaction. The application according to claim 1 is characterized in that, The detection reagent includes primers and / or probes that target the nucleic acid biomarker. The application according to any one of the preceding claims is characterized in that, The isothermal amplification reaction is one or more of the following: recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, multiple substitution amplification, cross primer amplification, recombinase-mediated amplification, novel recombinase-dependent amplification, chain substitution amplification, and helicase-dependent amplification. The application according to any one of the preceding claims is characterized in that, The reagent or kit contains a strand displacement DNA polymerase and / or recombinase system. The application according to any one of claims 1-4 is characterized in that, The reagent or kit also includes an isothermal amplification reaction product detection reagent to determine the presence or absence of Mycobacterium tuberculosis. The application according to any one of the preceding claims is characterized in that, The isothermal amplification reaction product detection reagent includes at least one of the following: a CRISPR-Cas detection system, a dye and / or auxiliary agent for detecting DNA double strands, a fluorescent probe for hybridizing with the isothermal amplification reaction product, and a pH colorimetric reagent. The application according to any one of the preceding claims is characterized in that, The CRISPR-Cas includes one or more of the following: a CRISPR-Cas12 detection system, a CRISPR-Cas13 detection system, and a Cas detection system having bypass cleavage activity similar to that of Cas12 and / or Cas13. The application according to any one of the preceding claims is characterized in that, At least one component of the reagent or kit is a solid, and the solid includes at least one of lyophilized microspheres, lyophilized cakes, lyophilized powders, and spots that depend on the presence of a solid medium. The application according to any one of the preceding claims is characterized in that, The biological sample is selected from one or more of the following: tongue swab, sputum, saliva, serum, plasma, blood, urine, mucus, sweat, tears or other eye fluids, ear fluids, blisters, sores, gastric juice, gastric juice, bronchoalveolar lavage fluid, feces, pancreatic juice or juice, pharyngeal swab, nasal swab, conjunctival swab, semen, breast milk, cerebrospinal fluid, liquid bone marrow, and lymph. The reagent or kit as defined in any one of claims 1-9. A diagnostic device for Mycobacterium tuberculosis, the diagnostic device comprising one or more independent modules, at least one independent module comprising the reagent or kit of claim 10. According to the diagnostic device of claim 11, the independent module includes one or more housing units, the housing units being provided with a nucleic acid marker amplification system and / or a detection system. The diagnostic device according to claim 12, wherein at least one housing unit contains at least a portion of the components of the reagent or kit. A method for detecting Mycobacterium tuberculosis in a sample includes: a) Obtain isolated nucleic acids from the sample; b) Amplify potential nucleic acid markers in the nucleic acids using an isothermal amplification reaction; The sequence of the nucleic acid biomarker is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; and c) the presence of the nucleic acid biomarker is detected. According to the method of claim 14, the isothermal amplification reaction includes one or more of the following: recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, multiple substitution amplification, cross-primer amplification, recombinase-mediated amplification, novel recombinase-dependent amplification, chain substitution amplification, and helicase-dependent amplification. According to the method of claim 14 or 15, the detection method in c) is selected from at least one of CRISPR-Cas method, fluorescence method, and lateral flow immunochromatography. The application of nucleic acid biomarkers in the preparation of reagents or kits for the diagnosis of Mycobacterium tuberculosis infection, wherein the sequences of the nucleic acid biomarkers are shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.

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